CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of International Patent Application Serial No. PCT/CN2016/073722, filed February 6, 2016, which is herein incorporated by reference in its entirety for all purposes.

FIELD OF INVENTION

[0002] The present invention relates to multivalent and multispecific binding proteins, and to methods of making and using multivalent and multispecific binding proteins.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0003] The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: EPBI_002_01WO_SeqList_ST25.txt, date recorded: February 3, 2017, file size 510KB).

BACKGROUND OF THE INVENTION

[0004] Bispecific or multispecific antibodies have been generated in attempts to prepare molecules useful for the treatment of various inflammatory diseases, cancers, and other disorders.

[0005] Bispecific antibodies have been produced using the quadroma technology (see Milstein, C. and A.C. Cuello, Nature, 1983. 305(5934): p. 537-40) based on the somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificities of the bispecific antibody. Bispecific antibodies can also be produced by chemical conjugation of two different mAbs (see Staerz, U.D., et al, Nature, 1985. 314(6012): p. 628-31). Other approaches have used chemical conjugation of two different monoclonal antibodies or smaller antibody fragments (see Brennan, M., et al., Science, 1985. 229(4708): p. 81-3).

[0006] Another method is the coupling of two parental antibodies with a hetero-bifunctional crosslinker. In particular, two different Fab fragments have been chemically crosslinked at their hinge cysteine residues in a site-directed manner (see Glennie, M.J., et al., J Immunol, 1987. 139(7): p. 2367-75).

[0007] Other recombinant bispecific antibody formats have been developed in the recent past (see Kriangkum, J., et al, Biomol Eng, 2001. 18(2): p. 31-40). Amongst them tandem single- chain Fv molecules and diabodies, and various derivatives thereof, have been used for the construction of recombinant bispecific antibodies. Normally, construction of these molecules starts from two single-chain Fv (scFv) fragments that recognize different antigens (see Economides, A.N., et al, Nat Med, 2003. 9(1): p. 47-52). Tandem scFv molecules (taFv) represent a straightforward format simply connecting the two scFv molecules with an additional peptide linker. The two scFv fragments present in these tandem scFv molecules form separate folding entities. Various linkers can be used to connect the two scFv fragments and linkers with a length of up to 63 residues (see Nakanishi, K., et al. Annu Rev Immunol,

2001. 19: p. 423-74).

[0008] In a recent study, in vivo expression by transgenic rabbits and cattle of a tandem scFv directed against CD28 and a melanoma-associated proteoglycan was reported (see Gracie, J.A., et al, J Clin Invest, 1999. 104(10): p. 1393-401). In this construct the two scFv molecules were connected by a CHI linker and serum concentrations of up to 100 mg/L of the bispecific antibody were found. A few studies have now reported expression of soluble tandem scFv molecules in bacteria (see Leung, B.P., et al, J Immunol, 2000. 164(12): p. 6495-502; Ito, A., et al, J Immunol, 2003. 170(9): p. 4802-9; Kami, A., et al, J Neuroimmunol, 2002. 125(1-2): p. 134-40) using either a very short Ala3 linker or long glycine/serine-rich linkers.

[0009] In a recent study, phage display of a tandem scFv repertoire containing randomized middle linkers with a length of 3 or 6 residues enriched those molecules which are produced in soluble and active form in bacteria. This approach resulted in the isolation of a preferred tandem scFv molecule with a 6 amino acid residue linker (see Arndt, M. and J. Krauss, Methods Mol Biol, 2003. 207: p. 305-21).

[0010] Bispecific diabodies (Db) utilize the diabody format for expression. Diabodies are produced from scFv fragments by reducing the length of the linker connecting the VH and VL domain to approximately 5 residues (see Peipp, M. and T. Valerius, Biochem Soc Trans,

2002. 30(4): p. 507-11). This reduction of linker size facilitates dimerization of two polypeptide chains by crossover pairing of the VH and VL domains. Bispecific diabodies are produced by expressing two polypeptide chains with either the structure VHA-VLB and VHB-VLA (VH-VL configuration) or VLA-VHB and VLB-VHA (VL-VH configuration) within the same cell. A recent comparative study demonstrates that the orientation of the variable domains can influence expression and formation of active binding sites (see Mack, M., G. Riethmuller, and P. Kufer, Proc Natl Acad Sci U S A, 1995. 92(15): p. 7021-5).

[0011] One approach to force the generation of bispecific diabodies is the production of knob-into-hole diabodies (see Holliger, P., T. Prospero, and G. Winter, Proc Natl Acad Sci U S A, 1993. 90(14): p. 6444-8.18). This was demonstrated for a bispecific diabody directed against HER2 and CD3. A large knob was introduced in the VH domain by exchanging Val37 with Phe and Leu45 with Trp and a complementary hole was produced in the VL domain by mutating Phe98 to Met and Tyr87 to Ala, either in the anti- HER2 or the anti-CD3 variable domains. By using this approach the production of bispecific diabodies could be increased from 72% by the parental diabody to over 90% by the knob-into-hole diabody.

[0012] Single-chain diabodies (scDb) represent an alternative strategy to improve the formation of bispecific diabody-like molecules (see Holliger, P. and G. Winter, Cancer Immunol Immunother, 1997. 45(3-4): p. 128-30; Wu, A.M., et al, Immunotechnology, 1996. 2(1): p. 21-36). Bispecific single-chain diabodies are produced by connecting the two diabody-forming polypeptide chains with an additional middle linker with a length of approximately 15 amino acid residues. Consequently, all molecules with a molecular weight corresponding to monomeric single-chain diabodies (50-60 kDa) are bispecific. Several studies have demonstrated that bispecific single chain diabodies are expressed in bacteria in soluble and active form with the majority of purified molecules present as monomers (see Holliger, P. and G. Winter, Cancer Immunol Immunother, 1997. 45(3-4): p. 128-30; Wu, AM., et al, Immunotechnology, 1996. 2(1): p. 21-36; Pluckthun, A. and P. Pack, Immunotechnology, 1997. 3(2): p. 83-105; Ridgway, IB., et al, Protein Eng, 1996. 9(7): p. 617-21).

[0013] Diabody have been fused to Fc to generate more Ig-like molecules, named di-diabody (see Lu, D., et al, J Biol Chem, 2004. 279(4): p. 2856-65). In addition, multivalent antibody construct comprising two Fab repeats in the heavy chain of an IgG and capable of binding four antigen molecules has been described (see US patent 8,722,859 B2, and Miller, K., et al, J Immunol, 2003. 170(9): p. 4854-61).

[0014] The most recent examples are tetravalent IgG-single-chain variable fragment (scFv) fusions (Dong J, et al. 2011 MAbs 3:273-288; Coloma MJ, Morrison SL 1997 Nat

Biotechnol 15: 159-163; Lu D, et al. 2002 J Immunol Methods 267:213-226), catumaxomab, a trifunctional rat/mouse hybrid bispecific epithelial cell adhesion molecule-CD3 antibody (Lindhofer H, et al 1995 J Immunol 155:219-225), the bispecific CD19-CD3 scFv antibody blinatumomab (Bargou R, et al. 2008 Science 321 :974-977), "dual- acting Fab" (DAF) antibodies (BostromJ, et al. 2009 Science 323: 1610-1614), covalently linked pharmacophore peptides to catalytic anti- bodies (Doppalapudi VR, et al. 2010 Proc Natl Acad Sci USA 107:22611-22616), use of the dynamic exchange between half IgG4 molecules to generate bispecific antibodies (van der Neut Kolfschoten M, et al. 2007 Science 317: 1554-1557; Stubenrauch K, et al. 2010 Drug Metab Dispos 38:84-91), or by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody (CrossMab format) (Schaefer Wet al 2011 Proc Natl Acad Sci 108: 11187- 92).

[0015] There is a need in the art for single molecular entities with dual antigen binding function, and for methods of generating such multivalent and multispecific binding proteins. The present invention addresses these and other needs.

SUMMARY OF THE INVENTION

[0016] The present invention provides multivalent and multispecific binding proteins, and methods of making and using such binding proteins. In one embodiment, the multivalent and multispecific binding proteins provided herein are Fabs-in-tandem immunoglobulins (FIT- Ig), and are capable of binding two or more antigens, or two or more epitopes of the same antigen, or two or more copies of the same epitope. The multivalent and multispecific binding proteins provided herein are useful for treatment and/or prevention of acute and chronic inflammatory diseases and disorders, autoimmune diseases, cancers, spinal cord injuries, sepsis, and other diseases, disorders, and conditions. Pharmaceutical compositions comprising the multivalent and multispecific binding proteins are provided herein. In addition, nucleic acids, recombinant expression vectors, and host cells for making such FIT- Igs are provided herein. Methods of using the FIT-Igs of the invention to detect specific antigens, in vivo or in vitro, are also encompassed by the invention.

[0017] The present invention provides a family of binding proteins that are capable of binding two or more antigens, e.g., with high affinity. In one aspect, the present invention provides an approach to construct a bispecific binding protein using two parental monoclonal antibodies: mAb A, which binds to antigen A, and mAb B, which binds to antigen B. The binding proteins disclosed herein, in one embodiment, are capable of binding antigens, cytokines, chemokines, cytokine receptors, chemokine receptors, cytokine- or chemokine- related molecules, or cell surface proteins.

[0018] Thus, in one aspect, binding proteins capable of binding two or more antigens are provided. In one embodiment, the present invention provides a binding protein comprising at least two polypeptide chains, wherein the polypeptide chains pair to form IgG-like molecules capable of binding two or more antigens. In one embodiment, the binding protein comprises two, three, four, five, or more polypeptide chains. In one embodiment, the binding protein comprises at least one VLA, at least one VLB, at least one VHA, at least one VHB, at least one CL, and at least one CHI, wherein VL is a light chain variable domain, VH is a heavy chain variable domain, CL is a light chain constant domain, CHI is the first constant domain of the heavy chain, A is a first antigen, and B is a second antigen. In a further embodiment, the first polypeptide chain comprises a VLA, a CL, a VHB, and a CHI . In a further embodiment, the binding protein further comprises an Fc. In another embodiment, the Fc region is a variant Fc region. In a further embodiment, the variant Fc region exhibits modified effector function, such as ADCC or CDC. In another embodiment, the variant Fc region exhibits modified affinity or avidity for one or more FcyR.

[0019] In one embodiment, the binding protein comprises three polypeptide chains, wherein the first polypeptide chain comprises a VLA, a CL, a VHB, and a CHI, the second polypeptide chain comprises VHA and CHI, and the third polypeptide chain comprises VLB and CL. In a further embodiment, the first polypeptide chain of the binding protein further comprises an Fc. In another embodiment, the binding protein comprises two polypeptide chains, wherein the first polypeptide chain comprises a VLA, a CL, a VHB, and a CHI, the second polypeptide chain comprises VHA, CHI, VLB, and CL. In a further embodiment, the first polypeptide chain further comprises an Fc.

[0020] In one embodiment, the binding protein comprises three polypeptide chains, and their corresponding cDNA during co-transfection are present at a molar ratio of first: second:third of 1 : 1 : 1, 1 : 1.5: 1, 1 :3: 1, 1 : 1 : 1.5, 1 : 1 :3, 1 : 1.5: 1.5, 1 :3: 1.5, 1 : 1.5:3, or 1 :3:3. In another embodiment, the binding protein comprises two polypeptide chains, and their corresponding cDNA during co-transfection are present at a molar ratio of firstsecond of 1 : 1, 1 : 1.5, or 1 :3, or any other ratios, through optimization, in an effort to maximize the monomeric FIT-Ig fraction in any given transfection. [0021] In one embodiment, the binding protein of the present invention does not comprise a peptide linker. In one embodiment, the binding protein of the present invention comprises at least one amino acid or polypeptide linker. In a further embodiment, the linker is selected from the group consisting of G, GS, SG, GGS, GSG, SGG, GGG, GGGS (SEQ ID NO: 489), SGGG (SEQ ID NO: 490), GGGGS (SEQ ID NO: 491), GGGGSGS (SEQ ID NO: 492), , GGGGSGGS (SEQ ID NO: 493), GGGGS GGGGS (SEQ ID NO: 494), GGGGS GGGGS GGGGS (SEQ ID NO: 495), AKTTPKLEEGEFSEAR (SEQ ID NO: 496), AKTTPKLEEGEFSEARV (SEQ ID NO: 497), AKTTPKLGG (SEQ ID NO: 498), SAKTTPKLGG (SEQ ID NO: 499), SAKTTP (SEQ ID NO: 500), RADAAP (SEQ ID NO: 501), RADAAPTVS (SEQ ID NO: 502), RADAAAAGGPGS (SEQ ID NO: 503), RADAAAA(G ₄ S) ₄ (SEQ ID NO: 504), S AKTTPKLEEGEFSEARV (SEQ ID NO: 505), ADAAP (SEQ ID NO: 506), ADAAPTVSIFPP (SEQ ID NO: 507), TVAAP (SEQ ID NO: 508), TVAAPSVFIFPP (SEQ ID NO: 509), QPKAAP (SEQ ID NO: 510), QPKAAPSVTLFPP (SEQ ID NO: 511), AKTTPP (SEQ ID NO: 512), AKTTPPSVTPLAP (SEQ ID NO: 513), AKTTAPSVYPLAP (SEQ ID NO: 514), ASTKGP (SEQ ID NO: 515), ASTKGPSVFPLAP (SEQ ID NO: 516), GENKVEYAPALMALS (SEQ ID NO: 517), GPAKELTPLKEAKVS (SEQ ID NO: 518), GHEAAAVMQVQYPAS (SEQ ID NO: 519), and AKTTAP (SEQ ID NO: 80). The linkers can also be in vivo cleavable peptide linkers, protease (such as MMPs) sensitive linkers, disulfide bond-based linkers that can be cleaved by reduction, etc., as previously described (Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges, edited by Stefan R. Schmidt), or any cleavable linkers known in the art. Such cleavable linkers can be used to release the top Fab in vivo for various purposes, in order to improve tissue/cell penetration and distribution, to enhance binding to targets, to reduce potential side effect, as well as to modulate in vivo functional and physical half-life of the 2 different Fab regions.

[0022] In one embodiment, the binding protein comprises a first polypeptide comprising, from amino to carboxyl terminus, VLA-CL-VHB-CH1-FC, a second polypeptide chain comprising, from amino to carboxyl terminus, VHA-CHI, and a third polypeptide chain comprising, from amino to carboxyl terminus, VLB-CL; wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CHI is the first constant domain of the heavy chain, A is a first epitope or antigen, and B is a second epitope or antigen. In one embodiment, the Fc region is human IgGl . In another embodiment, the Fc region is a variant Fc region. In a further embodiment, the amino acid sequence of the Fc region is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 20. In a further embodiment, the CL of the first polypeptide chain is fused directly to VHB. In another embodiment, the CL of the first polypeptide chain is linked to VHB via an amino acid or an oligopeptide linker. In a further embodiment, the linker is GSG (SEQ ID NO: 26) or GGGGSGS (SEQ ID NO: 28).

[0023] In another embodiment, the binding protein comprises a first polypeptide comprising, from amino to carboxyl terminus, VHB-CH1-VLA-CL-FC, a second polypeptide chain comprising, from amino to carboxyl terminus, VHA-CHI, and a third polypeptide chain comprising, from amino to carboxyl terminus, VLB-CL; wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CHI is the first constant domain of the heavy chain, A is a first epitope or antigen, and B is a second epitope or antigen. In one embodiment, the Fc region is human IgGl . In another embodiment, the Fc region is a variant Fc region. In a further embodiment, the amino acid sequence of the Fc region is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 20. In one embodiment, the CHI of the first polypeptide chain is fused directly to VLA. In another embodiment, the CHI of the first polypeptide chain is linked to VLA via an amino acid or an oligopeptide linker. In a further embodiment, the linker is GSG (SEQ ID NO: 26) or GGGGSGS (SEQ ID NO: 28).

[0024] In another embodiment, the binding protein comprises a first polypeptide comprising, from amino to carboxyl terminus, VLA-CL-VHB-CH1-FC, and a second polypeptide chain comprising, from amino to carboxyl terminus, VHA-CH1-VLB-CL; wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CHI is the first constant domain of the heavy chain, A is a first epitope or antigen, and B is a second epitope or antigen. In one embodiment, the Fc region is human IgGl . In another embodiment, the Fc region is a variant Fc region. In a further embodiment, the amino acid sequence of the Fc region is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 20. In a further embodiment, the CL of the first polypeptide chain is fused directly to VHB. In another embodiment, the CL of the first polypeptide chain is linked to VHB via an amino acid or an oligopeptide linker. In a further embodiment, the linker is GSG (SEQ ID NO: 26) or GGGGSGS (SEQ ID NO: 28).

[0025] In another embodiment, binding protein comprises a first polypeptide comprising, from amino to carboxyl terminus, VHB-CH1-VLA-CL-FC, and a second polypeptide chain comprising, from amino to carboxyl terminus, VLB-CL-VHA-CH1; wherein VL is a light chain variable domain, CL is a light chain constant domain, VH is a heavy chain variable domain, CHI is the first constant domain of the heavy chain, A is a first epitope or antigen, and B is a second epitope or antigen. In one embodiment, the Fc region is human IgGl . In another embodiment, the Fc region is a variant Fc region. In a further embodiment, the amino acid sequence of the Fc region is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 20. In one embodiment, the CHI of the first polypeptide chain is fused directly to VLA. In another embodiment, the CHI of the first polypeptide chain is linked to VLA via an amino acid or an oligopeptide linker. In a further embodiment, the linker is GSG (SEQ ID NO: 26) or GGGGSGS (SEQ ID NO: 28).

[0026] The binding proteins of the present invention are capable of binding pairs of cytokines. For example, the binding proteins of the present invention are capable of binding pairs of cytokines selected from the group consisting of IL-loc and IL-Ι β; IL-12 and IL-18, TNF a and IL-23, TNF a and IL-13; TNF and IL-18; TNF and IL-12; TNF and IL-lbeta; TNF and MIF; TNF and IL-6, TNF and IL-6 Receptor, TNF and IL-17; IL-17 and IL-20; IL-17 and IL-23; TNF and IL-15; TNF and VEGF; VEGFR and EGFR; PDGFR and VEGF, IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAM8; and TNF a and PGE4, IL-13 and PED2, TNF and PEG2. In one embodiment, the binding proteins of the present invention are capable of binding IL-17 and IL-20. The binding proteins of the present invention, in one embodiment, are capable of binding IL-17 and IL-20 and comprise variable heavy and light chains derived from the anti-IL-17 antibody LY and the anti-IL-20 antibody 15D2. In one embodiment, the binding proteins of the present invention are capable of binding IL-17 and TNF. The binding proteins of the present invention, in one embodiment, are capable of binding IL-17 and TNF and comprise variable heavy and light chains derived from the anti- IL-17 antibody LY and the TNF antibody golimumab.

[0027] In one embodiment, the binding proteins of the present invention bind IL-17 and IL- 20 and comprise a first polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 25, and 27; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 21 ; and a third polypeptide chain comprising, consisting essentially of, or consisting of a sequence according to SEQ ID NO: 23. In another embodiment, the binding proteins of the present invention bind IL-27 and IL-20 and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 25, and 27, and a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 30, and 31.

[0028] In one embodiment, the binding proteins of the present invention bind TNF and IL-17 and comprise a first polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NOs: 87; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 89; and a third polypeptide chain comprising, consisting essentially of, or consisting of a sequence according to SEQ ID NO: 91. In another embodiment, the binding protein is capable of binding pairs of targets selected from the group consisting of CD137 and CD20, CD137 and EGFR, CD137 and Her-2, CD137 and PD-1, CD137 and PDL-1, VEGF and PD- Ll, Lag-3 and TIM-3, OX40 and PD-1, TIM-3 and PD-1, TIM-3 and PDL-1, EGFR and DLL-4, CD138 and CD20; CD138 and CD40; CD 19 and CD20; CD20 and CD3; CD3 and CD33; CD3 and CD133; CD47 and CD20, CD38 and CD138; CD38 and CD20; CD20 and CD22; CD38 and CD40; CD40 and CD20; CD-8 and IL-6; CSPGs and RGM A; CTLA-4 and BTN02; IGF1 and IGF2; IGF1/2 and Erb2B; IGF-1R and EGFR; EGFR and CD13; IGF- 1R and ErbB3; EGFR-2 and IGFR; VEGFR-2 and Met; VEGF- A and Angiopoietin-2 (Ang- 2); IL-12 and TWEAK; IL-13 and IL-lbeta; PDGFR and VEGF,, EpCAM and CD3, Her2 and CD3, CD19 and CD3, EGFR and Her3, CD16a and CD30, CD30 and PSMA, EGFR and CD3, CEA and CD3, TROP-2 and HSG, TROP-2 and CD3, MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; PDL-1 and CTLA-4; CTLA-4 and PD-1 ; PD-1 and TIM-3; RGM A and RGM B; Te38 and TNFoc; TNFoc and Blys; TNFoc and CD-22; TNF a and CTLA-4 domain; TNF a and GP130; TNFoc and IL-12p40; and TNF a and RANK ligand, Factor IXa and Factor X; EGFR and PD-L1; EGFR and cMet; Her3 and IGF- IR; DLL-4 and VEGF; PD-1 and PD-L1 ; and Her3 and PD-1.

[0029] In one embodiment, the binding proteins of the present invention are capable of binding CD3 and CD20. The binding proteins of the present invention, in one embodiment, are capable of binding CD3 and CD20 and comprise variable heavy and light chains derived from the anti-CD3 antibody OKT3 or the anti-CD3 antibody disclosed in U.S. 2009/0252683, which is incorporated herein by reference in it entirety; and the anti-CD20 antibody ofatumumab. In some embodiments, the polypeptide derived from CD3 antibody is in the upper domain and the polypeptide derived from CD20 antibody is in the lower domain. As used herein, the upper domain is the N-terminal or "amino proximal" domain, and the lower domain is the C-terminal domain or the domain closer to the Fc, if present. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1- Fc, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is CD3, and antigen B is CD20. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is CD3, and antigen A is CD20. In some embodiments, polypeptide derived from CD3 antibody is in the lower domain and polypeptide derived from CD20 antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is CD20, and antigen B is CD-3. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is CD20, and antigen A is CD3.

[0030] In one embodiment, the binding proteins of the present invention bind CD3 and CD20 and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 and 48; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 44; and a third polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 46. In another embodiment, the binding proteins of the present invention bind CD20 and CD3 and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 114; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 115; and a third polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 116.

[0031] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of CD20 and the same epitope of CD3 as that of bispecific binding protein

FIT018a, wherein the bispecific binding protein FIT018a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 316; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 325; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 330.

[0032] In one embodiment, the binding proteins of the present invention bind CD3 and CD20 and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 318, a VLA CDR2 of SEQ ID NO: 319, and a VLA CDR3 of SEQ ID NO: 320.

[0033] In one embodiment, the binding proteins of the present invention bind CD3 and CD20 and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 322, a VHB CDR2 of SEQ ID NO: 323, and a VHB CDR3 of SEQ ID NO: 324.

[0034] In one embodiment, the binding proteins of the present invention bind CD3 and CD20 and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 327, a VHA CDR2 of SEQ ID NO: 328, and a VHA CDR3 of SEQ ID NO: 329.

[0035] In one embodiment, the binding proteins of the present invention bind CD3 and CD20 and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 332, a VLB CDR2 of SEQ ID NO: 333, and a VLB CDR3 of SEQ ID NO: 334.

[0036] In one embodiment, the binding proteins of the present invention bind CD3 and CD20 and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 318, a VLA CDR2 of SEQ ID NO: 319, and a VLA CDR3 of SEQ ID NO: 320; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 322, a VHB CDR2 of SEQ ID NO: 323, and a VHB CDR3 of SEQ ID NO: 324, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 327, a VHA CDR2 of SEQ ID NO: 328, and a VHA CDR3 of SEQ ID NO: 329; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 332, a VLB CDR2 of SEQ ID NO: 333, and a VLB CDR3 of SEQ ID NO: 334.

[0037] In one embodiment, the binding proteins of the present invention bind CD3 and CD20 and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 317, and a VHB having the sequence of SEQ ID NO: 321, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 326, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 331.

[0038] In one embodiment, the binding proteins of the present invention bind CD3 and CD20 and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 316; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 325; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 330.

[0039] In one embodiment, the binding proteins of the present invention bind CD3 and CD20, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[0040] In one embodiment, the binding proteins of the present invention are capable of binding CTLA-4 and PD-1. The binding proteins of the present invention, in one embodiment, are capable of binding CTLA-4 and PD-1 and comprise variable heavy and light chains derived from the CTLA-4 antibody ipilimumab and the PD-1 antibody nivolumab.

[0041] In one embodiment, the binding proteins of the present invention bind CTLA-4 and

PD-1 and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 92; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 95; and a third polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 97. In one embodiment, the binding protein provided herein is capable of binding one or more epitopes on CTLA-4. In one embodiment, the binding protein provided herein is capable of binding one or more epitopes on PD-1. In some embodiments, polypeptide derived from CTLA-4 antibody is in the upper domain and polypeptide derived from PD-1 antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is CTLA-4, and antigen B is PD-1. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-

CH1-VLA-CL-FC, a second polypeptide of VLB-CL, and a third polypeptide of VHA-CHI, wherein antigen B is CTLA-4, and antigen A is PD-1. In some embodiments, polypeptide derived from CTLA-4 antibody is in the lower domain and polypeptide derived from PD-1 antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is PD-1, and antigen B is CTLA-4. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB-CL, and a third polypeptide of VHA- CHI, wherein antigen B is PD-1, and antigen A is CTLA-4.

[0042] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of CTLA-4 and the same epitope of PD-1 as that of bispecific binding proteins NBS3, NBS3R, NBS3-C, or NBS3R-C, as described herein.

[0043] The bispecific binding protein NBS3 comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 126; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 135; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 140.

[0044] The bispecific binding protein NBS3R comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 145; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 154; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 159.

[0045] The bispecific binding protein NBS3-C comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 164; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 173; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 178.

[0046] The bispecific binding protein NBS3R-C comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 183; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 192; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 197.

[0047] In one embodiment, the binding proteins of the present invention bind CTLA4 and

PD-1 and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 128, a VLA CDR2 of SEQ ID NO: 129, and a VLA

CDR3 of SEQ ID NO: 130 (e.g., those on NBS3). In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprises a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 147, a VLA CDR2 of SEQ ID NO: 148, and a VLA CDR3 of SEQ ID NO: 149 (e.g., those on NBS3R).

In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprises a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 166, a VLA CDR2 of SEQ ID NO: 167, and a VLA CDR3 of

SEQ ID NO: 168 (e.g., those on NBS3-C). In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprises a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 185, a VLA

CDR2 of SEQ ID NO: 186, and a VLA CDR3 of SEQ ID NO: 187 (e.g., those on NBS3R-C).

[0048] In one embodiment, the binding proteins of the present invention bind CTLA4 and

PD-1 and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 132, a VHB CDR2 of SEQ ID NO: 133, and a VHB

CDR3 of SEQ ID NO: 134 (e.g., those on NBS3). In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 151, a VHB

CDR2 of SEQ ID NO: 152, and a VHB CDR3 of SEQ ID NO: 153 (e.g., those on NBS3R). In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 166, a VHB CDR2 of SEQ ID NO: 167, and a VHB CDR3 of

SEQ ID NO: 168 (e.g., those on NBS3-C). In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprise a VHB CDRl of SEQ ID NO: 185, a VHB

CDR2 of SEQ ID NO: 186, and a VHB CDR3 of SEQ ID NO: 187 (e.g., those on NBS3R-C).

[0049] In one embodiment, the binding proteins of the present invention bind CTLA4 and

PD-1 and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 137, a VHA CDR2 of SEQ ID NO: 138, and a VHA CDR3 of SEQ ID NO: 139 (e.g., those on NBS3). In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprises a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of

SEQ ID NO: 156, a VHA CDR2 of SEQ ID NO: 157, and a VHA CDR3 of SEQ ID NO: 158

(e.g., those on NBS3R). In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprises a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 175, a VHA CDR2 of SEQ

ID NO: 176, and a VHA CDR3 of SEQ ID NO: 177 (e.g., those on NBS-C). In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprises a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 194, a VHA CDR2 of SEQ ID NO: 195, and a VHA CDR3 of SEQ ID NO: 196 (e.g., those on NBS3R-C).

[0050] In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 142, a VLB CDR2 of SEQ ID NO: 143, and a VLB CDR3 of SEQ ID NO: 144 (e.g., those on NBS3). In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprises a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 161, a VLB CDR2 of SEQ ID NO: 162, and a VLB CDR3 of SEQ ID NO: 163 (e.g., those on NBS3R). In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprises a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 180, a VLB CDR2 of SEQ ID NO: 181, and a VLB CDR3 of SEQ ID NO: 182 (e.g., those on NBS3-C). In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprises a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 199, a VLB CDR2 of SEQ ID NO: 200, and a VLB CDR3 of SEQ ID NO: 201 (e.g., those on NBS3R-C).

[0051] In one embodiment, the binding proteins of the present invention bind CTLA4 and

PD-1 and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 128, a VLA CDR2 of SEQ ID NO: 129, and a VLA

CDR3 of SEQ ID NO: 130; wherein the VHB of the first polypeptide comprises a VHB

CDRl of SEQ ID NO: 132, a VHB CDR2 of SEQ ID NO: 133, and a VHB CDR3 of SEQ ID

NO: 134; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID

NO: 137, a VHA CDR2 of SEQ ID NO: 138, and a VHA CDR3 of SEQ ID NO: 139; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 142, a VLB

CDR2 of SEQ ID NO: 143, and a VLB CDR3 of SEQ ID NO: 144 (e.g., those on NBS3).

[0052] In one embodiment, the binding proteins of the present invention bind CTLA4 and

PD-1 and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 147, a VLA CDR2 of SEQ ID NO: 148, and a VLA CDR3 of SEQ ID NO: 149; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 151, a VHB CDR2 of SEQ ID NO: 152, and a VHB CDR3 of SEQ ID NO: 153; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 156, a VHA CDR2 of SEQ ID NO: 157, and a VHA CDR3 of SEQ ID NO: 158; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 161, a VLB CDR2 of SEQ ID NO: 162, and a VLB CDR3 of SEQ ID NO: 163 (e.g., those on NBS3R).

[0053] In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 166, a VLA CDR2 of SEQ ID NO: 167, and a VLA CDR3 of SEQ ID NO: 168; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 170, a VHB CDR2 of SEQ ID NO: 171, and a VHB CDR3 of SEQ ID NO: 172; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 175, a VHA CDR2 of SEQ ID NO: 176, and a VHA CDR3 of SEQ ID NO: 177; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 180, a VLB CDR2 of SEQ ID NO: 181, and a VLB CDR3 of SEQ ID NO: 182 (e.g., those on NBS3-C).

[0054] In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 166, a VLA CDR2 of SEQ ID NO: 167, and a VLA CDR3 of SEQ ID NO: 168; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 170, a VHB CDR2 of SEQ ID NO: 171, and a VHB CDR3 of SEQ ID NO: 172; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 175, a VHA CDR2 of SEQ ID NO: 176, and a VHA CDR3 of SEQ ID NO: 177; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 180, a VLB CDR2 of SEQ ID NO: 181, and a VLB CDR3 of SEQ ID NO: 182 (e.g., those on NBS3R-C).

[0055] In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 127, and a VHB having the sequence of SEQ ID NO: 131, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 136, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 141 (e.g., those on NBS3). [0056] In one embodiment, the binding proteins of the present invention bind CTLA4 and

PD-1 and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ

ID NO: 146, and a VHB having the sequence of SEQ ID NO: 150, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ

ID NO: 155, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 160 (e.g., those on NBS3R).

[0057] In one embodiment, the binding proteins of the present invention bind CTLA4 and

PD-1 and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ

ID NO: 165, and a VHB having the sequence of SEQ ID NO: 169, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ

ID NO: 174, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 179 (e.g., those on NBS3-C).

[0058] In one embodiment, the binding proteins of the present invention bind CTLA4 and

PD-1 and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ

ID NO: 184, and a VHB having the sequence of SEQ ID NO: 188, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ

ID NO: 193, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 198 (e.g., those on NBS3R-C).

[0059] In one embodiment, the binding proteins of the present invention bind CTLA4 and

PD-1 and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 126; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 135; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 140 (e.g., those on NBS3).

[0060] In one embodiment, the binding proteins of the present invention bind CTLA4 and

PD-1 and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 145; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 154; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 159 (e.g., those on NBS3R).

[0061] In one embodiment, the binding proteins of the present invention bind CTLA4 and

PD-1 and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 164; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 173; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 178 (e.g., those on NBS3-C). [0062] In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 183; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 192; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 197 (e.g., those on NBS3R-C).

[0063] In one embodiment, the binding proteins of the present invention bind CTLA4 and PD-1 , and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution.

[0064] In one embodiment, the binding proteins of the present invention are capable of binding EGFR and PD-L1. The binding proteins of the present invention, in one embodiment, are capable of binding EGFR and PD-L1 and comprise variable heavy and light chains derived from the EGFR antibody panitumumab and the PD-L1 antibody IB 12. In some embodiments, polypeptide derived from EGFR antibody is in the upper domain and polypeptide derived from PD-L1 antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is EGFR, and antigen B is PD-L1. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is EGFR, and antigen A is PD- Ll . In some embodiments, polypeptide derived from EGFR antibody is in the lower domain and polypeptide derived from PD-L1 antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is PD-L1, and antigen B is EGFR. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is PD-L1, and antigen A is EGFR.

[0065] In one embodiment, the binding proteins of the present invention bind EGFR and PD-

Ll and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 99; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 100; and a third polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 101. In one embodiment, the binding proteins of the present invention are capable of binding the same epitope of EGFR and the same epitope of PD-Ll as that of bispecific binding protein FIT012a, wherein the bispecific binding protein FIT012a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 99; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 100; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 101 (e.g., those on FIT012a).

[0066] In one embodiment, the binding proteins of the present invention are capable of binding the same epitope of EGFR and the same epitope of PD-Ll as that of bispecific binding protein FIT012b, wherein the bispecific binding protein FIT012b comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 202; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 211; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 216 (e.g., those on FIT012b).

[0067] In one embodiment, the binding proteins of the present invention are capable of binding the same epitope of EGFR and the same epitope of PD-Ll as that of bispecific binding protein FIT012d, wherein the bispecific binding protein FIT012d comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 221; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 230; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 235 (e.g., those on FIT012d).

[0068] In one embodiment, the binding proteins of the present invention bind EGFR and PD- Ll and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 204, a VLA CDR2 of SEQ ID NO: 205, and a VLA CDR3 of SEQ ID NO: 206 (e.g., those on FIT012b). In one embodiment, the binding proteins of the present invention bind EGFR and PD-Ll and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 223, a VLA CDR2 of SEQ ID NO: 224, and a VLA CDR3 of SEQ ID NO: 225 (e.g., those on FIT012d).

[0069] In one embodiment, the binding proteins of the present invention bind EGFR and PD- Ll and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 208, a VHB CDR2 of SEQ ID NO: 209, and a VHB CDR3 of SEQ ID NO: 210 (e.g., those on FIT012b). In one embodiment, the binding proteins of the present invention bind EGFR and PD-Ll and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 227, a VHB CDR2 of SEQ ID NO: 228, and a VHB CDR3 of SEQ ID NO: 229 (e.g., those on FIT012d).

[0070] In one embodiment, the binding proteins of the present invention bind EGFR and PD- Ll and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 213, a VHA CDR2 of SEQ ID NO: 214, and a VHA CDR3 of SEQ ID NO: 215 (e.g., those on FIT012b). In one embodiment, the binding proteins of the present invention bind EGFR and PD-Ll and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 232, a VHA CDR2 of SEQ ID NO: 233, and a VHA CDR3 of SEQ ID NO: 234 (e.g., those on FIT012d).

[0071] In one embodiment, the binding proteins of the present invention bind EGFR and PD- Ll and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 218, a VLB CDR2 of SEQ ID NO: 219, and a VLB CDR3 of SEQ ID NO: 220 (e.g., those on FIT012b). In one embodiment, the binding proteins of the present invention bind EGFR and PD-Ll and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 237, a VLB CDR2 of SEQ ID NO: 238, and a VLB CDR3 of SEQ ID NO: 239 (e.g., those on FIT012d).

[0072] In one embodiment, the binding proteins of the present invention bind EGFR and PD- Ll and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA comprises a VLA CDRl of SEQ ID NO: 204, a VLA CDR2 of SEQ ID NO: 205, and a VLA CDR3 of SEQ ID NO: 206, the VHB comprises a VHB CDRl of SEQ ID NO: 208, a VHB CDR2 of SEQ ID NO: 209, and a VHB CDR3 of SEQ ID NO: 210, the VHA comprises a VHA CDRl of SEQ ID NO: 213, a VHA CDR2 of SEQ ID NO: 214, and a VHA CDR3 of SEQ ID NO: 215, and the VLB comprises a VLB CDRl of SEQ ID NO: 218, a VLB CDR2 of SEQ ID NO: 219, and a VLB CDR3 of SEQ ID NO: 220 (e.g., those on FIT012b).

[0073] In one embodiment, the binding proteins of the present invention bind EGFR and PD- Ll and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 223, a VLA CDR2 of SEQ ID NO: 224, and a VLA CDR3 of

SEQ ID NO: 225; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ

ID NO: 227, a VHB CDR2 of SEQ ID NO: 228, and a VHB CDR3 of SEQ ID NO: 229; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 232, a

VHA CDR2 of SEQ ID NO: 233, and a VHA CDR3 of SEQ ID NO: 234; and wherein the

VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 237, a VLB CDR2 of

SEQ ID NO: 239, and a VLB CDR3 of SEQ ID NO: 239 (e.g., those on FIT012d).

[0074] In one embodiment, the binding proteins of the present invention bind EGFR and PD-

Ll and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID

NO: 203, and a VHB having the sequence of SEQ ID NO: 207, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID

NO: 212, and wherein the binding protein comprises a third polypeptide chain comprising a

VLB having the sequence of SEQ ID NO: 217 (e.g., those on FIT012b).

[0075] In one embodiment, the binding proteins of the present invention bind EGFR and PD-

Ll and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID

NO: 222, and a VHB having the sequence of SEQ ID NO: 226, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID

NO: 231, and wherein the binding protein comprises a third polypeptide chain comprising a

VLB having the sequence of SEQ ID NO: 236 (e.g., those on FIT012d).

[0076] In one embodiment, the binding proteins of the present invention bind EGFR and PD-

Ll and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 202; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 211; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 216 (e.g., those on FIT012b).

[0077] In one embodiment, the binding proteins of the present invention bind EGFR and PD-

Ll and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 221 ; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 230; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 235 (e.g., those on FIT012d).

[0078] In one embodiment, the binding proteins of the present invention bind EGFR and PD-

Ll, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5, 6, 7, 8,

9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[0079] In one embodiment, the binding proteins of the present invention are capable of binding cMet and EGFR. The binding proteins of the present invention, in one embodiment, are capable of binding cMet and EGFR and comprise variable heavy and light chains derived from the cMet antibody (hl332 (13.3.2L-A91T,H-42K,S97T)) and the EGFR antibody panitumumab. In some embodiments, polypeptide derived from cMet antibody is in the upper domain and polypeptide derived from EGFR antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA- CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is cMet, and antigen B is EGFR. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB-CL, and a third polypeptide of VHA-CHI, wherein antigen B is cMet, and antigen A is EGFR. In some embodiments, polypeptide derived from cMet antibody is in the lower domain and polypeptide derived from EGFR antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is EGFR, and antigen B is cMet. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB- CH1-VLA-CL-FC, a second polypeptide of VLB-CL, and a third polypeptide of VHA-CHI, wherein antigen B is EGFR, and antigen A is cMet.

[0080] In one embodiment, the binding proteins of the present invention bind cMet and

EGFR and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 102; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 103; and a third polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 104.

[0081] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of cMet and the same epitope of EGFR as that of bispecific binding protein

FIT013a, wherein the bispecific binding protein FIT013a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 240; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 249; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 254. [0082] In one embodiment, the binding proteins of the present invention bind cMet and EGFR and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 242, a VLA CDR2 of SEQ ID NO: 243, and a VLA CDR3 of SEQ ID NO: 244.

[0083] In one embodiment, the binding proteins of the present invention bind cMet and EGFR and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 246, a VHB CDR2 of SEQ ID NO: 247, and a VHB CDR3 of SEQ ID NO: 248.

[0084] In one embodiment, the binding proteins of the present invention bind cMet and EGFR and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 251, a VHA CDR2 of SEQ ID NO: 252, and a VHA CDR3 of SEQ ID NO: 253.

[0085] In one embodiment, the binding proteins of the present invention bind cMet and EGFR and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 256, a VLB CDR2 of SEQ ID NO: 257, and a VLB CDR3 of SEQ ID NO: 258.

[0086] In one embodiment, the binding proteins of the present invention bind cMet and EGFR and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 242, a VLA CDR2 of SEQ ID NO: 243, and a VLA CDR3 of SEQ ID NO: 244; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 246, a VHB CDR2 of SEQ ID NO: 247, and a VHB CDR3 of SEQ ID NO: 248; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 251, a VHA CDR2 of SEQ ID NO: 252, and a VHA CDR3 of SEQ ID NO: 253; wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 256, a VLB CDR2 of SEQ ID NO: 257, and a VLB CDR3 of SEQ ID NO: 258.

[0087] In one embodiment, the binding proteins of the present invention bind cMet and EGFR and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 241, and a VHB having the sequence of SEQ ID NO: 245, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 250, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 255. [0088] In one embodiment, the binding proteins of the present invention bind cMet and EGFR and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 240; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 249; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 254.

[0089] In one embodiment, the binding proteins of the present invention bind cMet and EGFR, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[0090] In one embodiment, the binding proteins of the present invention are capable of binding Factor IXa and Factor X. The binding proteins of the present invention, in one embodiment, are capable of binding Factor IXa and Factor X and comprise variable heavy and light chains derived from an anti-Factor IXa antibody and variable light and heavy chains derived from an anti-Factor X antibody. In some embodiments, polypeptide derived from Factor IXa antibody is in the upper domain and polypeptide derived from Factor X antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is Factor IXa, and antigen B is Factor X. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB-CL, and a third polypeptide of VHA- CHI, wherein antigen B is Factor IXa, and antigen A is Factor X. In some embodiments, polypeptide derived from Factor IXa antibody is in the lower domain and polypeptide derived from Factor X antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is Factor X, and antigen B is Factor IXa. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB-CL, and a third polypeptide of VHA-CHI, wherein antigen B is Factor X, and antigen A is Factor IXa.

[0091] In one embodiment, the binding proteins of the present invention bind Factor IXa and Factor X and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 105; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 106; and a third polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 107.

[0092] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of Factor IXa and the same epitope of Factor X as that of bispecific binding protein FIT014a, wherein the bispecific binding protein FIT014a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 259; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 268; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 273.

[0093] In one embodiment, the binding proteins of the present invention bind Factor IXa and Factor X and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 261, a VLA CDR2 of SEQ ID NO: 262, and a VLA CDR3 of SEQ ID NO: 263.

[0094] In one embodiment, the binding proteins of the present invention bind Factor IXa and Factor X and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 265, a VHB CDR2 of SEQ ID NO: 266, and a VHB CDR3 of SEQ ID NO: 267.

[0095] In one embodiment, the binding proteins of the present invention bind Factor IXa and Factor X and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 270, a VHA CDR2 of SEQ ID NO: 271, and a VHA CDR3 of SEQ ID NO: 272.

[0096] In one embodiment, the binding proteins of the present invention bind Factor IXa and Factor X and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 275, a VLB CDR2 of SEQ ID NO: 276, and a VLB CDR3 of SEQ ID NO: 277.

[0097] In one embodiment, the binding proteins of the present invention bind Factor IXa and Factor X and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 261, a VLA CDR2 of SEQ ID NO: 262, and a VLA CDR3 of SEQ ID NO: 263; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 265, a VHB CDR2 of SEQ ID NO: 266, and a VHB CDR3 of SEQ ID NO: 267; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 270, a VHA CDR2 of SEQ ID NO: 271, and a VHA CDR3 of SEQ ID NO: 272; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 275, a VLB CDR2 of SEQ ID NO: 276, and a VLB CDR3 of SEQ ID NO: 277.

[0098] In one embodiment, the binding proteins of the present invention bind Factor IXa and Factor X and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 260, and a VHB having the sequence of SEQ ID NO: 264, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 269, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 274.

[0099] In one embodiment, the binding proteins of the present invention bind Factor IXa and Factor X and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 259; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 268; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 273.

[00100] In one embodiment, the binding proteins of the present invention bind Factor

IXa and Factor X, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[00101] In one embodiment, the binding proteins of the present invention are capable of binding Her3 and IGF-1R. The binding proteins of the present invention, in one embodiment, are capable of binding Her3 and IGF-1R and comprise variable heavy and light chains derived from the Her3 antibody patritumab and the IGF-1R antibody figitumumab. In some embodiments, polypeptide derived from Her3 antibody is in the upper domain and polypeptide derived from IGF-1R antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is Her3, and antigen B is IGF-1R. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is Her3, and antigen A is IGF- 1R. In some embodiments, polypeptide derived from Her3 antibody is in the lower domain and polypeptide derived from IGF-1R antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is IGF-IR, and antigen B is Her3. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is IGF-IR, and antigen A is Her3.

[00102] In one embodiment, the binding proteins of the present invention bind Her3 and IGF-IR and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 108; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 109; and a third polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 110.

[00103] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of Her3 and the same epitope of IGF-IR as that of bispecific binding protein FIT016a, wherein the bispecific binding protein FIT016a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 278; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 287; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 292.

[00104] In one embodiment, the binding proteins of the present invention bind Her3 and IGF-IR and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 280, a VLA CDR2 of SEQ ID NO: 281, and a VLA CDR3 of SEQ ID NO: 282.

[00105] In one embodiment, the binding proteins of the present invention bind Her3 and IGF-IR and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 284, a VHB CDR2 of SEQ ID NO: 285, and a VHB CDR3 of SEQ ID NO: 286.

[00106] In one embodiment, the binding proteins of the present invention bind Her3 and IGF-IR and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 289, a VHA CDR2 of SEQ ID NO: 290, and a VHA CDR3 of SEQ ID NO : 291.

[00107] In one embodiment, the binding proteins of the present invention bind Her3 and IGF-IR and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 294, a VLB CDR2 of SEQ ID NO: 295, and a VLB CDR3 of SEQ ID NO: 296. [00108] In one embodiment, the binding proteins of the present invention bind Her3 and IGF-IR and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 280, a VLA CDR2 of SEQ ID NO: 281, and a VLA CDR3 of SEQ ID NO: 282; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 284, a VHB CDR2 of SEQ ID NO: 285, and a VHB CDR3 of SEQ ID NO: 286; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 289, a VHA CDR2 of SEQ ID NO: 290, and a VHA CDR3 of SEQ ID NO: 291; wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 294, a VLB CDR2 of SEQ ID NO: 295, and a VLB CDR3 of SEQ ID NO: 296.

[00109] In one embodiment, the binding proteins of the present invention bind Her3 and IGF-IR and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 279, and a VHB having the sequence of SEQ ID NO: 283, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 288, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 293.

[00110] In one embodiment, the binding proteins of the present invention bind Her3 and IGF-IR and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 278; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 287; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 292.

[00111] In one embodiment, the binding proteins of the present invention bind Her3 and IGF-IR, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[00112] In one embodiment, the binding proteins of the present invention are capable of binding DLL-4 and VEGF. The binding proteins of the present invention, in one embodiment, are capable of binding DLL-4 and VEGF and comprise variable heavy and light chains derived from the DLL-4 antibody demcizumab and the VEGF antibody bevicizumab.

In some embodiments, polypeptide derived from DLL-4 antibody is in the upper domain and polypeptide derived from VEGF antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is DLL-4, and antigen B is VEGF. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is DLL-4, and antigen A is VEGF. In some embodiments, polypeptide derived from DLL-4 antibody is in the lower domain and polypeptide derived from VEGF antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB- CHl-Fc, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is VEGF, and antigen B is DLL-4. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB-CL, and a third polypeptide of VHA-CHI, wherein antigen B is VEGF, and antigen A is DLL-4.

[00113] In one embodiment, the binding proteins of the present invention bind DLL-4 and VEGF and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 111; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 112; and a third polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 113.

[00114] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of DLL-4 and the same epitope of VEGF as that of bispecific binding protein FIT017a, wherein the bispecific binding protein FIT017a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 297; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 306; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 311.

[00115] In one embodiment, the binding proteins of the present invention bind DLL-4 and VEGF and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 299, a VLA CDR2 of SEQ ID NO: 300, and a VLA CDR3 of SEQ ID NO: 301.

[00116] In one embodiment, the binding proteins of the present invention bind DLL-4 and VEGF and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 303, a VHB CDR2 of SEQ ID NO: 304, and a VHB CDR3 of SEQ ID NO: 305. [00117] In one embodiment, the binding proteins of the present invention bind DLL-4 and VEGF and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 308, a VHA CDR2 of SEQ ID NO: 309, and a VHA CDR3 of SEQ ID NO : 310.

[00118] In one embodiment, the binding proteins of the present invention bind DLL-4 and VEGF and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 313, a VLB CDR2 of SEQ ID NO: 314, and a VLB CDR3 of SEQ ID NO: 315.

[00119] In one embodiment, the binding proteins of the present invention bind DLL-4 and VEGF and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 299, a VLA CDR2 of SEQ ID NO: 300, and a VLA CDR3 of SEQ ID NO: 301; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 303, a VHB CDR2 of SEQ ID NO: 304, and a VHB CDR3 of SEQ ID NO: 305; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 308, a VHA CDR2 of SEQ ID NO: 309, and a VHA CDR3 of SEQ ID NO: 310; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 313, a VLB CDR2 of SEQ ID NO: 314, and a VL _B CDR3 of SEQ ID NO: 315.

[00120] In one embodiment, the binding proteins of the present invention bind DLL-4 and VEGF and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 298, and a VHB having the sequence of SEQ ID NO: 302, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 307, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 312.

[00121] In one embodiment, the binding proteins of the present invention bind DLL-4 and VEGF and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 297; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 306; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 311.

[00122] In one embodiment, the binding proteins of the present invention bind DLL-4 and VEGF, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5,

6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,

200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[00123] In one embodiment, the binding proteins of the present invention are capable of binding Her3 and EGFR. The binding proteins of the present invention, in one embodiment, are capable of binding Her3 and EGFR and comprise variable heavy and light chains derived from the Her3 antibody patritumab and the EGFR antibody panitumumab. In some embodiments, polypeptide derived from Her3 antibody is in the upper domain and polypeptide derived from EGFR antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is Her3, and antigen B is EGFR. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is Her3, and antigen A is EGFR. In some embodiments, polypeptide derived from Her3 antibody is in the lower domain and polypeptide derived from EGFR antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is EGFR, and antigen B is Her3. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is EGFR, and antigen A is Her3.

[00124] In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 117; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 118; and a third polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 119.

[00125] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of Her 3 and the same epitope of EGFR as that of bispecific binding protein FIT019a, wherein the bispecific binding protein FIT019a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 335; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 344; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 349. In one embodiment, the binding protein of the present invention is capable of binding the same epitope of Her3 and the same epitope of EGFR as that of bispecific binding protein FIT019b, wherein the bispecific binding protein FIT019b comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 354; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 363; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 368.

[00126] In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 337, a VLA CDR2 of SEQ ID NO: 338, and a VLA CDR3 of SEQ ID NO: 339 (e.g., those on FIT019a). In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 356, a VLA CDR2 of SEQ ID NO: 357, and a VLA CDR3 of SEQ ID NO: 358 (e.g., those on FIT019b).

[00127] In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 341, a VHB CDR2 of SEQ ID NO: 342, and a VHB CDR3 of SEQ ID NO: 343 (e.g., those on FIT019a). In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 360, a VHB CDR2 of SEQ ID NO: 361, and a VHB CDR3 of SEQ ID NO: 362 (e.g., those on FIT019b).

[00128] In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 346, a VHA CDR2 of SEQ ID NO: 347, and a VHA CDR3 of SEQ ID NO: 348 (e.g., those on FIT019a). In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 365, a VHA CDR2 of SEQ ID NO: 366, and a VHA CDR3 of SEQ ID NO: 367 (e.g., those on FIT019b).

[00129] In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 351, a VLB CDR2 of SEQ ID NO: 352, and a VLB CDR3 of SEQ ID NO: 353 (e.g., those on FIT019a). In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 370, a VLB CDR2 of SEQ ID NO: 371, and a VLB CDR3 of SEQ ID NO: 372 (e.g., those on FIT019b).

[00130] In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 337, a VLA CDR2 of SEQ ID NO: 338, and a VLA CDR3 of SEQ ID NO: 339; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 341, a VHB CDR2 of SEQ ID NO: 342, and a VHB CDR3 of SEQ ID NO: 343; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 346, a VHA CDR2 of SEQ ID NO: 347, and a VHA CDR3 of SEQ ID NO: 348; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 351, a VLB CDR2 of SEQ ID NO: 352, and a VLB CDR3 of SEQ ID NO: 353 (e.g., those on FIT019a).

[00131] In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 356, a VLA CDR2 of SEQ ID NO: 357, and a VLA CDR3 of SEQ ID NO: 358; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 360, a VHB CDR2 of SEQ ID NO: 361, and a VHB CDR3 of SEQ ID NO: 362; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 365, a VHA CDR2 of SEQ ID NO: 366, and a VHA CDR3 of SEQ ID NO: 367; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 370, a VLB CDR2 of SEQ ID NO: 371, and a VLB CDR3 of SEQ ID NO: 372 (e.g., those on FIT019b).

[00132] In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprises a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 336, and a VHB having the sequence of SEQ ID NO: 340, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 345, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 350 (e.g., those on FIT019a). In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprises a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 355, and a VHB having the sequence of SEQ ID NO: 359, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 364, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 369 (e.g., those on FIT019b).

[00133] In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 335; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 344; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 349 (e.g., those on FIT019a). In one embodiment, the binding proteins of the present invention bind Her3 and EGFR and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 354; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 363; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 368 (e.g., those on FIT019b).

[00134] In one embodiment, the binding proteins of the present invention bind Her3 and EGFR, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[00135] In one embodiment, the binding proteins of the present invention are capable of binding PD-1 and PD-L1. The binding proteins of the present invention, in one embodiment, are capable of binding PD-1 and PD-L1 and comprise variable heavy and light chains derived from the PD-1 antibody nivolumab and the PD-L1 antibody 1B12. In some embodiments, polypeptide derived from PD-1 antibody is in the upper domain and polypeptide derived from PD-L1 antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is PD-1, and antigen B is PD-L1. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is PD-1, and antigen A is PD- Ll . In some embodiments, polypeptide derived from PD-1 antibody is in the lower domain and polypeptide derived from PD-L1 antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is PD-Ll, and antigen B is PD-1. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is PD-Ll, and antigen A is PD- 1.

[00136] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 120; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 121; and a third polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 122.

[00137] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of PD-1 and the same epitope of PD-Ll as that of bispecific binding protein FIT020a, wherein the bispecific binding protein FIT020a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 120; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 121; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 122.

[00138] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of PD-1 and the same epitope of PD-Ll as that of bispecific binding protein FIT020b, wherein the bispecific binding protein FIT020b comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 297; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 306; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 311.

[00139] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 389, a VLA CDR2 of SEQ ID NO: 390, and a VLA CDR3 of SEQ ID NO: 391 (e.g., those on FIT020a).

[00140] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a VLA CDRl of SEQ ID NO: 375, a VLA CDR2 of SEQ ID NO: 376, and a VLA CDR3 of SEQ ID NO: 377 (e.g., those on FIT020b).

[00141] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 384, a VHB CDR2 of SEQ ID NO: 385, and a VHB CDR3 of SEQ ID NO: 386 (e.g., those on FIT020a).

[00142] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a VHB CDRl of SEQ ID NO: 379, a VHB CDR2 of SEQ ID NO: 380, and a VHB CDR3 of SEQ ID NO: 381 (e.g., those on FIT020b).

[00143] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 379, a VHA CDR2 of SEQ ID NO: 380, and a VHA CDR3 of SEQ ID NO: 381 (e.g., those on FIT020a).

[00144] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 384, a VHA CDR2 of SEQ ID NO: 385, and a VHA CDR3 of SEQ ID NO: 386 (e.g., those on FIT020b).

[00145] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 375, a VLB CDR2 of SEQ ID NO: 376, and a VLB CDR3 of SEQ ID NO: 377 (e.g., those on FIT020a).

[00146] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 389, a VLB CDR2 of SEQ ID NO: 390, and a VLB CDR3 of SEQ ID NO: 391 (e.g., those on FIT020b).

[00147] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 389, a VLA CDR2 of SEQ ID NO: 390, and a VLA

CDR3 of SEQ ID NO: 391; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 384, a VHB CDR2 of SEQ ID NO: 385, and a VHB CDR3 of SEQ ID NO:

386; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO:

379, a VHA CDR2 of SEQ ID NO: 380, and a VHA CDR3 of SEQ ID NO: 381 ; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 375, a VLB CDR2 of SEQ ID NO: 376, and a VLB CDR3 of SEQ ID NO: 377 (e.g., those on FIT020a).

[00148] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 375, a VLA CDR2 of SEQ ID NO: 376, and a VLA

CDR3 of SEQ ID NO: 377; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 379, a VHB CDR2 of SEQ ID NO: 380, and a VHB CDR3 of SEQ ID NO:

381; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO:

384, a VHA CDR2 of SEQ ID NO: 385, and a VHA CDR3 of SEQ ID NO: 386; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 389, a VLB CDR2 of SEQ ID NO: 390, and a VLB CDR3 of SEQ ID NO: 391 (e.g., those on FIT020b).

[00149] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a first polypeptide chain comprising a VLA having the sequence of

SEQ ID NO: 388, and a VHB having the sequence of SEQ ID NO: 383, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ

ID NO: 378, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 374 (e.g., those on FIT020a).

[00150] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprise a first polypeptide chain comprising a VLA having the sequence of

SEQ ID NO: 374, and a VHB having the sequence of SEQ ID NO: 378, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ

ID NO: 383, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 388 (e.g., those on FIT020b).

[00151] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 120; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 121; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 122 (e.g., those on FIT020a).

[00152] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 373; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 382; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 387 (e.g., those on FIT020b).

[00153] In one embodiment, the binding proteins of the present invention bind PD-1 and PD-Ll, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5,

6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[00154] In one embodiment, the binding proteins of the present invention are capable of binding Her3 and PD-1. The binding proteins of the present invention, in one embodiment, are capable of binding Her3 and PD-1 and comprise variable heavy and light chains derived from the Her3 antibody patritumab and the EGFR antibody nivolumab. In some embodiments, polypeptide derived from Her3 antibody is in the upper domain and polypeptide derived from PD-1 antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is Her3, and antigen B is PD-1. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is Her3, and antigen A is PD-1. In some embodiments, polypeptide derived from Her3 antibody is in the lower domain and polypeptide derived from PD-1 antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is PD-1, and antigen B is Her3. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is PD-1, and antigen A is Her3.

[00155] In one embodiment, the binding proteins of the present invention bind Her3 and PD-1 and comprise a first polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 123; a second polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 124; and a third polypeptide chain comprising, consisting essentially of, or consisting of an amino acid sequence according to SEQ ID NO: 125.

[00156] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of Her3 and the same epitope of PD-1 as that of bispecific binding protein FIT022a, wherein the bispecific binding protein FIT022a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 411; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 420; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 425. [00157] In one embodiment, the binding proteins of the present invention bind Her3 and PD-1 and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 413, a VLA CDR2 of SEQ ID NO: 414, and a VLA CDR3 of SEQ ID NO: 415.

[00158] In one embodiment, the binding proteins of the present invention bind Her3 and PD-1 and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 417, a VHB CDR2 of SEQ ID NO: 418, and a VH _B CDR3 of SEQ ID NO: 419.

[00159] In one embodiment, the binding proteins of the present invention bind Her3 and PD-1 and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 422, a VHA CDR2 of SEQ ID NO: 423, and a VHA CDR3 of SEQ ID NO: 424.

[00160] In one embodiment, the binding proteins of the present invention bind Her3 and PD-1 and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 427, a VLB CDR2 of SEQ ID NO: 428, and a VLB CDR3 of SEQ ID NO: 429.

[00161] In one embodiment, the binding proteins of the present invention bind Her3 and PD-1 and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 413, a VLA CDR2 of SEQ ID NO: 414, and a VLA CDR3 of SEQ ID NO: 415; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 417, a VHB CDR2 of SEQ ID NO: 418, and a VHB CDR3 of SEQ ID NO: 419; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 422, a VHA CDR2 of SEQ ID NO: 423, and a VHA CDR3 of SEQ ID NO: 424; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 427, a VLB CDR2 of SEQ ID NO: 428, and a VLB CDR3 of SEQ ID NO: 429.

[00162] In one embodiment, the binding proteins of the present invention bind Her3 and PD-1 and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 412, and a VHB having the sequence of SEQ ID NO: 416, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 421, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 426. [00163] In one embodiment, the binding proteins of the present invention bind Her3 and PD-1 and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 411 ; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 420; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 425.

[00164] In one embodiment, the binding proteins of the present invention bind Her3 and PD-1, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[00165] In one embodiment, the binding proteins of the present invention are capable of binding cMet and PD-L1. The binding proteins of the present invention, in one embodiment, are capable of binding cMet and PD-L1 and comprise variable heavy and light chains derived from the cMet antibody hl332 and the PD-L1 antibody 1B12. In some embodiments, polypeptide derived from cMet antibody is in the upper domain and polypeptide derived from PD-L1 antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is cMet, and antigen B is PD-L1. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is cMet, and antigen A is PD- Ll . In some embodiments, polypeptide derived from cMet antibody is in the lower domain and polypeptide derived from PD-L1 antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is PD-L1, and antigen B is cMet. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is PD-L1, and antigen A is cMet.

[00166] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of cMet and the same epitope of PD-L1 as that of bispecific binding protein FIT023a, wherein the bispecific binding protein FIT023a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 430; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 439; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 444.

[00167] In one embodiment, the binding proteins of the present invention bind cMet and PD-Ll and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 432, a VLA CDR2 of SEQ ID NO: 433, and a VLA CDR3 of SEQ ID NO: 434.

[00168] In one embodiment, the binding proteins of the present invention bind cMet and PD-Ll and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 436, a VHB CDR2 of SEQ ID NO: 437, and a VHB CDR3 of SEQ ID NO: 438.

[00169] In one embodiment, the binding proteins of the present invention bind cMet and PD-Ll and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 441, a VHA CDR2 of SEQ ID NO: 442, and a VHA CDR3 of SEQ ID NO: 443.

[00170] In one embodiment, the binding proteins of the present invention bind cMet and PD-Ll and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 446, a VLB CDR2 of SEQ ID NO: 447, and a VLB CDR3 of SEQ ID NO: 448.

[00171] In one embodiment, the binding proteins of the present invention bind cMet and PD-Ll and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 432, a VLA CDR2 of SEQ ID NO: 433, and a VLA CDR3 of SEQ ID NO: 434; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 436, a VHB CDR2 of SEQ ID NO: 437, and a VHB CDR3 of SEQ ID NO: 438; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 441, a VHA CDR2 of SEQ ID NO: 442, and a VHA CDR3 of SEQ ID NO: 443; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 446, a VLB CDR2 of SEQ ID NO: 447, and a VLB CDR3 of SEQ ID NO: 448.

[00172] In one embodiment, the binding proteins of the present invention bind cMet and PD-Ll and comprise a first polypeptide chain comprising a VLA having the sequence of

SEQ ID NO: 431, and a VHB having the sequence of SEQ ID NO: 435, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 440, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 445.

[00173] In one embodiment, the binding proteins of the present invention bind cMet and PD-L1 and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 430; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 439; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 444.

[00174] In one embodiment, the binding proteins of the present invention bind cMet and PD-L1, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[00175] In one embodiment, the binding proteins of the present invention are capable of binding BTLA and PD-1. The binding proteins of the present invention, in one embodiment, are capable of binding BTLA and PD-1 and comprise variable heavy and light chains derived from the BTLA antibody 6A5 and the PD-1 antibody Nivolumab. In some embodiments, polypeptide derived from BTLA antibody is in the upper domain and polypeptide derived from PD-1 antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is BTLA, and antigen B is PD-1. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is BTLA, and antigen A is PD- 1. In some embodiments, polypeptide derived from BTLA antibody is in the lower domain and polypeptide derived from PD-1 antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is PD-1, and antigen B is BTLA. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is PD-1, and antigen A is BTLA. [00176] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of BTLA and the same epitope of PD-1 as that of bispecific binding protein FIT024a, wherein the bispecific binding protein FIT024a comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 449; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 458; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 463.

[00177] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of BTLA and the same epitope of PD-1 as that of bispecific binding protein FIT024b, wherein the bispecific binding protein FIT024b comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 468; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 477; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 482.

[00178] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 451, a VLA CDR2 of SEQ ID NO: 452, and a VLA CDR3 of SEQ ID NO: 453 (e.g., those of FIT024a).

[00179] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 470, a VLA CDR2 of SEQ ID NO: 471, and a VLA CDR3 of SEQ ID NO: 472 (e.g., those of FIT024b).

[00180] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 455, a VHB CDR2 of SEQ ID NO: 456, and a VHB CDR3 of SEQ ID NO: 457 (e.g., those of FIT024a).

[00181] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 474, a VHB CDR2 of SEQ ID NO: 475, and a VHB CDR3 of SEQ ID NO: 476 (e.g., those of FIT024b).

[00182] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 460, a VHA CDR2 of SEQ ID NO: 461, and a VHA CDR3 of SEQ ID NO: 462 (e.g., those of FIT024a). [00183] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 479, a VHA CDR2 of SEQ ID NO: 480, and a VHA CDR3 of SEQ ID NO: 481 (e.g., those of FIT024b).

[00184] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 465, a VLB CDR2 of SEQ ID NO: 466, and a VLB CDR3 of SEQ ID NO: 467 (e.g., those of FIT024a).

[00185] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 484, a VLB CDR2 of SEQ ID NO: 485, and a VLB CDR3 of SEQ ID NO: 486 (e.g., those of FIT024b).

[00186] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 451, a VLA CDR2 of SEQ ID NO: 452, and a VLA CDR3 of SEQ ID NO: 453; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 455, a VHB CDR2 of SEQ ID NO: 456, and a VHB CDR3 of SEQ ID NO: 457; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 460, a VHA CDR2 of SEQ ID NO: 461, and a VHA CDR3 of SEQ ID NO: 462; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 465, a VLB CDR2 of SEQ ID NO: 466, and a VLB CDR3 of SEQ ID NO: 467 (e.g., those of FIT024a).

[00187] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 470, a VLA CDR2 of SEQ ID NO: 471, and a VLA CDR3 of SEQ ID NO: 472; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 474, a VHB CDR2 of SEQ ID NO: 475, and a VHB CDR3 of SEQ ID NO: 476; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 479, a VHA CDR2 of SEQ ID NO: 480, and a VHA CDR3 of SEQ ID NO: 481; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 484, a VLB CDR2 of SEQ ID NO: 485, and a VLB CDR3 of SEQ ID NO: 486 (e.g., those of FIT024b). [00188] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 450, and a VHB having the sequence of SEQ ID NO: 454, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 459, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 464 (e.g., those of FIT024a).

[00189] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 469, and a VHB having the sequence of SEQ ID NO: 473, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 478, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 483 (e.g., those of FIT024b).

[00190] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 449; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 458; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 463 (those of FIT024a).

[00191] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1 and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 468; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 477; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 482 (those of FIT024b).

[00192] In one embodiment, the binding proteins of the present invention bind BTLA and PD-1, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[00193] In one embodiment, the binding proteins of the present invention are capable of binding CD20 and CD22. The binding proteins of the present invention, in one embodiment, are capable of binding CD20 and CD22 and comprise variable heavy and light chains derived from the CD20 antibody Ofatumumab and the CD22 antibody Epratuzumab.

In some embodiments, polypeptide derived from CD20 antibody is in the upper domain and polypeptide derived from CD22 antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is CD20, and antigen B is CD22. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is CD20, and antigen A is CD22. In some embodiments, polypeptide derived from CD20 antibody is in the lower domain and polypeptide derived from CD22 antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1- Fc, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is CD22, and antigen B is CD20. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB- CL, and a third polypeptide of VHA-CHI, wherein antigen B is CD22, and antigen A is CD20.

[00194] In one embodiment, the binding protein of the present invention is capable of binding the same epitope of CD20 and the same epitope of CD22 as that of bispecific binding protein FIT021b, wherein the bispecific binding protein FIT021b comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 392; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 401; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 406.

[00195] In one embodiment, the binding proteins of the present invention bind CD20 and CD22 and comprise a VLA on the first polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 394, a VLA CDR2 of SEQ ID NO: 395, and a VLA CDR3 of SEQ ID NO: 396.

[00196] In one embodiment, the binding proteins of the present invention bind CD20 and CD22 and comprise a VHB on the first polypeptide, wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 398, a VHB CDR2 of SEQ ID NO: 399, and a VHB CDR3 of SEQ ID NO: 400.

[00197] In one embodiment, the binding proteins of the present invention bind CD20 and CD22 and comprise a VHA on the second polypeptide, wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 403, a VHA CDR2 of SEQ ID NO: 404, and a VHA CDR3 of SEQ ID NO: 405. [00198] In one embodiment, the binding proteins of the present invention bind CD20 and CD22 and comprise a VLB on the third polypeptide, wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 408, a VLB CDR2 of SEQ ID NO: 409, and a VLB CDR3 of SEQ ID NO: 410.

[00199] In one embodiment, the binding proteins of the present invention bind CD20 and CD22 and comprise a VLA and VHB on the first polypeptide, a VHA on the second polypeptide, and a VLB on the third polypeptide, wherein the VLA of the first polypeptide comprises a VLA CDRl of SEQ ID NO: 394, a VLA CDR2 of SEQ ID NO: 395, and a VLA CDR3 of SEQ ID NO: 396; wherein the VHB of the first polypeptide comprises a VHB CDRl of SEQ ID NO: 398, a VHB CDR2 of SEQ ID NO: 399, and a VHB CDR3 of SEQ ID NO: 400; wherein the VHA of the second polypeptide comprises a VHA CDRl of SEQ ID NO: 403, a VHA CDR2 of SEQ ID NO: 404, and a VHA CDR3 of SEQ ID NO: 405; and wherein the VLB of the third polypeptide comprises a VLB CDRl of SEQ ID NO: 408, a VLB CDR2 of SEQ ID NO: 409, and a VLB CDR3 of SEQ ID NO: 410.

[00200] In one embodiment, the binding proteins of the present invention bind CD20 and CD22 and comprise a first polypeptide chain comprising a VLA having the sequence of SEQ ID NO: 393, and a VHB having the sequence of SEQ ID NO: 397, wherein the binding protein comprises a second polypeptide chain comprising a VHA having the sequence of SEQ ID NO: 402, and wherein the binding protein comprises a third polypeptide chain comprising a VLB having the sequence of SEQ ID NO: 407.

[00201] In one embodiment, the binding proteins of the present invention bind CD20 and CD22 and comprising, consisting essentially of, or consisting of a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 392; a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 401; and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 406.

[00202] In one embodiment, the binding proteins of the present invention bind CD20 and CD22, and are derived from binding proteins described herein by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more (inclusive of all values therebetween) amino acids with conservative amino acid substitution, while still maintaining equivalent activity as the corresponding binding proteins without the substitution(s).

[00203] In one embodiment, the binding proteins of the present invention are capable of binding PD-L1 and TIM3. In some embodiments, polypeptide derived from PD-L1 antibody is in the upper domain and polypeptide derived from TIM3 antibody is in the lower domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is PD-L1, and antigen B is TIM3. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB- CH1-VLA-CL-FC, a second polypeptide of VLB-CL, and a third polypeptide of VHA-CHI, wherein antigen B is PD-L1, and antigen A is TIM3. In some embodiments, polypeptide derived from PD-L1 antibody is in the lower domain and polypeptide derived from TIM3 antibody is in the upper domain. For example, in some embodiments, the binding proteins comprise a first polypeptide of VLA-CL-VHB-CH1-FC, a second polypeptide of VHA-CHI, and a third polypeptide of VLB-CL, wherein antigen A is TIM3, and antigen B is PD-L1. For another example, in some embodiments, the binding proteins comprise a first polypeptide of VHB-CH1-VLA-CL-FC, a second polypeptide of VLB-CL, and a third polypeptide of VHA- CHI, wherein antigen B is TIM3, and antigen A is PD-L1.

[00204] In one embodiment, the binding protein is capable of binding one or more epitopes on one or more immune checkpoint protein on T cells such as, for example, TIM-3, Lag3, ICOS, BTLA, CD160, 2B4, KIR, CD137, CD27, OX40, CD40L, and A2aR. In another embodiment, the binding protein is capable of binding one or more epitopes on one or more tumor cell surface protein that is involved with immune checkpoint pathways, such as, for example, PD-L1, PD-L2, Galectin9, HVEM, CD48, B7-1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CD70, and CD40.

[00205] In one aspect, the present invention provides pharmaceutical compositions comprising the binding proteins described herein. In one embodiment, provided herein are pharmaceutical compositions comprising the binding protein of any one of the preceding claims and one or more pharmaceutically acceptable carrier.

[00206] In another aspect, the present invention provides methods of treating or preventing an inflammatory disease, autoimmune disease, neurodegenerative disease, cancer, sepsis, or spinal cord injury in a subject in need thereof. In one embodiment, the method comprises administering to a subject an effective amount of one or more of the binding proteins provided herein, or one or more pharmaceutical compositions comprising the binding proteins provided herein and a pharmaceutically acceptable carrier. Uses of the binding proteins described herein in the manufacture of a medicament for treatment or prevention of an inflammatory disease, autoimmune disease, neurodegenerative disease, cancer, spinal cord injury, or other conditions are also provided herein. In one embodiment, the inflammatory disease, autoimmune disease, cancer, neurodegenerative disease, and other conditions include, but are not limited to, asthma, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, Alzheimer's disease, Parkinson's disease, infectious diseases and disorders, such as psoriasis, psoriatic arthritis, dermatitis, systemic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, Raynaud's syndrome, Sjogren's syndrome, juvenile onset diabetes, Reiter's disease, Behcet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathies, immune-mediated thrombocytopenias, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, atopic dermatitis, pemphigus, Graves' disease, severe acute respiratory distress syndrome, choreoretinitis, Hashimoto's thyroiditis,

Wegener's granulomatosis, Omenn's syndrome, chronic renal failure, acute infectious mononucleosis, HIV, herpes virus associated diseases, type 1 diabetes, graft versus host disease (GVHD); immune disorders associated with graft transplantation rejection; T cell lymphoma, T cell acute lymphoblastic leukemia, testicular angiocentric T cell lymphoma, benign lymphocytic angiitis, primary myxedema, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin dependent diabetes mellitis, good pasture's syndrome, sympathetic ophthalmia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatoid arthritis, polymyositis, scleroderma, mixed connective tissue disease, pemphigus vulgaris, pemphigoid, ankylosing spondylitis, aplastic anemia, autoimmune hepatitis, coeliac disease, dermatomyositis,

Goodpasture's syndrome, Guillain-Barre syndrome, idiopathic leucopenia, idiopathic thrombocytopenic purpura, male infertility, phacogenic uveitis, primary myxoedema, Reiter's syndrome, stiff man syndrome, thyrotoxicosis, ulceritive colitis, breast cancer, ovarian cancer, lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer, vaginal cancer, cervical cancer, cancer of the spleen, testicular cancer, cancer of the thymus, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, blastoma, sarcom, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, dermal cancer, dermal or intraocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, male/female genital tract cancer, nerve cancer, chronic or acute leukemia, lymphocyte lymphoma, hepatoma, stomach cancer, glioblastoma, ovarian cancer, liver cancer, hepatic tumor, colon cancer, large intestine cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulvar cancer, thyroid cancer, gestational diabetes, chronic thromboembolic diseases or disorders associated with fibrin formation including vascular disorders such as deep venous thrombosis, arterial thrombosis, stroke, tumor metastasis, thrombolysis, arteriosclerosis and restenosis following angioplasty, septic shock, septicemia, hypotension, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulopathy (DIC), sarcoidosis, arterial arteriosclerosis, peptic ulcers, burns, pancreatitis, polycystic ovarian disease (POD), endometriosis, uterine fibroid, benign prostate hypertrophy, T-cell acute lymphoblastic leukemia (T-ALL), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), tetralogy of Fallot (TOF), Alagille syndrome (AS), macular degeneration and age-related macular degeneration diseases, inflammatory fibrosis (e.g., scleroderma, lung fibrosis, and cirrhosis), osteoarthritis, osteoporosis, asthma (including allergic asthma), allergies, chronic obstructive pulmonary disease (COPD), juvenile early-onset Type I diabetes, transplant rejection, and SLE.

[00207] In one embodiment, the present disclosure provides methods for treating or preventing rheumatoid arthritis, psoriasis, osteoporosis, stroke, liver disease, or oral cancer to a subject in need thereof, the method comprising administering to the subject a FIT-Ig binding protein described herein, wherein the binding protein is capable of binding IL-17 and IL-20. In a further embodiment, the FIT-Ig binding protein comprises an amino acid sequence selected from SEQ ID NOs: 15, 25, and 27; and amino acid sequence according to SEQ ID NO: 21; and an amino acid sequence according to SEQ ID NO: 23. In another embodiment, the FIT-Ig binding protein comprises an amino acid sequence selected from SEQ ID NOs: 15, 25, and 27; and an amino acid sequence selected from SEQ ID NOs: 29, 30 and 31.

[00208] In one embodiment, the present disclosure provides methods for treating or preventing a B cell cancer in a subject in need thereof, the method comprising administering to the subject a FIT-Ig binding protein, wherein the FIT-Ig binding protein is capable of binding one or more B cell antigen. In a further embodiment, the FIT-Ig binding protein is capable of binding CD20. In a further embodiment, the FIT-Ig binding protein is capable of binding CD20 and another antigen. In a further embodiment, the binding protein is capable of binding CD3 and CD20. In a further embodiment, the cancer is a B cell cancer. In a still further embodiment, the B cell cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma [NHL], precursor B cell lymphoblastic leukemia/lymphoma, mature B cell neoplasms, B cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, cutaneous follicle center lymphoma, marginal zone B cell lymphoma, hairy cell leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large-cell lymphoma. In one embodiment, the present disclosure provides methods for treating or preventing a B cell cancer in a subject in need thereof, the method comprising administering to the subject a FIT-Ig binding protein, wherein the FIT-Ig binding protein comprises an amino acid sequence according to SEQ ID NOs: 41 or 48; and amino acid sequence according to SEQ ID NO: 44, and an amino acid sequence according to SEQ ID NO: 46.

[00209] In one embodiment, the present disclosure provides methods for treating or preventing an autoimmune disease, inflammatory disease, or infection in a subject in need thereof, the method comprising administering to the subject a FIT-Ig binding protein described herein, wherein the binding protein is capable of binding TNF and IL-17. In a further embodiment, the FIT-Ig binding protein comprises sequences according to SEQ ID

NOs: 87, 89, and 91. In another embodiment, the present disclosure provides methods for treating or preventing an autoimmune or inflammatory disease, the method comprising administering to the subject a FIT-Ig binding protein, wherein the binding protein is capable of binding TNF and IL-17, and wherein the autoimmune or inflammatory disease is selected from the group consisting of Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoratic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, ankylosing spondylitis, spondylosing arthropathy, systemic lupus erythematosus, uveitis, sepsis, neurodegenerative diseases, neuronal regeneration, spinal cord injury, primary and metastatic cancers, a respiratory disorder; asthma; allergic and nonallergic asthma; asthma due to infection; asthma due to infection with respiratory syncytial virus (RSV); chronic obstructive pulmonary disease (COPD); a condition involving airway inflammation; eosinophilia; fibrosis and excess mucus production; cystic fibrosis; pulmonary fibrosis; an atopic disorder; atopic dermatitis; urticaria; eczema; allergic rhinitis; allergic enterogastritis; an inflammatory and/or autoimmune condition of the skin; an inflammatory and/or autoimmune condition of gastrointestinal organs; inflammatory bowel diseases (IBD); ulcerative colitis; an inflammatory and/or autoimmune condition of the liver; liver cirrhosis; liver fibrosis; and liver fibrosis caused by hepatitis B and/or C virus; scleroderma. In another embodiment, In another embodiment, the present disclosure provides methods for treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject a FIT-Ig binding protein described herein, wherein the binding protein is capable of binding TNF and IL-17. In a further embodiment, the cancer is hepatocellular carcinoma; glioblastoma; lymphoma; or Hodgkin's lymphoma. In another embodiment, the present disclosure provides methods for treating or preventing and infection in a subject in need thereof, the method comprising administering to the subject a FIT-Ig binding protein described herein, wherein the infection is a viral infection, a bacterial infection, a parasitic infection, HTLV- 1 infection. In one embodiment, the present disclosure provides methods for suppression of expression of protective type 1 immune responses, and suppression of expression of a protective type 1 immune response during vaccination.

[00210] In one embodiment, the present disclosure provides methods for treating rheumatoid arthritis in a subject in need thereof, the method comprising administering to the subject a FIT-Ig binding protein, wherein the binding protein comprises sequences according to SEQ ID NOs: 87, 89, and 91.

[00211] In one embodiment, the present disclosure provides methods for treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject a FIT-Ig binding protein described herein, wherein the binding protein is capable of binding CTLA-4 and PD-1. In a further embodiment, the FIT-Ig binding protein comprises an amino acid sequence comprising SEQ ID NOs: 92, 95, and 97. In another embodiment, the present disclosure provides methods for treating or preventing cancer in a subject in need thereof, wherein the binding protein is capable of binding CTLA-4 and PD-1, and wherein the cancer is a cancer typically responsive to immunotherapy. In another embodiment, the cancer is a cancer that has not been associated with immunotherapy. In another embodiment, the cancer is a cancer that is a refractory or recurring malignancy. In another embodiment, the binding protein inhibits the growth or survival of tumor cells. In another embodiment, the cancer is selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g. non-small cell lung cancer), esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies. [00212] In one embodiment, the present disclosure provides methods for treating or preventing melanoma in a subject in need thereof, the method comprising administering to the subject a FIT-Ig binding protein described herein, wherein the binding protein is capable of binding CTLA-4 and PD-1. In a further embodiment, the present disclosure provides methods for treating or preventing melanoma in a subject in need thereof, wherein the method comprises administering to the subject a FIT-Ig binding protein comprising amino acid sequences according to SEQ ID NOs: 92, 95, and 97.

[00213] In another embodiment, the present disclosure provides methods for treating or preventing infections or infectious disease in a subject in need thereof, the method comprising administering to the subject a FIT-Ig binding protein described herein, wherein the binding protein is capable of binding CTLA-4 and PD- 1. In one embodiment, the FIT-Ig binding protein is administered alone, or in combination with vaccines, to stimulate the immune response to pathogens, toxins, and self-antigens. Therefore, in one embodiment, the binding proteins provided herein can be used to stimulate immune response to viruses infectious to humans, such as, but not limited to, human immunodeficiency viruses, hepatitis viruses class A, B and C, Epstein Barr virus, human cytomegalovirus, human papilloma viruses, herpes viruses, bacteria, fungal parasites, or other pathogens.

BRIEF DESCRIPTION OF THE DRAWINGS

[00214] Figure 1A shows the structure of FIT-Igs that are made up of three constructs, such as FITl-Ig, FIT2-Ig, and FIT3-Ig. Figure IB shows the three constructs used to prepare such FITl-Igs.

[00215] Figure 2A shows the basic structure of FIT-Igs that are made up of two constructs. Figure 2B shows the two constructs used to prepare such FIT-Igs.

[00216] Figure 3 provides the dual-specific antigen binding of FITl-Ig as measured by

Biacore. The top panel of Figure 3 shows the results of the Biacore binding assay in which

FITl-Ig was first saturated by IL-17, followed by IL-20. The bottom panel of Figure 3 shows the results of the Biacore assay in which FITl-Ig was first saturated by IL-20, followed by

IL-17.

[00217] Figure 4. Shows the solubility at a range of pH of anti-IL-17/IL-20 FIT Ig or monoclonal antibody rituximab, as measured by PEG-induced precipitation.

[00218] Figure 5 shows the binding to CTLA-4 (Figure 5A) or PD-1 (Figure 5B) by

FITIO-Ig or the parental antibodies Ipilimumab and Nivolumab, as assessed by ELISA. [00219] Figure 6 shows a multiple binding study of FITI O-Ig against both CTLA-4 and PD-1. Binding to CTLA-4 followed by PD-1 ; and binding by PD-1 followed by CTLA-4 are both shown as indicated in Figure 6.

[00220] Figures 7a-7i show the SEC profiles for FIT12a-Ig (EGFR/PD-L1 ; Figure

7a), FIT13a-Ig (cMet/EGFR; Figure 7b), FIT14-Ig (Factor IXa/Factor X; Figure 7c), FIT16a-Ig (Her3/IGF-1R; Figure 7d), FIT17a-Ig (DLL-4/VEGF; Figure 7e), FIT18a-Ig (CD20/CD3; Figure If), FIT19a-Ig (Her3/EGFR; Figure 7g), FIT20a-Ig (PD-1/PD-L1 ; Figure 7h), and FIT22a-Ig (Her3/PD-1 ; Figure 7i).

[00221] Figure 8 shows a multiple binding study of FIT13a-Ig against both cMet and

EGFR.

[00222] Figure 9A and Figure 9B show the binding of NBSR or the parental antibody

Nivolumab to PD-1 (Figure 9A) or CTLA-4 (Figure 9B), as assessed by ELISA. Human IgGl was included as a control.

[00223] Figure 10 shows a multiple binding study of NBS3 against both CTLA-4 and

PD-1. Binding to CTLA-4 followed by PD-1 ; and binding by PD-1 followed by CTLA-4 are both shown as indicated.

[00224] Figure 11A shows mean serum concentration-time profiles of 5mg/kg NBS3 administered intravenously (IV) or subcutaneously (SC). Figure 11B provides the detail PK parameters of this study.

[00225] Figure 12 shows cell-based receptor blocking assay of NBS3, NBS3-C,

NBS3R-C compared to the parental antibody Nivolumab. Human IgG4 was included as a control.

[00226] Figure 13A shows functional activity of NBS3, NBS3-C, and NBS3R-C in

MLR assays, when compared to the parental antibody Nivolumab at a concentration of 0, 0.01 , 0.1, 1 , or 10 μg/ml. The induction of IFN-γ was measured for each antibody. Human IgG was included as a control. Figure 13B shows the induction of IL-2 by these antibodies.

[00227] Figure 14 shows functional activity of NBS3, NBS3-C, and NBS3R-C in

PBMC SEB-stimulation assays when compared to the parental antibody Ipilimumab, at a concentration of 0.016. 0.08, 0.4, 2, or 10 μg/ml. The IL-2 cytokine production in the supernatant was detected by ELISA.

[00228] Figure 15 shows a multiple binding study of FIT012b against both EGFR and

PD-L1. Binding to human EGFR followed by human PD-L1 ; and binding by human PD-L1 followed by human EGFR are both shown as indicated. [00229] Figure 16A and Figure 16B show a multiple binding study of FIT012d against both human EGFR and human PD-L1. Binding to human EGFR followed by human PD-L1 (Figure 16A); and binding by human PD-L1 followed by human EGFR (Figure 16B) are both shown as indicated.

[00230] Figure 17 shows a multiple binding study of FIT013a against both cMet and

EGFR. Binding to cMet followed by EGFR; and binding by EGFR followed by cMet are both shown as indicated.

[00231] Figure 18A to Figure 18C show FACS assays in which FIT013a's binding activity to membrane c-Met and EGFR was tested. Figure 18A (right panel) shows dual binding to MKN-45 cell as measured by a BD FACSVerse flow cytometer. Figure 18A (left panel) indicates that in MKN-45 cell, membrane expression level c-Met is much higher than EGFR, so c-Met binding cite of FIT013a and FIT013a-Fab can be occupied by membrane c- Met, the free EGFR binding cite of FIT013a and FIT013a-Fab can be detected by biotinylated EGFR. Figure 18B (right panel) shows dual binding to SGC-7901 cell as measured by a BD FACSVerse flow cytometer. Figure 18B (left panel) indicates that in SGC-7901 cell, membrane expression level EGFR is much higher than c-Met, so EGFR binding cite of FIT013a and FIT013a-Fab can be occupied by membrane EGFR, the free c- Met binding cite of FIT013a and FIT013a-Fab can be detected by biotinylated c-Met. Figure 18C (right panel) shows dual binding to NCI-H1975 cell as measured by a BD FACSVerse flow cytometer. Figure 18C (left panel) indicates that in NCI-H1975 cell, membrane expression level c-Met is equal to EGFR , so c-Met and EGFR binding cites of FIT013a and FIT013a-Fab are occupied simultaneously, and no free EGFR or c-Met binding cite of FIT013a and FIT013a-Fab can be detected.

[00232] Figure 19 shows FIT013a's ability of inhibiting HGF induced AKT phosphorylation in NCI-H292 cells. FIT013a, hl332, panitumumab, a combination of hl332/panitumumab, and human IgGl were added to cells and incubated for 30mins and then 40ng/ml HGF was added to the assay plate for 5mins. The cells were lysed and AKT phosphorylation was detected by ERK phospho-T202/Y204 kit.

[00233] Figure 20 shows FIT013a's agonist effect in the absence of HGF as measured by AKT phosphorylation. Serially diluted FIT013a or other Abs (hi 332, a combination of hl332/panitumumab, emibetuzumab, 9.1.2, and human IgGl) were added to cells and incubate for 30 mins. The cells were lysed and AKT phosphorylation was detected by ERK phospho-T202/Y204 kit. [00234] Figure 21A shows individual serum concentration-time profiles of FIT013a

(c-met/EGFR) after an IV dose of 5 mg/kg in male SD rats(N=4/time point, c-met plate). Figure 21B shows individual serum concentration-time profiles of FIT013a (c-met/EGFR) after a SC dose of 5 mg/kg in male SD rats(N=4/time point, c-met plate). Figure 21C shows individual serum concentration-time profiles of FIT013a (c-Met/EGFR) after an IV dose of 5 mg/kg in male SD rats (N=4/time point, EGFR plate). Figure 21D shows individual serum concentration-time profiles of FIT013a (c-met/EGFR) after a SC dose of 5 mg/kg in male SD rats (N=4/time point, EGFR plate).

[00235] Figure 22 shows distributions of FIT013a, panitumumab, and H1332 in the serum and tumor in nude BALB/c mice (N=3 animal/group) inoculated with NCI-H1975- HGF tumor cells.

[00236] Figure 23 shows the efficacy of FIT013a, panitumumab, H1332, vehicle in inhibiting tumor size in nude BALB/c mice (N=8 animal/group) inoculated with NCI-H1975- HGF tumor cells. The antibodies were dosed two times/week i.p. for three weeks. The dosing for FIT013a was 16mg/kg, for HI 332 or Panitumumab was lOmg/kg.

[00237] Figure 24 shows enzyme assay for Factor Villa-like activity using BIOPHEN

FVIILC kit(Hyphen-Biomed). Samples containing mAb FIX, mAb FX, a combination of mAb FIX and mAb FX, Emicizumab, FIT014a, and purified FVIIIa were analyzed.

[00238] Figure 25 shows a multiple binding study of FIT014a against both FIX and

FX. Binding to FIX followed by FX; and binding by FX followed by FIX are both shown as indicated.

[00239] Figure 26 shows mean serum concentration-time profiles of 5mg/kg FIT014a administered intravenously (IV) or subcutaneously (SC) on either hlgG plate or Factor X plate. Antibody concentrations in rat serum samples were detected by ELISA with LLOQ of 62.5 ng/mL. On hlgG plate, the coating protein is anti-hlgG Fc, and the detection antibody is anti-hlgG Fab. On Factor X plate, the coating protein is Factor X, while the detection antibody is anti-human-IgG Fc.

[00240] Figure 27 shows a multiple binding study of FIT016a against both Her3 and

IGF1R Binding to Her3 followed by IGF1R; and binding by IGF1R followed by Her3 are both shown as indicated.

[00241] Figure 28 shows a multiple binding study of FIT017a against both DLL4 and

VEGF Binding to DLL4 followed by VEGF; and binding by VEGF followed by DLL4 are both shown as indicated. [00242] Figure 29A and Figure 29B show cell based FACS binding assays of

FIT018a compared to its related parental antibodies (CD3 mAb, Ofatumumab, and a combination of CD3 mAb and Ofatumumab), and human IgGl for their ability of binding to CD20 and CD3 on human B cells and human T cells. In the assay of Figure 29A, human B cell line Raji was used. In the assay of Figure 29B, human T cell line Jurkat was used.

[00243] Figure 30A shows cell based FACS binding assays of FIT018a compared to ofatumumab (CD20) and anti-RAC-human IgGl for their ability of binding to cynomologus CD20 cells. Figure 30B shows cell based FACS binding assays of FIT018a compared to ofatumumab (CD20), CD3 mAb for their ability of binding to cynomolgus T cells.

[00244] Figure 31A and Figure 31B show the ability of FIT018a, Ofatumumab, CD3 mAb, and a combination ofatumumab and CD3 mAb (1 : 1) in inducing apoptosis of human B cell (Raji) at day 2 (Figure 31A) and day 3 (Figure 31B), as measured in B-cell depletion assays.

[00245] Figure 32 shows a multiple binding study of FIT019a against both Her3 and

EGFR. Binding to Her3 followed by EGFR, and binding by EGFR followed by Her3 are both shown as indicated.

[00246] Figure 33A and Figure 33B show a multiple binding study of FIT019b against both Her3 and hEGFR. Binding to EGFR followed by Her3 (Figure 33A); and binding by Her3 followed by EGFR (Figure 33B) are both shown as indicated.

[00247] Figure 34A shows functional activity of FIT020b in MLR assays, when compared to the parental antibody Nivolumab, 1B12 and a combination of Nivolumab and lB12 (l: l) at a concentration of 0, 0.01, 0.1, 1, 10, or 100 nM, as measured by the level of induced IL-2. Figure 34B shows the induction of IL-2 at a concentration of 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 nM by these antibodies.

[00248] Figure 35 shows a multiple binding study of FIT020b against both PD-L1 and

PD-1. Binding to PD-L1 followed by PD-1; and binding by PD-1 followed by PD-L1 are both shown as indicated.

[00249]

[00250] Figure 36A and Figure 36B show cell based FACS binding assays of

FIT021b compared to its related parental antibodies (Ofatumumab, Epratuzumab, and a combination ofatumumab and Epratuzumab (1 : 1)), and human IgGl for its ability of binding to human B cells. In the assay of Figure 36A, human B cell line Raji was used. In the assay of Figure 36B, human B cell line Daudi was used. [00251] Figure 37 shows a multiple binding study of FIT022a against both Her3 and human PD-1. Binding to Her3 followed by human PD-1; and binding by human PD-1 followed by Her3 are both shown as indicated

[00252] Figure 38A shows functional activity of FIT022a in MLR assays, when compared to the parental antibody Nivolumab and Patritumab at a concentration of 0, 0.01, 0.1, 1, or 10 μg/ml. The induction of LI-2 was measured for each antibody. Human IgGl and human IgG4 were included as controls. Figure 38B shows the induction of IFN-γ by these antibodies.

[00253] Figure 39A and Figure 39B show a multiple binding study of FIT023a against both cMet-his and PD-Ll-his. Binding to cMet-his followed by PD-Ll-his (Figure 39A); and binding by PD-Ll-his followed by cMet-his (Figure 39B) are both shown as indicated.

[00254] Figure 40A and Figure 40B show a multiple binding study of FIT024a against both BTLA-his and PDl-his. Binding to BTLA-his followed by PDl-his (Figure 40A); and binding by PDl-his followed by BTLA-his (Figure 40B) are both shown as indicated.

[00255] Figure 41A and Figure 41B show a multiple binding study of FIT024b against both BTLA-his and PDl-his. Binding to BT:A-his followed by PDl-his (Figure 41A); and binding by PDl-his followed by BTLA-his (Figure 41B) are both shown as indicated.

[00256] Figure 42A shows functional activity of FIT024a and FIT024b in MLR assays, when compared to the parental antibody Nivolumab at a concentration of 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, or 100 nM, as measured by the level of induced IL-2. Human IgGl was included as a control. Figure 42B shows the induction of IL-2 at a concentration of 0, 0.01, 0.1, 1, 10, or 100 by these antibodies.

DETAILED DESCRIPTION

[00257] The present invention relates to multivalent and multispecific binding proteins, methods of making the binding proteins, and to their uses in the prevention and/or treatment of acute and chronic inflammatory diseases and disorders, cancers, and other diseases. This invention pertains to multivalent and/or multispecific binding proteins capable of binding two or more antigens. Specifically, the invention relates to Fabs-in-tandem immunoglobulins (FIT-Ig), and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such FIT-Igs. Methods of using the FIT-Igs of the invention to detect specific antigens, either in vitro or in vivo are also encompassed by the invention.

[00258] The novel family of binding proteins provided herein are capable of binding two or more antigens, e.g., with high affinity. Specifically, the present invention provides an approach to construct a bispecific binding protein using 2 parental monoclonal antibodies: mAb A, which binds to antigen a; and mAb B, which binds to antigen b.

[00259] In one aspect, the present invention provides a binding protein comprising a variable light chain specific for a first antigen or epitope, a first light chain constant domain, a variable heavy chain specific for a second antigen or epitope, a first heavy chain CHI, a variable heavy chain specific for the first antigen or epitope, a second heavy chain CHI, a variable heavy chain specific for the second antigen or epitope, and a second light chain constant domain. In one embodiment, the binding protein further comprises an Fc region. The binding protein may further comprise one or more amino acid or polypeptide linker linking two or more of the components of the binding protein. For example, the binding protein may comprise a polypeptide linker linking the light chain variable region to the light chain constant region.

[00260] In one embodiment, the present disclosure provides a binding protein comprising a polypeptide chain comprising VLA-CL-(Xl)n-VHB-CHl-(X2)n, wherein VLA is the light chain variable domain of mAb A, CL is a light chain constant domain, XI represents an amino acid or an oligopeptide linker, VHB is the heavy chain variable domain of mAb B, CHI is the first constant domain of the heavy chain, X2 represents an Fc region or a different dimerization domain, and n is 0 or 1.

[00261] In one embodiment, the invention provides a binding protein comprising three different polypeptide chains (Figure 1), wherein the first polypeptide chain (construct # 1) comprises VLA-CL-(Xl)n-VHB-CHl-(X2)n, wherein VLA is the light chain variable domain of mAb A, CL is a light chain constant domain, XI represents an amino acid or an oligopeptide linker, VHB is the heavy chain variable domain of mAb B, CHI is the first constant domain of the heavy chain, X2 represents an Fc region or a different dimerization domain, and n is 0 or 1. The second polypeptide chain (construct #2) comprises VHA-CHI, wherein VHA is the heavy chain variable domain of mAb A, and CHI is the first constant domain of the heavy chain. The third polypeptide chain (construct #3) comprises VLB-CL, wherein VLB is the light chain variable domain of mAb B, and CL is the constant domain of the light chain. [00262] In another embodiment, the invention provides a binding protein comprising three different polypeptide chains with the overall molecular design similar to the previous embodiment except the order of the variable domains are reversed. In the embodiment the first polypeptide chain comprises VHB-CHl-(Xl)n-VLA-CL-(X2)n, wherein VLA is a light chain variable domain of mAb A, CL is a light chain constant domain, XI represents an amino acid or an oligopeptide linker, VHB is the heavy chain variable domain of mAb B, CHI is the first constant domain of the heavy chain, X2 represents an Fc region or a different dimerization domain, and n is 0 or 1. The second polypeptide chain comprises VHA-CHI, wherein VHA is the heavy chain variable domain of mAb A and CHI is the first constant domain of the heavy chain. The third polypeptide chain comprises VLB-CL, wherein VLB is the light chain variable domain of mAb B and CL is the constant domain of the light chain.

[00263] In another embodiment the invention provides a binding protein comprising two different polypeptide chains (Figure 2), wherein the first polypeptide chain (construct #1) comprises VLA-CL-(Xl)n-VHB-CHl-(X2)n, wherein VLA is a light chain variable domain of mAb A, CL is a light chain constant domain, XI represents an amino acid or an oligopeptide linker, VHB is the heavy chain variable domain of mAb B, CHI is the first constant domain of the heavy chain, X2 represents an Fc region or a different dimerization domain, and n is 0 or 1. The second polypeptide chain (construct #4) comprises VHA-CHI- (X3)n-VLB-CL, wherein VHA is the heavy chain variable domain of mAb A, CHI is the first constant domain of the heavy chain, X3 represents an amino acid or polypeptide that is not a constant domain, n is 0 or 1, VLB is the light chain variable domain of mAb B, and CL is the constant domain of the light chain.

[00264] In another embodiment the invention provides a binding protein comprising two polypeptide chains with the overall molecular design similar to the previous embodiment except the order of the variable domains are reversed. In this embodiment the first polypeptide chain comprises VHB-CHl-(Xl)n-VLA-CL-(X2)n, wherein VLA is a light chain variable domain of mAb A, CL is a light chain constant domain, XI represents an amino acid or an oligopeptide linker, VHB is the heavy chain variable domain of mAb B, CHI is the first constant domain of the heavy chain, X2 represents an Fc region or a different dimerization domain, and n is 0 or 1. The second polypeptide chain comprises VLB-CL-(X3)n-VHA-CHl, wherein VHA is the heavy chain variable domain of mAb A, CHI is the first constant domain of the heavy chain, X3 represents an amino acid or an oligopeptide linker, n is 0 or 1, VLB is the light chain variable domain of mAb B, and CL is the constant domain of the light chain. [00265] In one embodiment, the VH and VL domains in the binding protein are selected from the group consisting of murine heavy /light chain variable domains, fully human heavy/light chain variable domains, CDR grafted heavy/light chain variable domains, humanized heavy/light chain variable domains, and mixtures thereof. In a preferred embodiment VHA/VLA and VHB/VLB are capable of binding the same antigen. In another embodiment VHA/VLA and VHB/VLB are capable of binding different antigens.

[00266] In one embodiment, the first polypeptide chain comprises VLA-CL-VHB-CH1-

Fc, and the CL and VHB of the first polypeptide chain are directly fused together. In another embodiment, the CL and VHB are linked by an amino acid or an oligopeptide linker. In another embodiment, the first polypeptide chain comprises VHB-CH1-VLA-CL-FC, and the

CHI and VLA are directly fused together. In another embodiment, the CHI and VLA are linked by an amino acid or an oligopeptide linker. In a further embodiment, the oligo- or poly-peptide linker comprises 1 or more amino acids of any reasonable sequence that provides flexibility. Preferably the linker is selected from the group consisting of G, GS, SG,

GGS, GSG, SGG, GGG, GGGS (SEQ ID NO: 489), SGGG (SEQ ID NO: 490), GGGGS

(SEQ ID NO: 491), GGGGS GS (SEQ ID NO: 492), , GGGGS GGS (SEQ ID NO: 493),

GGGGS GGGGS (SEQ ID NO: 494), GGGGS GGGGS GGGGS (SEQ ID NO: 495),

AKTTPKLEEGEF S EAR (SEQ ID NO: 496), AKTTPKLEEGEFSEARV (SEQ ID NO: 497),

AKTTPKLGG (SEQ ID NO: 498), SAKTTPKLGG (SEQ ID NO: 499), SAKTTP (SEQ ID

NO: 500), RADAAP (SEQ ID NO: 501), RADAAPTVS (SEQ ID NO: 502),

RADAAAAGGPGS (SEQ ID NO: 503), RADAAAA(G ₄ S)4 (SEQ ID NO: 504),

S AKTTPKLEEGEFSEARV (SEQ ID NO: 505), ADAAP (SEQ ID NO: 506),

ADAAPTVSIFPP (SEQ ID NO: 507), TVAAP (SEQ ID NO: 508), TVAAPSVFIFPP (SEQ

ID NO: 509), QPKAAP (SEQ ID NO: 510), QPKAAPSVTLFPP (SEQ ID NO: 511),

AKTTPP (SEQ ID NO: 512), AKTTPP S VTPLAP (SEQ ID NO: 513), AKTTAPSVYPLAP

(SEQ ID NO: 514), ASTKGP (SEQ ID NO: 515), ASTKGPSVFPLAP (SEQ ID NO: 516),

GENKVEYAPALMALS (SEQ ID NO: 517), GPAKELTPLKEAKVS (SEQ ID NO: 518),

GHEAAAVMQVQYPAS (SEQ ID NO: 519), and AKTTAP (SEQ ID NO: 80). In one embodiment, the amino acid sequence of the linker may be selected from the group consisting of SEQ ID NOs. 26, 28, and 49-86. In one embodiment, the linker is GSG (SEQ

ID NO: 26) or GGGGS GS (SEQ ID NO: 28). The linkers can also be in vivo cleavable peptide linkers, protease (such as MMPs) sensitive linkers, disulfide bond-based linkers that can be cleaved by reduction, etc., as previously described (Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges, edited by Stefan R. Schmidt), or any cleavable linkers known in the art. Such cleavable linkers can be used to release the top Fab in vivo for various purposes, in order to improve tissue/cell penetration and distribution, to enhance binding to targets, to reduce potential side effect, as well as to modulate in vivo functional and physical half-life of the 2 different Fab regions. In one embodiment, the binding protein comprises an Fc region. As used herein, the term "Fc region" refers to the C- terminal region of an IgG heavy chain. An example of the amino acid sequence containing the human IgGl Fc region is SEQ ID NO: 20. The Fc region of an IgG comprises two constant domains, CH2 and CH3.

[00267] In one embodiment, the Fc region is a variant Fc region. In one embodiment, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions, or insertions, relative to the parent Fc region. In a further embodiment, amino acid modifications of the Fc region alter the effector function activity relative to the parent Fc region activity. For example, in one embodiment, the variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding. In another embodiment, amino acid modifications of the Fc region may alter (i.e., increase or decrease) the affinity of the variant Fc region for an FcyR relative to the parent Fc region. For example, the variant Fc region may alter the affinity for FcyRI, FcyRII, FcyRIII.

[00268] In one preferred embodiment, the binding proteins provided herein are capable of binding one or more targets. In one embodiment, the target is selected from the group consisting of cytokines, cell surface proteins, enzymes and receptors. Preferably the binding protein is capable of modulating a biological function of one or more targets. More preferably the binding protein is capable of neutralizing one or more targets.

[00269] In one embodiment, the binding protein of the invention is capable of binding cytokines selected from the group consisting of lymphokines, monokines, and polypeptide hormones. In a further embodiment, the binding protein is capable of binding pairs of cytokines selected from the group consisting of IL-loc and IL-Ι β; IL-12 and IL-18, TNFoc and IL-23, TNFoc and IL-13; TNF and IL-18; TNF and IL-12; TNF and IL-lbeta; TNF and MIF; TNF and IL-6, TNF and IL-6 Receptor, TNF and IL-17; IL-17 and IL-20; IL-17 and IL-23; TNF and IL-15; TNF and VEGF; VEGFR and EGFR; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAM8; and TNFa and PGE4, IL-13 and PED2, TNF and PEG2.

[00270] In another embodiment, the binding protein of the invention is capable of binding pairs of targets selected from the group consisting of CD 137 and CD20, CD 137 and EGFR, CD137 and Her-2, CD137 and PD-1, CD137 and PDL-1, VEGF and PD-L1, Lag-3 and TIM-3, OX40 and PD-1, TIM-3 and PD-1, TIM-3 and PDL-1, EGFR and DLL-4, VEGF and EGFR, HGF and VEGF, VEGF and VEGF (same or a different epitope), VEGF and Ang2, EGFR and cMet, PDGF and VEGF, VEGF and DLL-4, OX40 and PD-L1, ICOS and PD-1, ICOS and PD-L1, Lag-3 and PD-1, Lag-3 and PD-L1, Lag-3 and CTLA-4, ICOS and CTLA-4, CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD3 and CD33; CD3 and CD133; CD38 & CD138; CD38 and CD20; CD20 and CD22; CD38 and CD40; CD40 and CD20; CD47 and CD20, CD-8 and IL-6; CSPGs and RGM A; CTLA-4 and BTN02; CTLA-4 and PD-1 ; IGFl and IGF2; IGFl/2 and Erb2B; IGF-IR and EGFR; EGFR and CD13; IGF-IR and ErbB3; EGFR-2 and IGFR; Her2 and Her2 (same or a different epitope); Factor IXa and Factor X ,VEGFR-2 and Met; VEGF-A and Angiopoietin-2 (Ang-2); IL-12 and TWEAK; IL-13 and IL-lbeta ; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; PDL-1 and CTLA-4; PD-1 and CTLA-4, PD-1 and TIM-3; RGM A and RGM B; Te38 and TNFa; TNFa and Blys; TNFa and CD-22; TNFa and CTLA-4 domain; TNFa and GP130; TNFa and IL-12p40; TNFa and RANK ligand; EGFR and PD-L1 ; EGFR and cMet; Her3 and IGF-IR; DLL-4 and VEGF; PD-1 and PD-L1; Her3 and PD-1, Her3 and EGFR, cMet and PD-L1, and BTLA and PD-1.

[00271] In some embodiments, the binding proteins contain variable regions or CDRs derived from CD20 antibodies including, but not limited to, ofatumumab, rituximab, iodine i 131 tositumomab, obinutuzumab, ibritumomab, and those described in U.S. Patent Nos. 9228008, 8206711, 7682612, 8562992, 7799900, 7422739, 7850962, 8097713, 8057793, 8592156, 6652852, 6893625, 6120767, 8084582, 8778339, 9184781, 7381560, 8101179, 9382327, 7151164, 7435803, 8529902, 9416187, 7812116, 8329181, 8034902, 9289479, 9234045, 4987084, 9173961, 9175086, 6410319, each of which is incorporated by reference in its entirety.

[00272] In some embodiments, the binding proteins contain variable regions or CDRs derived from CD3 antibodies including, but not limited to, muromonab-CD3, otelixizumab, teplizumab and visilizumab, and those described in U.S. Patent Nos. 8569450, 7635472,5585097, 6706265, 5834597, 9056906, 9486475, 7728114, 8551478, 9226962, 9192665, 9505849, 8394926, 6306575, 5795727, 8840888, 5627040, 9249217, 8663634, 6491917, 5877299, each of which is incorporated by reference in its entirety.

[00273] In some embodiments, the binding proteins contain variable regions or CDRs derived from CTLA-4 antibodies including, but not limited to, ipilimumab and those described in U.S. Patent Nos. 5434131, 5968510, 5844095, 7572772, 6090914, 7311910, 5885796, 5885579, 5770197, 5851795, 5977318, 7161058, 6875904, 7504554, 7034121, 6719972, 7592007, each of which is incorporated by reference in its entirety.

[00274] In some embodiments, the binding proteins contain variable regions or CDRs derived from PD-1 antibodies including, but are not limited to, pembrolizumab, nivolumab, atezolizumab, and those described in U.S. Patent Nos. 8741295, 7029674, 7722868, 9243052, 8927697, 9181342, 8552154, 9102727, 9220776, 9084776, 8008449, 9387247, 9492540, 8779105, 9358289, 9492539, 9205148, 8900587, 8952136, 8460886, 7414171, 8287856, 8580247, 7488802, 7521051, 8088905, 7709214, 8617546, 9381244, 8993731, 8574872, 7432059, 8216996, 9499603, 9102728, 9212224, each of which is incorporated by reference in its entirety.

[00275] In some embodiments, the binding proteins contain variable regions or CDRs derived from PD-Ll antibodies including, but not limited to, durvalumab, avelumab, and those described in U.S. Patent Nos. 8741295, 9102725, 8168179, 8952136, 8552154, 8617546, 9212224, 8217149, 8383796, each of which is incorporated by reference in its entirety.

[00276] In some embodiments, the binding proteins contain variable regions or CDRs derived from EGFR antibodies including, but not limited to, gefitinib, erlotinib, lapatinib, cetuximab, panitumumab, vandetanib, necitumumab, osimertinib, and those described in U.S. Patent Nos. 7723484, 9044460, 9226964, 8658175, 7618631, 8748175, 9499622, 9527913, 9493568, 8580263, 7514240, 9314536, 9051370, 9233171, 9029513, 8592152, 8597652, 9327035, 8628773, 9023356, 9132192, 8637026, 9283276, 9540440, 9545442, 8758756, 9120853, 7981605, 8546107, 7598350, 5212290, 8017321, 7589180, 9260524, 8790649, 9125896, 9238690, 8071093, each of which is incorporated by reference in its entirety.

[00277] In some embodiments, the binding proteins contain variable regions or CDRs derived from TIM3 (CD366) antibodies including, but not limited to, 4C4G3, 7D3, B8.2C12, F38-2E2, and those described in U.S. Patent Nos. 8841418, 8552156, 9556270, each of which is incorporated by reference in its entirety.

[00278] In some embodiments, the binding proteins contain variable regions or CDRs derived from cMet antibodies including, but not limited to, hl332, 71-8000, ab74217, and those described in U.S. Patent Nos. 8673302, 9120852, 7476724, 7892550, 9249221, 9535055, 9487589, 8329173, 9101610, 8101727, 9068011, 9260531, 9296817, 8481689, 8546544, 8563696, 8871909, 8889832, 8871910, 9107907, 8747850, 9469691, 8765128, 8729249, 8741290, 8637027, 8900582, 9192666, 9201074, 9505843, 8821869, 8163280, 7498420, 8562985, 8545839, 9213031, 9213032, 8217148, 8398974, 9394367, 9364556, 8623359, 9011865, 9375425, 9233155, 9169329, 9150655, each of which is incorporated by reference in its entirety.

[00279] In some embodiments, the binding proteins contain variable regions or CDRs derived from Factor IXa antibodies including, but not limited to, ab97619, abl28048, abl28038, and those described in U.S. Patent Nos. 7279161, 7033590, 4786726, 6624295,

7049411, 7297336, each of which is incorporated by reference in its entirety.

[00280] In some embodiments, the binding proteins contain variable regions or CDRs derived from Factor X antibodies including, but not limited to, PA5-22059, ab97632,

B122M.

[00281] In some embodiments, the binding proteins contain variable regions or CDRs derived from Her3 (ErbB3) antibodies including, but not limited to, duligotumab, elgemtumab, lumretuzumab, patritumab, seribantumab, and those described in U.S. Patent Nos. 9346883, 9321839, 8859737, 8362215, 8828388, 9220775, 9217039, 9527913, 9085622, 9192663, 8735551, 9011851, 7846440, 9284380, 8791244, 8691225, 9487588, 8961966, 9034328, 5968511, 9346889, 9217039, 9346890, each of which is incorporated by reference in its entirety.

[00282] In some embodiments, the binding proteins contain variable regions or CDRs derived from IGF-1R (CD221) antibodies including, but not limited to cixutumumab, dalotuzumab, figitumumab, ganitumab, robatumumab, teprotumumab, and those described in U.S. Patent Nos. 7572897, 7579157, 7968093, 7638605, 7329745, 7037498, 7982024, 8642037, 7700742, 9234041, 7815907, 8945871, 8361461, 9056907, 8168410, 7241444, 7914784, 9150644, 7985842, 7538195, 8268617, 8034904, 8344112, 7553485, 8101180, 8105598, 7824681, 8124079, 8420085, 7854930, each of which is incorporated by reference in its entirety. [00283] In some embodiments, the binding proteins contain variable regions or CDRs derived from DLL4 antibodies including, but not limited to demcizumab, enoticumab, navicixizumab, and those described in U.S. Patent Nos. 9469689, 9115195, 8623358, 9132190, 9469688, 9403904, 8663636, 8192738, 750124, each of which is incorporated by reference in its entirety.

[00284] In some embodiments, the binding proteins contain variable regions or CDRs derived from VEGF antibodies including, but not limited to Bevacizumab, Brolucizumab, Ranibizumab, and those described in U.S. Patent Nos. 8921537, 7910098, 7365166, 7060269, 7169901, 6884879, 7297334, 7375193, 9388239, 8834883, 8287873, 7998931, 8007799, 7785803, 9102720, 8486397, 6730489, 6383484, 9441034, 7097986, 9079953, 8945552, 8236312, 7740844, 6403088, 9018357, 8975381, 7691977, 7758859, 8512699, 8492527, 9353177, 8092797, 7811785, 8101177, 8592563, 9090684, 8349322, each of which is incorporated by reference in its entirety.

[00285] In some embodiments, the binding proteins contain variable regions or CDRs derived from CD22 antibodies including, but not limited to bectumomab, epratuzumab, inotuzumab, moxetumomab, pinatuzumab, and those described in U.S. Patent Nos. 9181343, 5484892, 9279019, 8591889, 9499632, 8481683, 7355012, 7777019, 8809502, 8389688, 7829086, each of which is incorporated by reference in its entirety.

[00286] In some embodiments, the binding proteins contain variable regions or CDRs derived from BTLA(CD272) antibodies including, but not limited to MIH26, AAP44003, MA5-16843, 6A5, and those described in U.S. Patent Nos. 8563694, 9346882, 8580259, 8247537, each of which is incorporated by reference in its entirety.

[00287] In some embodiments, the binding proteins contain antibodies as described in

WO2015103072, which is herein incorporated by reference in its entirety.

[00288] In one embodiment, the binding protein is capable of binding human IL-17 and human IL-20. In a further embodiment, the binding protein is capable of binding human IL-17 and human IL-20 and comprises a FIT-Ig polypeptide chain #1 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO. 15, 25, and 27; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 21 ; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about

85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 23. In another embodiment, the binding protein is capable of binding human IL-17 and human IL-20 and comprises FIT-Ig polypeptide chain #1 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO. 15, 25, and 27; and a polypeptide chain #4 that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO. 29, 30, and 31.

[00289] In one embodiment, the binding protein is capable of binding human CD3 and human CD20. In a further embodiment, the binding protein comprises a FIT-Ig polypeptide chain #1 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO. 41, 48 and 316; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID N0.44 or SEQ ID NO. 325; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 46 or SEQ ID NO. 330.

[00290] In one embodiment, the binding protein is capable of binding human IL-17 and human TNF. In a further embodiment, the binding protein is capable of binding human IL-17 and human TNF and comprises a FIT-Ig polypeptide chain #1 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 87; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 89; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 91.

[00291] In one embodiment, the binding protein is capable of binding human CTLA-4 and human PD-1. In a further embodiment, the binding protein is capable of binding human CTLA-4 and human PD-1 and comprises a FIT-Ig polypeptide chain #1 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 92, SEQ ID NO. 126, SEQ ID NO. 145, SEQ ID NO. 164, or SEQ ID NO. 183, a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 95, SEQ ID NO. 135, SEQ ID NO. 154, SEQ ID NO. 173, or SEQ ID NO. 192; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 97, SEQ ID NO. 140, SEQ ID NO. 159, SEQ ID NO. 178, or SEQ ID NO. 197.

[00292] In one embodiment, the binding protein is capable of binding EGFR and PD-

Ll . In a further embodiment, the binding protein is capable of binding EGFR and PD-L1 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 99, SEQ ID NO. 202 or SEQ ID NO. 221 ; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 100, SEQ ID NO. 211, or SEQ ID NO. 230; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 101, SEQ ID NO. 216, or SEQ ID NO. 235.

[00293] In one embodiment, the binding protein is capable of binding cMet and EGFR.

In a further embodiment, the binding protein is capable of binding cMET and EGFR and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 102 or SEQ ID NO. 240; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 103 or SEQ ID NO. 249; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 104 or SEQ ID NO. 254.

[00294] In one embodiment, the binding protein is capable of binding Factor IXa and

Factor X. In a further embodiment, the binding protein is capable of binding Factor IXa and Factor X and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 105, or SEQ ID NO. 259; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 106 or SEQ ID NO. 268; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 107 or SEQ ID NO. 273. [00295] In one embodiment, the binding protein is capable of binding Her3 and IGF-

1R. In a further embodiment, the binding protein is capable of binding Her3 and IGF-1R and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 108, SEQ ID NO. 278; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 109 or SEQ ID NO. 287; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 110 or SEQ ID NO. 292.

[00296] In one embodiment, the binding protein is capable of binding DLL-4 and

VEGF. In a further embodiment, the binding protein is capable of binding DLL-4 and VEGF and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. I l l, or SEQ ID NO. 297; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 112, or SEQ ID NO. 306; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 113 or SEQ ID NO. 311.

[00297] In one embodiment, the binding protein is capable of binding CD20 and CD3.

In a further embodiment, the binding protein is capable of binding CD20 and CD3 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 114 or SEQ ID NO. 316; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 115 or SEQ ID NO. 325; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 116 or SEQ ID NO. 330.

[00298] In one embodiment, the binding protein is capable of binding Her3 and EGFR.

In a further embodiment, the binding protein is capable of binding Her3 and EGFR and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about

75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ

ID NO. 117, SEQ ID NO. 335, or SEQ ID NO. 354; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 118, SEQ ID NO. 344, or SEQ ID NO. 363; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 119, SEQ ID NO. 349, or SEQ ID NO. 368.

[00299] In one embodiment, the binding protein is capable of binding PD-1 and PD-

Ll . In a further embodiment, the binding protein is capable of binding PD-1 and PD-L1 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 120 or SEQ ID NO. 373; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 121 or SEQ ID NO. 382; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 122 or SEQ ID NO. 387.

[00300] In one embodiment, the binding protein is capable of binding Her3 and PD-1.

In a further embodiment, the binding protein is capable of binding Her3 and PD-1 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 123 or SEQ ID NO. 411; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 124 or SEQ ID NO. 420; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 125 or SEQ ID NO. 425.

[00301] In one embodiment, the binding protein is capable of binding cMet and PD-

Ll . In a further embodiment, the binding protein is capable of binding cMet and PD-L1 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about

75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ

ID NO. 430; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO.

439; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about

80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 444.

[00302] In one embodiment, the binding protein is capable of binding BTLA and PD-

1. In a further embodiment, the binding protein is capable of binding BTLA and PD-1 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 449 or SEQ ID NO. 468; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 458 or SEQ ID NO. 477; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 463 or SEQ ID NO. 482.

[00303] In one embodiment, the binding protein is capable of binding CD20 and

CD22. In a further embodiment, the binding protein is capable of binding CD20 and CD22 and comprises a FIT-Ig polypeptide chain #1 sequence that that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 392; a polypeptide chain #2 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 401 ; and a polypeptide chain #3 sequence that is about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% identical to SEQ ID NO. 406.

[00304] Biologically active variants or functional variants of the exemplary binding proteins described herein are also a part of the present invention. As used herein, the phrase "a biologically active variant" or "functional variant" with respect to a protein refers to an amino acid sequence that is altered by one or more amino acids with respect to a reference sequence, while still maintains substantial biological activity of the reference sequence. The variant can have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. The following table shows exemplary conservative amino acid substitutions. In some embodiments, the variant has one or more amino acid substitutions, wherein one or more or all substitutions are acidic amino acid, such as Aspartic acid, Asparagine, Glutamc acid, or Glutamine.

Original Very Highly - Highly Conserved Conserved Substitutions

Residue Conserved Substitutions (from the (from the Blosum65 Matrix)

Substitutions Blosum90 Matrix)

Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, Val

Arg Lys Gin, His, Lys Asn, Gin, Glu, His, Lys

Asn Gin; His Asp, Gin, His, Lys, Ser, Thr Arg, Asp, Gin, Glu, His, Lys, Ser, Thr

Asp Glu Asn, Glu Asn, Gin, Glu, Ser

Cys Ser None Ala Gin Asn Arg, Asn, Glu, His, Lys, Met Arg, Asn, Asp, Glu, His, Lys, Met, Ser

Glu Asp Asp, Gin, Lys Arg, Asn, Asp, Gin, His, Lys, Ser

Gly Pro Ala Ala, Ser

His Asn; Gin Arg, Asn, Gin, Tyr Arg, Asn, Gin, Glu, Tyr

He Leu; Val Leu, Met, Val Leu, Met, Phe, Val

Leu He; Val He, Met, Phe, Val He, Met, Phe, Val

Lys Arg; Gin; Glu Arg, Asn, Gin, Glu Arg, Asn, Gin, Glu, Ser,

Met Leu; He Gin, He, Leu, Val Gin, He, Leu, Phe, Val

Phe Met; Leu; Tyr Leu, Tip, Tyr He, Leu, Met, Tip, Tyr

Ser Thr Ala, Asn, Thr Ala, Asn, Asp, Gin, Glu, Gly, Lys, Thr

Thr Ser Ala, Asn, Ser Ala, Asn, Ser, Val

Tip Tyr Phe, Tyr Phe, Tyr

Tyr Tip; Phe His, Phe, Tip His, Phe, Tip

Val He; Leu He, Leu, Met Ala, He, Leu, Met, Thr

Alternatively, a variant can have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations can also include amino acid deletion or insertion, or both. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software.

[00305] Binding proteins that are capable of binding the same epitopes on a given group of targets as that of an exemplary bispecific binding protein described herein are also a part of the present invention. The epitopes can be linear epitopes, conformational epitopes, or a mixture thereof. In some embodiments, such same epitopes can be identified by a suitable epitope mapping technique, including but not limited to, X-ray co-crystallography, array-based oligo-peptide scanning, site-directed mutagenesis, high throughput mutagenesis mapping, bacteriophage surface display, and hydrogen-deuterium exchange. Additional methods are described in U.S. Patent Nos. 5955264, 65796676, 6984488, and 8802375, each of which is incorporated by reference in its entirety for all purposes.

[00306] In another embodiment, the binding protein of the invention is capable of binding one or two cytokines, cytokine-related proteins, and cytokine receptors selected from the group consisting of BMP1, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP 8, CSF1 (M-

CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (aFGF), FGF2 (bFGF), FGF3 (int-2),

FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B,

FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFNG, IFNW1, FIL1, FIL1 (EPSILON), FIL1 (ZETA), ILIA, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, FGER1, FGFR2, FGFR3, EGFR, ROR1, 2B4, KIR, CD137, CD27, OX40, CD40L, A2aR, CD48, B7-1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CD70, CD40, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a ), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, FIGF (VEGFD), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.

[00307] The binding protein of the invention is capable of binding one or more chemokines, chemokine receptors, and chemokine-related proteins selected from the group consisting of CCL1 (1-309), CCL2 (MCP -1 / MCAF), CCL3 (MIP-la), CCL4 (MIP-lb),

CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCL11 (eotaxin), CCL13 (MCP-4),

CCL15 (MIP-ld), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b),

CCL20 (MIP-3a), CCL21 (SLC / exodus-2), CCL22 (MDC / STC-1), CCL23 (MPIF-1),

CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK / ILC),

CCL28, CXCL1 (GROl), CXCL2 (GR02), CXCL3 (GR03), CXCL5 (ENA-78), CXCL6

(GCP-2), CXCL9 (MIG), CXCL10 (IP 10), CXCL11 (I-TAC), CXCL12 (SDF1), CXCL13,

CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1), SCYE1, XCL1

(lymphotactin), XCL2 (SCM-lb), BLRl (MDR15), CCBP2 (D6 / JAB61), CCRl (CKRl /

HM145), CCR2 (mcp-lRB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 /

ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EBI1), CCR8

(CMKBR8 / TER1 / CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR),

XCRl (GPR5 / CCXCRl), CMKLRl, CMKORl (RDCl), CX3CR1 (V28), CXCR4, GPR2

(CCRIO), GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6 (TYMSTR

/STRL33 / Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R (GPR16), TCP10,

CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3, GRCC10 (CIO), EPO, FY (DARC), GDF5, HIF1A, IL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL.

[00308] In another embodiment, a binding protein of the invention is capable of binding cell surface protein such as, for example, integrins. In another embodiment, the binding protein of the invention is capable of binding enzymes selected from the group consisting of kinases and proteases. In yet another embodiment, the binding protein of the invention is capable of binding receptors selected from the group consisting of lymphokine receptors, monokine receptors, and polypeptide hormone receptors.

[00309] In one embodiment, the binding protein is multivalent. In another embodiment, the binding protein is multispecific. The multivalent and or multispecific binding proteins described above have desirable properties particularly from a therapeutic standpoint. For instance, the multivalent and or multispecific binding protein may (1) be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind; (2) be an agonist antibody; and/or (3) induce cell death and/or apoptosis of a cell expressing an antigen which the multivalent antibody is capable of binding to. The "parent antibody" which provides at least one antigen binding specificity of the multivalent and or multispecific binding proteins may be one which is internalized (and/or catabolized) by a cell expressing an antigen to which the antibody binds; and/or may be an agonist, cell death-inducing, and/or apoptosis-inducing antibody, and the multivalent and or multispecific binding protein as described herein may display improvement(s) in one or more of these properties. Moreover, the parent antibody may lack any one or more of these properties, but may be endowed with them when constructed as a multivalent binding protein as herein described.

[00310] In another embodiment a binding protein of the invention has an on rate constant (Kon) to one or more targets selected from the group consisting of: at least about K^M- ¹ ; at least about K^M' ¹ ; at least about lO^T ¹ ; at least about K^M' ¹ ; and at least about lC^M' ¹ (inclusive of all values therebetween), as measured by surface plasmon resonance. Preferably, the binding protein of the invention has an on rate constant (Kon) to one or more targets between lC^M ^{' 1} to lC M ^{' 1} ; between 10 M ^"1 s ^"1 to lC^M ^{' 1} ; between K^M- ¹ to K^M ^{' 1} ; or between K^M ^{' 1} to lO'TVTV ¹ (inclusive of all values therebetween), as measured by surface plasmon resonance.

[00311] In another embodiment a binding protein has an off rate constant (Koff) for one or more targets selected from the group consisting of: at most about lO'V ¹ ; at most about 10 ^' V ¹ ; at most about 10 ^"5 s ^_1 ; and at most about 10 ^"6 s ^_1 , as measured by surface plasmon resonance (inclusive of all values therebetween). Preferably, the binding protein of the invention has an off rate constant (Kofi) to one or more targets of 10 ^" s ^_1 to 10 ^"4 s ^_1 ; of 10 ^"4 s ^_1 to 10 ^"5 s ^_1 ; or of 10 ^"5 s ^_1 to 10 ^"6 s ^_1 , as measured by surface plasmon resonance (inclusive of all values therebetween).

[00312] In another embodiment a binding protein has a dissociation constant (KD) to one or more targets selected from the group consisting of: at most about 10 ^"7 M; at most about 10 ^"8 M; at most about 10 ^"9 M; at most about 10 ^"10 M; at most about 10 ^"11 M; at most about 10 ^"12 M; and at most 10 ^"13 M (inclusive of all values therebetween). Preferably, the binding protein of the invention has a dissociation constant (KD) to IL-12 or IL-23 of 10 ^"7 M to 10- ⁸ M; of 10- ⁸ M to 10 ^"9 M; of 10 ^"9 M to 10 ^"10 M; of 10 ^"10 to 10 ^"11 M; of 10 ^"11 M to 10 ^"12 M; or of 10 ^"12 M to 10 ^"13 M (inclusive of all values therebetween).

[00313] In another embodiment a binding protein of the invention has a monomer % of at least about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 80%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, or more, or 100% (inclusive of all values therebetween) in a one-step Protein A purification using Size- exclusion chromatography (SEC)- HPLC.

[00314] In another embodiment a binding protein of the invention has an expression level at least about 0.01 mg/L, 0.05 mg/L, 0.1 mg/L, about 0.2 mg/L, about 0.3 mg/L, about 0.4 mg/L, about 0.5 mg/L, about 0.6 mg/L, about 0.7 mg/L, about 0.8 mg/L, about 0.9 mg/L, about 1.0 mg/L, about 2 mg/L, about 3 mg/L, about 4 mg/L, about 5 mg/L, about 6 mg/L, about 7 mg/L, about 8 mg/L, about 9 mg/L, about 10 mg/L, about 11 mg/L, about 12 mg/L, about 13 mg/L, about 14 mg/L, about 15 mg/L, about 16 mg/L, about 17 mg/L, about 18 mg/L, about 19 mg/L, about 20 mg/L, about 21 mg/L, about 22 mg/L, about 23 mg/L, about 24 mg/L, about 25 mg/L, about 26 mg/L, about 27 mg/L, about 28 mg/L, about 29 mg/L, about 30 mg/L, about 40 mg/L, about 50 mg/L, about 60 mg/L, about 70 mg/L, about 80 mg/L, about 90 mg/L, about 100 mg/L, about 200 mg/L, about 300 mg/L, about 400 mg/L, about 500 mg/L, about 600 mg/L, about 700 mg/L, about 800 mg/L, about 900 mg/L, about 1000 mg/L (inclusive of all values therebetween) or more, under optimized conditions. In some embodiments, the binding protein of the invention is expressed in 293E cells or in any other cells suitable for the purpose.

[00315] In another embodiment a binding protein of the invention has an Tml transition temperature as measured by Differential scanning calorimetry (DSC) which is at least about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, about 80°C, about 81°C, about 82°C, about 83°C, about 84°C, about 85°C, about 86°C, about 87°C, about 88°C, about 89°C, about 90°C, about 95°C, about 99°C (inclusive of all values therebetween) or more.

[00316] A binding protein of the invention has great stability in freeze-thaw test. In some embodiments, when a binding protein sample of the invention is thawed and incubated at 4 °C, 25 °C and 40 °C for 1 day, 3 days or 7 days, reduction of intact binding protein due to aggregates is less than about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, about 0.1%, about 0.05%, about 0.01% (inclusive of all values therebetween), or less, as measured by SEC-HPLC.

[00317] In another embodiment, when a binding protein of the invention is administered intravenously, it has one or more of the following PK parameters (1) an Apparent total body clearance of the drug from plasma (CL, mL/day/kg) of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 (inclusive of all values therebetween) or more; (2) an Apparent volume of distribution at steady state (Vss, mL/kg) of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 (inclusive of all values therebetween), or more; (3) an Apparent volume of the central or plasma compartment in a two-compartment model (VI, mL/kg) of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 (inclusive of all values therebetween), or more; (4) an initial or disposition half-life (Alpha tl/2, day) of about 0.01, 0.05, 0.1, 0.2 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 (inclusive of all values therebetween) or more; (5) a Terminal elimination half-life (Beta tl/2, day) of about 0.01, 0.05, 0.1, 0.2 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 (inclusive of all values therebetween) or more; (6) an area under the plasma concentration-time curve (AUC, day x μg/mL) of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 (inclusive of all values therebetween) or more; and (6) a Mean residence time (MRT, day) of about 0.01, 0.05, 0.1, 0.2 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 (inclusive of all values therebetween) or more. In some embodiments, the above parameters are associated with a dosage of about 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg (inclusive of all values therebetween) or more.

[00318] In another embodiment, when a binding protein of the invention is administered subcutaneously, it has one or more of the following PK parameters (1) a Time to reach maximum (peak) plasma concentration following drug administration (Tmax, day) of about 0.05, 0.1, 0.2 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 (inclusive of all values therebetween) or more; (2) a Maximum (peak) plasma drug concentration (Cmax, μg/ml) of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500 (inclusive of all values therebetween) or more; (3) an Elimination half-life (Terminal ti/2, day) of about 0.01, 0.05, 0.1, 0.2 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 (inclusive of all values therebetween) or more; (4) an Area under the plasma concentration-time curve from time zero to time of last measurable concentration (AUClast, day x μg/ml) of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 (inclusive of all values therebetween) or more; (5) an Area under the plasma concentration-time curve from time zero to infinity (AUCinf, day x μg/ml) of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 (inclusive of all values therebetween) or more; (6) a Formation clearance of drug to metabolite (CL/F, mL/day/kg) of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 (inclusive of all values therebetween) or more; (7) a Bioavailability (F, %) of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 (inclusive of all values therebetween), or more. In some embodiments, the above parameters are associated with a dosage of about 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg (inclusive of all values therebetween) or more.

[00319] In another embodiment, the binding protein described above is a conjugate further comprising an agent selected from the group consisting of an immunoadhesion molecule, an imaging agent, a therapeutic agent, and a cytotoxic agent. In one embodiment, the imaging agent is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. In a further embodiment, the imaging agent is a radiolabel selected from the group consisting of: H, ¹⁴ C, ⁵ S, ⁹⁰ Y, "Tc, ^m In, ¹²⁵ I, ^{1 1} I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵ Sm. In one embodiment, the therapeutic or cytotoxic agent is selected from the group consisting of an immunosuppressive agent, an immuno-stimulatory agent, an anti-metabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an anti-angiogenic agent, an anti-mitotic agent, an anthracycline, a toxin, and an apoptotic agent. In one embodiment, the binding protein is conjugated directly to the agent. In another embodiment, the binding protein is conjugated to the agent via a linker. Suitable linkers include, but are not limited to, amino acid and polypeptide linkers disclosed herein. Linkers may be cleavable or non-cleavable.

[00320] In another embodiment the binding protein described above is a crystallized binding protein and exists as a crystal. Preferably the crystal is a carrier-free pharmaceutical controlled release crystal. More preferably the crystallized binding protein has a greater half life in vivo than the soluble counterpart of said binding protein. Most preferably the crystallized binding protein retains biological activity.

[00321] In another embodiment the binding protein described above is glycosylated.

Preferably, the glycosylation is a human glycosylation pattern.

[00322] One aspect of the invention pertains to an isolated nucleic acid encoding any one of the binding protein disclosed above. A further embodiment provides a vector comprising the isolated nucleic acid disclosed above wherein said vector is selected from the group consisting of pcDNA; pTT (Durocher et al, Nucleic Acids Research 2002, Vol 30, No.2); pTT3 (pTT with additional multiple cloning site; pEFBOS (Mizushima, S. and Nagata, S., (1990) Nucleic acids Research Vol 18, No. 17); pBV; pJV; pcDNA3.1 TOPO, pEF6 TOPO and pBJ. The multi-specific binding proteins and methods of making the same are provided. The binding protein can be generated using various techniques. Expression vectors, host cells and methods of generating the binding proteins are provided in this disclosure.

[00323] The antigen-binding variable domains of the binding proteins of this disclosure can be obtained from parent binding proteins, including polyclonal Abs, monoclonal Abs, and or receptors capable of binding antigens of interest. These parent binding proteins may be naturally occurring or may be generated by recombinant technology.

The person of ordinary skill in the art is well familiar with many methods for producing antibodies and/or isolated receptors, including, but not limited to using hybridoma techniques, selected lymphocyte antibody method (SLAM), use of a phage, yeast, or RNA- protein fusion display or other library, immunizing a non-human animal comprising at least some of the human immunoglobulin locus, and preparation of chimeric, CDR-grafted, and humanized antibodies. See, e.g., US Patent Publication No. 20090311253 Al . Variable domains may also be prepared using affinity maturation techniques. The binding variable domains of the binding proteins can also be obtained from isolated receptor molecules obtained by extraction procedures known in the art (e.g., using solvents, detergents, and/or affinity purifications), or determined by biophysical methods known in the art (e.g., X-ray crystallography, NMR, interferometry, and/or computer modeling).

[00324] An embodiment is provided comprising selecting parent binding proteins with at least one or more properties desired in the binding protein molecule. In an embodiment, the desired property is one or more of those used to characterize antibody parameters, such as, for example, antigen specificity, affinity to antigen, potency, biological function, epitope recognition, stability, solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross reactivity, or orthologous antigen binding. See, e.g., US Patent Publication No. 20090311253.

[00325] The multi-specific antibodies may also be designed such that one or more of the antigen binding domain are rendered non-functional. The variable domains may be obtained using recombinant DNA techniques from parent binding proteins generated by any one of the methods described herein. In an embodiment, a variable domain is a murine heavy or light chain variable domain. In another embodiment, a variable domain is a CDR grafted or a humanized variable heavy or light chain domain. In an embodiment, a variable domain is a human heavy or light chain variable domain.

[00326] In an embodiment, one or more constant domains are linked to the variable domains using recombinant DNA techniques. In an embodiment, a sequence comprising one or more heavy chain variable domains is linked to a heavy chain constant domain and a sequence comprising one or more light chain variable domains is linked to a light chain constant domain. In an embodiment, the constant domains are human heavy chain constant domains and human light chain constant domains, respectively. In an embodiment, the heavy chain is further linked to an Fc region. The Fc region may be a native sequence Fc region or a variant Fc region. In another embodiment, the Fc region is a human Fc region. In another embodiment, the Fc region includes Fc region from IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD. [00327] Additionally, the binding proteins provided herein can be employed for tissue- specific delivery (target a tissue marker and a disease mediator for enhanced local PK thus higher efficacy and/or lower toxicity), including intracellular delivery (targeting an internalizing receptor and an intracellular molecule), delivering to inside brain (targeting transferrin receptor and a CNS disease mediator for crossing the blood-brain barrier). The binding proteins can also serve as a carrier protein to deliver an antigen to a specific location via binding to a non-neutralizing epitope of that antigen and also to increase the half-life of the antigen. Furthermore, the binding proteins can be designed to either be physically linked to medical devices implanted into patients or target these medical devices (see Burke et al. (2006) Advanced Drug Deliv. Rev. 58(3): 437-446; Hildebrand et al. (2006) Surface and Coatings Technol. 200(22-23): 6318-6324; Drug/device combinations for local drug therapies and infection prophylaxis, Wu (2006) Biomaterials 27(l l):2450-2467; Mediation of the cytokine network in the implantation of orthopedic devices, Marques (2005) Biodegradable Systems in Tissue Engineer. Regen. Med. 377-397). Directing appropriate types of cell to the site of medical implant may promote healing and restoring normal tissue function. Alternatively, inhibition of mediators (including but not limited to cytokines), released upon device implantation by a receptor antibody fusion protein coupled to or target to a device is also provided.

[00328] In one aspect, a host cell is transformed with the vector disclosed above. In one embodiment, the host cell is a prokaryotic cell. In a further embodiment, the host cell is Escherecia coli. In another embodiment, the host cell is an eukaryotic cell. In a further embodiment, the eukaryotic cell is selected from the group consisting of protist cell, animal cell, plant cell and fungal cell. In one embodiment, the host cell is a mammalian cell including, but not limited to, 293, COS, NS0, and CHO and; or a fungal cell such as Saccharomyces cerevisiae; or an insect cell such as Sf9.

[00329] Another aspect of the invention provides a method of producing a binding protein disclosed above comprising culturing any one of the host cells also disclosed above in a culture medium under conditions sufficient to produce the binding protein. Preferably 50%-75% of the binding protein produced by this method is a dual specific tetravalent binding protein. More preferably 75%-90% of the binding protein produced by this method is a dual specific tetravalent binding protein. Most preferably 90%-95% of the binding protein produced is a dual specific tetravalent binding protein. [00330] Another embodiment provides a binding protein produced according to the method disclosed above.

[00331] One embodiment provides a composition for the release of a binding protein wherein the composition comprises a formulation which in turn comprises a crystallized binding protein, as disclosed above and an ingredient; and at least one polymeric carrier. Preferably the polymeric carrier is a polymer selected from one or more of the group consisting of: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid), poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride- alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polyeaccharides, blends and copolymers thereof. Preferably the ingredient is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β- cyclodextrin, methoxypolyethylene glycol and polyethylene glycol. Another embodiment provides a method for treating a mammal comprising the step of administering to the mammal an effective amount of the composition disclosed above.

[00332] The invention also provides a pharmaceutical composition comprising a binding protein, as disclosed above and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers include, but are not limited to, phosphate buffer or saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on

Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In some cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00333] In a further embodiment the pharmaceutical composition comprises at least one additional therapeutic agent for treating a disorder. In one embodiment, the additional agent is selected from the group consisting of: therapeutic agents, imaging agents, cytotoxic agent, angiogenesis inhibitors (including but not limited to anti-VEGF antibodies or VEGF- trap); kinase inhibitors (including but not limited to KDR and TIE-2 inhibitors); co- stimulation molecule blockers (including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig, anti-PD-1 , anti-CD20); adhesion molecule blockers (including but not limited to anti-LFA-1 Abs, anti-E/L selectin Abs, small molecule inhibitors); anti-cytokine antibody or functional fragment thereof (including but not limited to anti-IL-18, anti-TNF, anti-IL-6/cytokine receptor antibodies); methotrexate; cyclosporin; rapamycin; FK506; detectable label or reporter; a TNF antagonist; an antirheumatic; a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or analog, a cytokine, and a cytokine antagonist.

[00334] In another aspect, the invention provides a method for treating a human subject suffering from a disorder in which the target, or targets, capable of being bound by the binding protein disclosed above is detrimental, comprising administering to the human subject a binding protein disclosed above such that the activity of the target or targets in the human subject is inhibited and treatment or preventions of the disorder is achieved. In one embodiment, the disease or disorder is an inflammatory condition, autoimmune disease, or cancer. In one embodiment, the disease or disorder is selected from the group comprising arthritis, osteoarthritis, juvenile chronic arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, inflammatory bowel disease, insulin dependent diabetes mellitus, thyroiditis, asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft versus host disease, organ transplant rej ection, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation,

Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, infectious diseases, parasitic diseases, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, malignancies, heart failure, myocardial infarction, Addison's disease, sporadic, polyglandular deficiency type I and polyglandular deficiency type II, Schmidt's syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic synovitis, chlamydia, yersinia and salmonella associated arthropathy, spondyloarthopathy, atheromatous disease/arteriosclerosis, atopic allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis B, Hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinemia), dilated cardiomyopathy, female infertility, ovarian failure, premature ovarian failure, fibrotic lung disease, cryptogenic fibrosing alveolitis, postinflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjogren's disease associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, psoriasis type 1, psoriasis type 2, idiopathic leucopaenia, autoimmune neutropaenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the kidneys, lyme disease, discoid lupus erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis

(all subtypes), sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Sjorgren's syndrome, Takayasu's disease/arteritis, autoimmune thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute liver disease, chronic liver diseases, alcoholic cirrhosis, alcohol-induced liver injury, choleosatatis, idiosyncratic liver disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis, allergy and asthma, group B streptococci (GBS) infection, mental disorders (e.g. , depression and schizophrenia), Th2

Type and Thl Type mediated diseases, acute and chronic pain (different forms of pain), and cancers such as lung, breast, stomach, bladder, colon, pancreas, ovarian, prostate and rectal cancer and hematopoietic malignancies (leukemia and lymphoma), Abetalipoprotemia,

Acrocyanosis, acute and chronic parasitic or infectious processes, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute or chronic bacterial infection, acute pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic beats, AIDS dementia complex, alcohol-induced hepatitis, allergic conjunctivitis, allergic contact dermatitis, allergic rhinitis, allograft rejection, alpha-1- antitrypsin deficiency, amyotrophic lateral sclerosis, anemia, angina pectoris, anterior horn cell degeneration, anti cd3 therapy, antiphospholipid syndrome, anti-receptor hypersensitivity reactions, aordic and peripheral aneuryisms, aortic dissection, arterial hypertension, arteriosclerosis, arteriovenous fistula, ataxia, atrial fibrillation (sustained or paroxysmal), atrial flutter, atrioventricular block, B cell lymphoma, bone graft rejection, bone marrow transplant (BMT) rejection, bundle branch block, Burkitt's lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome, cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation response, cartilage transplant rejection, cerebellar cortical degenerations, cerebellar disorders, chaotic or multifocal atrial tachycardia, chemotherapy associated disorders, chronic myelocytic leukemia (CML), chronic alcoholism, chronic inflammatory pathologies, chronic lymphocytic leukemia (CLL), chronic obstructive pulmonary disease (COPD), chronic salicylate intoxication, colorectal carcinoma, congestive heart failure, conjunctivitis, contact dermatitis, cor pulmonale, coronary artery disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic fibrosis, cytokine therapy associated disorders, Dementia pugilistica, demyelinating diseases, dengue hemorrhagic fever, dermatitis, dermatologic conditions, diabetes, diabetes mellitus, diabetic ateriosclerotic disease, Diffuse Lewy body disease, dilated congestive cardiomyopathy, disorders of the basal ganglia, Down's Syndrome in middle age, drug- induced movement disorders induced by drugs which block CNS dopamine receptors, drug sensitivity, eczema, encephalomyelitis, endocarditis, endocrinopathy, epiglottitis, epstein-barr virus infection, erythromelalgia, extrapyramidal and cerebellar disorders, familial hematophagocytic lymphohistiocytosis, fetal thymus implant rejection, Friedreich's ataxia, functional peripheral arterial disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular nephritis, graft rejection of any organ or tissue, gram negative sepsis, gram positive sepsis, granulomas due to intracellular organisms, hairy cell leukemia, Hallerrorden-Spatz disease, hashimoto's thyroiditis, hay fever, heart transplant rejection, hemachromatosis, hemodialysis, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, hemorrhage, hepatitis (A), His bundle arrythmias, HIV infection/HIV neuropathy, Hodgkin's disease, hyperkinetic movement disorders, hypersensitity reactions, hypersensitivity pneumonitis, hypertension, hypokinetic movement disorders, hypothalamic-pituitary-adrenal axis evaluation, idiopathic

Addison's disease, idiopathic pulmonary fibrosis, antibody mediated cytotoxicity, Asthenia, infantile spinal muscular atrophy, inflammation of the aorta, influenza a, ionizing radiation exposure, iridocyclitis/uveitis/optic neuritis, ischemia- reperfusion injury, ischemic stroke, juvenile rheumatoid arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma, kidney transplant rejection, legionella, leishmaniasis, leprosy, lesions of the corticospinal system, lipedema, liver transplant rejection, lymphederma, malaria, malignamt Lymphoma, malignant histiocytosis, malignant melanoma, meningitis, meningococcemia, metabolic/idiopathic, migraine headache, mitochondrial multi-system disorder, mixed connective tissue disease, monoclonal gammopathy, multiple myeloma, multiple systems degenerations (Mencel

Dejerine- Thomas Shi-Drager and Machado- Joseph), myasthenia gravis, mycobacterium avium intracellulare, mycobacterium tuberculosis, myelodyplastic syndrome, myocardial infarction, myocardial ischemic disorders, nasopharyngeal carcinoma, neonatal chronic lung disease, nephritis, nephrosis, neurodegenerative diseases, neurogenic I muscular atrophies, neutropenic fever, non- Hodgkin's lymphoma, occlusion of the abdominal aorta and its branches, occlusive arterial disorders, okt3 therapy, orchitis/epidydimitis, orchitis/vasectomy reversal procedures, organomegaly, osteoporosis, pancreas transplant rejection, pancreatic carcinoma, paraneoplastic syndrome/hypercalcemia of malignancy, parathyroid transplant rejection, pelvic inflammatory disease, perennial rhinitis, pericardial disease, peripheral atherlosclerotic disease, peripheral vascular disorders, peritonitis, pernicious anemia, Pneumocystis carinii pneumonia, pneumonia, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), post perfusion syndrome, post pump syndrome, post-MI cardiotomy syndrome, preeclampsia, Progressive supranucleo Palsy, primary pulmonary hypertension, radiation therapy, Raynaud's phenomenon and disease, Raynoud's disease, Refsum's disease, regular narrow QRS tachycardia, renovascular hypertension, reperfusion injury, restrictive cardiomyopathy, sarcomas, scleroderma, senile chorea, Senile Dementia of Lewy body type, seronegative arthropathies, shock, sickle cell anemia, skin allograft rejection, skin changes syndrome, small bowel transplant rejection, solid tumors, specific arrythmias, spinal ataxia, spinocerebellar degenerations, streptococcal myositis, structural lesions of the cerebellum, Subacute sclerosing panencephalitis, Syncope, syphilis of the cardiovascular system, systemic anaphalaxis, systemic inflammatory response syndrome, systemic onset juvenile rheumatoid arthritis, T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans, thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type III hypersensitivity reactions, type IV hypersensitivity, unstable angina, uremia, urosepsis, urticaria, valvular heart diseases, varicose veins, vasculitis, venous diseases, venous thrombosis, ventricular fibrillation, viral and fungal infections, vital encephalitis/aseptic meningitis, vital-associated hemaphagocytic syndrome, Wernicke- Korsakoff syndrome, Wilson's disease, xenograft rejection of any organ or tissue.

[00335] In another aspect the invention provides a method of treating a patient suffering from a disorder comprising the step of administering any one of the binding proteins disclosed above before, concurrent, or after the administration of a second agent, as discussed above. In a preferred embodiment the second agent is selected from the group consisting of budenoside, epidermal growth factor, corticosteroids, cyclosporin, sulfasalazine, aminosalicylates, 6-mercaptopurine, azathioprine, metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine, balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor antagonists, anti-IL-Ιβ monoclonal antibodies, anti-IL-6 monoclonal antibodies, growth factors, elastase inhibitors, pyridinyl-imidazole compounds, antibodies or agonists of TNF,

LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-23, EMAP-II, GM-

CSF, FGF, and PDGF, antibodies of CD2, CD3, CD4, CD8, CD-19, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands, methotrexate, cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, ibuprofen, corticosteroids, prednisolone, phosphodiesterase inhibitors, adens-osine agonists, antithrombotic agents, complement inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase inhibitors, IL-Ι β converting enzyme inhibitors, TNFoc converting enzyme inhibitors, T-cell signalling inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin converting enzyme inhibitors, soluble cytokine receptors, soluble p55 TNF receptor, soluble p75 TNF receptor, sIL-lRI, sIL-lRII, sIL-6R, antiinflammatory cytokines, IL-4, IL-10, IL-11, IL-13 and TGF .

[00336] In one embodiment, the pharmaceutical compositions disclosed above are administered to the subject by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

[00337] One aspect of the invention provides at least one anti-idiotype antibody to at least one binding protein of the present invention. The anti-idiotype antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule such as, but not limited to, at least one complementarily determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or; any portion thereof, that can be incorporated into a binding protein of the present invention.

[00338] In another embodiment the binding proteins of the invention are capable of binding one or more targets selected from the group consisting of ABCF1 ; ACVR1 ; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AIF1 ; AIG1 ; AKAP1 ; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-glycoprotein); B7.1 ; B7.2; BAD; BAFF; BAGl; BAIl ; BCL2; BCL6; BDNF; BLNK; BLRl (MDR15); BlyS; BMPl ; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1 ; C19orfl0 (IL27w); C3; C4A; C5; C5R1; CANTl; CASPl ; CASP4; CAVl ; CCBP2 (D6 /

JAB61); CCL1 (1-309); CCL11 (eotaxin); CCL13 (MCP-4); CCL15 (MIP-ld); CCL16

(HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP -1); MCAF;

CCL20 (MIP-3a); CCL21 (MIP-2); SLC; exodus-2; CCL22 (MDC / STC-1); CCL23 (MPIF-

1); CCL24 (MPIF-2 / eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK /

ILC); CCL28; CCL3 (MIP-la); CCL4 (MIP-lb); CCL5 (RANTES); CCL7 (MCP-3); CCL8

(mcp-2); CCNA1 ; CCNA2; CCND1; CCNE1 ; CCNE2; CCR1 (CKR1 / HM145); CCR2

(mcp-lRB / RA);CCR3 (CKR3 / CMKBR3); CCR4; CCR5 (CMKBR5 / ChemR13); CCR6

(CMKBR6 / CKR-L3 / STRL22 / DRY6); CCR7 (CKR7 / EBIl); CCR8 (CMKBR8 / TERl /

CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C;

CD20; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4;

CD40; CD40L; CD44; CD45RB; CD47, CD48, CD52; CD69; CD70, CD72; CD74; CD79A;

CD79B; CD8; CD80; CD81; CD83; CD86; CD137, CD138, B7-1, B7-2, ICOSL, B7-H3, B7-

H4, CD137L, OX40L, CDHl (E-cadherin); CDHIO; CDHl 2; CDH13; CDH18; CDHl 9;

CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7;

CDK9; CDKN1A (p21Wapl/Cipl); CDKN1B (p27Kipl); CDKN1C; CDKN2A

(pl6INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1 ; CHGA; CHGB; Chitinase;

CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8;

CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); cMet; CMKLR1; CMKOR1 (RDC1);

CNR1 ; COL18A1; COL1A1 ; COL4A3; COL6A1; CR2; CRP; CSF1 (M-CSF); CSF2 (GM-

CSF); CSF3 (GCSF); CTLA-4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1

(SCYD1); CX3CR1 (V28); CXCL1 (GROl); CXCL10 (IP-10); CXCL11 (I-TAC / IP-9);

CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GR02); CXCL3 (GR03);

CXCL5 (ENA-78 / LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2);

CXCR4; CXCR6 (TYMSTR /STRL33 / Bonzo); CYB5; CYC1; CYSLTR1 ; DAB2IP; DES;

DKFZp451J0118; DLL-4; DNCL1; DPP4; E2F1 ; ECGF1; EDG1 ; EFNA1 ; EFNA3; EFNB2;

EGF; EGFR; ELAC2; ENG; ENOl ; EN02; EN03; EPHB4; EPO; ERBB2 (Her-2); EREG;

ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FGF;

FGF1 (aFGF); FGF 10; FGF11; FGF 12; FGF12B; FGF13; FGF 14; FGF 16; FGF 17; FGF18;

FGF 19; FGF2 (bFGF); FGF20; FGF21 ; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5;

FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FILl (EPSILON);

FILl (ZETA); FLJ12584; FLJ25530; FLRTl (fibronectin); FLTl ; FOS; FOSLl (FRA-1); FY

(DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GAT A3; GDF5; GFI1 ; GGT1 ; GM-CSF; GNAS1 ; GNRH1 ; GPR2 (CCR10); GPR31 ; GPR44; GPR81

(FKSG80); GRCC10 (CIO); GRP; GSN (Gelsolin); GSTP1 ; HAVCR2; HDAC4; HDAC5;

HDAC7A; HDAC9; HGF; HIF1A; HIP1 ; histamine and histamine receptors; Her3; HLA-A;

HLA-DRA; HM74; HMOX1 ; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNA1 ;

IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; IFNW1 ; IGBP1; IGF1;

IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1 ; IL10; IL10RA; IL10RB; IL11 ; IL11RA; IL-

12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2; IL14; IL15; IL15RA;

IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; ILIA; IL1B;

IL1F10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1 ; IL1R1; IL1R2; IL1RAP; IL1RAPL1 ;

IL1RAPL2;IL1RL1 ;IL1RL2 IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2;

IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30;

IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); IL7; IL7R; IL8;

IL8RA; IL8RB; IL8RB; IL9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAKI; IRAK2;

ITGA1 ; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1 ;

JAK1 ; JAK3; JU ; K6HF; KAI1 ; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLK10;

KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRTl; KRTl 9

(Keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); LAMA5; LEP (leptin);

Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR;

MACMARCKS; MAG or Omgp ; MAP2K7 (c-Jun); MDK; MIB1 ; midkine; MIF; MIP-2;

MKI67 (Ki-67); MMP2; MMP9; MS4A1 ; MSMB; MT3 (metallothionectin-III); MTSS1 ;

MUC1 (mucin); MYC; MYD88; NCK2; neurocan; NFKB1 ; NFKB2; NGFB (NGF); NGFR;

NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NMEl (NM23A); NOX5; NPPB;

NROBl; NR0B2; NR1D1 ; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1 ;

NR2C2; NR2E1; NR2E3; NR2F1 ; NR2F2; NR2F6; NR3C1 ; NR3C2; NR4A1 ; NR4A2;

NR4A3; NR5A1 ; NR5A2; NR6A1; NRPl ; NRP2; NT5E; NTN4; ODZl; OPRDl ; PCSK9;

P2RX7; PAP; PARTI ; PATE; PAWR; PC A3; PCNA; PD-1 ; PD-L1 ; alpha4beta7, OX40,

GITR, TIM-3, Lag-3, B7-H3, B7-H4, GDF8, CGRP, Lingo-1, Factor IXa, Factor X, ICOS,

GARP, BTLA, CD 160, ROR1, 2B4, KIR, CD27, OX40, CD40L, A2aR, PDGFA; PDGFB;

PEC AMI; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG;

PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1 ; PRL; PROC; PROK2; PSAP;

PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS 1; RGS13;

RGS3; RNF110 (ZNF144); ROB02; S100A2; SCGB1D2 (lipophilin B); SCGB2A1

(mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial Monocyte-activating cytokine); SDF2; SERPINA1 ; SERPINA3; SERPINB5 (maspin); SERPINE1 (PAI-1); SERPINF1; SHBG; SLA2; SLC2A2; SLC33A1 ; SLC43A1 ; SLIT2; SPP1; SPRR1B (Sprl); ST6GAL1 ; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21 ; TCP10; TDGF1; TEK; TGFA; TGFB1 ; TGFB1I1 ; TGFB2; TGFB3; TGFBI; TGFBR1 ; TGFBR2; TGFBR3; TH1L; THBS1 (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TIMP3; tissue factor; TLR10; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21 ; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSFIO (TRAIL); TNFSF11 (TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase Iia); TP53; TPM1 ; TPM2; TRADD; TRAF1 ; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCL1 (lymphotactin); XCL2 (SCM- lb); XCR1 (GPR5 / CCXCR1); YY1; and ZFPM2.

[00339] Given their ability to bind to two or more antigens, the binding proteins of the present invention can be used to detect the antigens (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry. The FIT-Ig is directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 3H, 14C, 35S, 90Y, 99Tc, l l lln, 1251, 1311, 177Lu, I66H0, or 153Sm.

[00340] The binding proteins of the invention, in one embodiment, are capable of neutralizing the activity of the antigens both in vitro and in vivo. Accordingly, such FIT-Igs can be used to inhibit antigen activity, e.g., in a cell culture containing the antigens, in human subjects or in other mammalian subjects having the antigens with which a binding protein of the invention cross-reacts. In another embodiment, the invention provides a method for reducing antigen activity in a subject suffering from a disease or disorder in which the antigen activity is detrimental. A binding protein of the invention can be administered to a human subject for therapeutic purposes.

[00341] As used herein, the term "a disorder in which antigen activity is detrimental" is intended to include diseases and other disorders in which the presence of the antigen in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which antigen activity is detrimental is a disorder in which reduction of antigen activity is expected to alleviate the symptoms and/or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of the antigen in a biological fluid of a subject suffering from the disorder (e.g., an increase in the concentration of antigen in serum, plasma, synovial fluid, etc. of the subject). Non-limiting examples of disorders that can be treated with the binding proteins of the invention include those disorders discussed below and in the section pertaining to pharmaceutical compositions of the antibodies of the invention.

[00342] The FIT-Igs of the invention may bind one antigen or multiple antigens. Such antigens include, but are not limited to, the targets listed in the following databases, which databases are incorporated herein by reference. These target databases include, but are not limited to, the following listings:

• Therapeutic targets (http://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp);

• Cytokines and cytokine receptors (http://www.cytokinewebfacts.com/, http://www. copewithcytokines.de/cope. cgi, and

• http://cmbi.bj mu. edu. cn/ cmbidata/ cgf/CGF Database/ cytokine, medic, kumamoto- u.ac.jp/CFC/indexR.html);

• Chemokines (http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.htm l);

• Chemokine receptors and GPCRs (http://csp.medic.kumamoto- u. ac.j p/C S P/Receptor . html , http : //www. gpcr . org/7tm/) ;

• Olfactory Receptors (http://senselab.med.yale.edu/senselab/ORDB/default.asp);

• Receptors (http://www.iuphar-db.org/iuphar-rd71ist/index.htm);

• Cancer targets (http://cged.hgc.jp/cgi-bin/input.cgi); • Secreted proteins as potential antibody targets (http://spd.cbi.pku.edu.cn/);

• Protein kinases (http://spd.cbi.pku.edu.cn/), and

• Human CD markers (http://content.labvelocity.eom/tools/6/1226/CD_table_final_ locked.pdf) and (Zola H, 2005 CD molecules 2005: human cell differentiation molecules Blood, 106:3123-6).

[00343] FIT-Igs are useful as therapeutic agents to simultaneously block two different targets to enhance efficacy/safety and/or increase patient coverage. Such targets may include soluble targets (e.g., IL-13 and TNF) and cell surface receptor targets (e.g., VEGFR and EGFR). It can also be used to induce redirected cytotoxicity between tumor cells and T cells (Her2 and CD3) for cancer therapy, or between autoreactive cell and effector cells for autoimmune/transplantation, or between any target cell and effector cell to eliminate disease- causing cells in any given disease.

[00344] In addition, FIT-Ig can be used to trigger receptor clustering and activation when it is designed to target two different epitopes on the same receptor. This may have benefit in making agonistic and antagonistic anti-GPCR therapeutics. In this case, FIT-Ig can be used to target two different epitopes on one cell for clustering/signaling (two cell surface molecules) or signaling (on one molecule). Similarly, a FIT-Ig molecule can be designed to trigger CTLA-4 ligation, and a negative signal by targeting two different epitopes (or 2 copies of the same epitope) of CTLA-4 extracellular domain, leading to down regulation of the immune response. CTLA-4 is a clinically validated target for therapeutic treatment of a number of immunological disorders. CTLA-4/B7 interactions negatively regulate T cell activation by attenuating cell cycle progression, IL-2 production, and proliferation of T cells following activation, and CTLA-4 (CD 152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by agonistic antibody engagement of CTLA-4 has been unsuccessful since CTLA-

4 activation requires ligation. The molecular interaction of CTLA-4/B7 is in "skewed zipper" arrays, as demonstrated by crystal structural analysis (Stamper 2001 Nature410:608).

However none of the currently available CTLA-4 binding reagents have ligation properties, including anti-CTLA-4 monoclonal antibodies. There have been several attempts to address this issue. In one case, a cell member-bound single chain antibody was generated, and significantly inhibited allogeneic rejection in mice (Hwang 2002 JI 169:633). In a separate case, artificial APC surface-linked single-chain antibody to CTLA-4 was generated and demonstrated to attenuate T cell responses (Griffin 2000 JI 164:4433). In both cases, CTLA- 4 ligation was achieved by closely localized member-bound antibodies in artificial systems. While these experiments provide proof-of-concept for immune down-regulation by triggering CTLA-4 negative signaling, the reagents used in these reports are not suitable for therapeutic use. To this end, CTLA-4 ligation may be achieved by using a FIT-Ig molecule, which target two different epitopes (or 2 copies of the same epitope) of CTLA-4 extracellular domain. The rationale is that the distance spanning two binding sites of an IgG, approximately 150- 170A, is too large for active ligation of CTLA-4 (30-50 A between 2 CTLA-4 homodimer). However the distance between the two binding sites on FIT-Ig (one arm) is much shorter, also in the range of 30-50 A, allowing proper ligation of CTLA-4.

[00345] Similarly, FIT-Ig can target two different members of a cell surface receptor complex (e.g. IL-12R alpha and beta). Furthermore, FIT-Ig can target CR1 and a soluble protein/pathogen to drive rapid clearance of the target soluble protein/pathogen.

[00346] Additionally, FIT-Igs of the invention can be employed for tissue-specific delivery (target a tissue marker and a disease mediator for enhanced local PK thus higher efficacy and/or lower toxicity), including intracellular delivery (targeting an internalizing receptor and a intracellular molecule), delivering to inside brain (targeting transferrin receptor and a CNS disease mediator for crossing the blood-brain barrier). FIT-Ig can also serve as a carrier protein to deliver an antigen to a specific location via biding to a non-neutralizing epitope of that antigen and also to increase the half-life of the antigen. Furthermore, FIT-Ig can be designed to either be physically linked to medical devices implanted into patients or target these medical devices (Burke, Sandra E.; Kuntz, Richard E.; Schwartz, Lewis B. Zotarolimus (ABT-578) eluting stents. Advanced Drug Delivery Reviews (2006), 58(3), 437-446. ; Surface coatings for biological activation and functionalization of medical devices. Hildebrand, H. F.; Blanchemain, N.; Mayer, G.; Chai, F.; Lefebvre, M.; Boschin, F. Surface and Coatings Technology (2006), 200(22-23), 6318-6324. ; Drug/ device combinations for local drug therapies and infection prophylaxis. Wu, Peng; Grainger, David W. Biomaterials (2006), 27(11), 2450-2467. ; Mediation of the cytokine network in the implantation of orthopedic devices. Marques, A. P.; Hunt, J. A.; Reis, Rui L. Biodegradable Systems in Tissue Engineering and Regenerative Medicine (2005), 377- 397; Page: 52

[00347] Mediation of the cytokine network in the implantation of orthopedic devices.

Marques, A. P.; Hunt, J. A.; Reis, Rui L. Biodegradable Systems in Tissue Engineering and

Regenerative Medicine (2005), 377-397.) Briefly, directing appropriate types of cell to the site of medical implant may promote healing and restoring normal tissue function. Alternatively, inhibition of mediators (including but not limited to cytokines), released upon device implantation by a FIT-Ig coupled to or target to a device is also provided. For example, Stents have been used for years in interventional cardiology to clear blocked arteries and to improve the flow of blood to the heart muscle. However, traditional bare metal stents have been known to cause restenosis (re-narrowing of the artery in a treated area) in some patients and can lead to blood clots. Recently, an anti-CD34 antibody coated stent has been described which reduced restenosis and prevents blood clots from occurring by capturing endothelial progenitor cells (EPC) circulating throughout the blood. Endothelial cells are cells that line blood vessels, allowing blood to flow smoothly. The EPCs adhere to the hard surface of the stent forming a smooth layer that not only promotes healing but prevents restenosis and blood clots, complications previously associated with the use of stents (Aoji et al . 2005 J Am Coll Cardiol. 45(10): 1574-9). In addition to improving outcomes for patients requiring stents, there are also implications for patients requiring cardiovascular bypass surgery. For example, a prosthetic vascular conduit (artificial artery) coated with anti- EPC antibodies would eliminate the need to use arteries from patients legs or arms for bypass surgery grafts. This would reduce surgery and anesthesia times, which in turn will reduce coronary surgery deaths. FIT-Ig are designed in such a way that it binds to a cell surface marker (such as CD34) as well as a protein (or an epitope of any kind, including but not limited to lipids and polysaccharides) that has been coated on the implanted device to facilitate the cell recruitment. Such approaches can also be applied to other medical implants in general. Alternatively, FIT-Igs can be coated on medical devices and upon implantation and releasing all FITs from the device (or any other need which may require additional fresh FIT-Ig, including aging and denaturation of the already loaded FIT-Ig) the device could be reloaded by systemic administration of fresh FIT-Ig to the patient, where the FIT-Ig is designed to binds to a target of interest (a cytokine, a cell surface marker (such as CD34) etc.) with one set of binding sites and to a target coated on the device (including a protein, an epitope of any kind, including but not limited to lipids, polysaccharides and polymers ) with the other. This technology has the advantage of extending the usefulness of coated implants.

[00348] FIT-Ig molecules of the invention are also useful as therapeutic molecules to treat various diseases. Such FIT-Ig molecules may bind one or more targets involved in a specific disease. Examples of such targets in various diseases are described below. [00349] Many proteins have been implicated in general autoimmune and inflammatory responses, including C5, CCL1 (1-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15 (MIP-ld), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP- 3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-la), CCL4 (MIP-lb), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp- 2), CXCL1, CXCL10 (IP-10), CXCL11 (I-TAC / IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78 / LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCRl (CCXCR1), IFNA2, IL10, IL13, IL17C, ILIA, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1 (endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5, IFNA2, ILIORA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAKI, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAFl, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, ACVRl, ACVRIB, ACVR2, ACVR2B, ACVRLl, CD28, CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA-4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCLl, XCL2, XCRl, AMH, AMHR2, BMPRIA, BMPRIB, BMPR2, C19orfl0 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, ILIA, IL1B, IL1R1, IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, ILIORA, IL10RB, IL11, IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGFl, TGFA, TGFBl, TGFBIII, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNFl lO (ZNF144). FIT-Igs capable of binding one or more of the targets listed above are also contemplated. [00350] Allergic asthma is characterized by the presence of eosinophilia, goblet cell metaplasia, epithelial cell alterations, airway hyperreactivity (AHR), and Th2 and Thl cytokine expression, as well as elevated serum IgE levels. It is now widely accepted that airway inflammation is the key factor underlying the pathogenesis of asthma, involving a complex interplay of inflammatory cells such as T cells, B cells, eosinophils, mast cells and macrophages, and of their secreted mediators including cytokines and chemokines. Corticosteroids are the most important anti-inflammatory treatment for asthma today, however their mechanism of action is non-specific and safety concerns exist, especially in the juvenile patient population. The development of more specific and targeted therapies is therefore warranted. There is increasing evidence that IL-13 in mice mimics many of the features of asthma, including AHR, mucus hypersecretion and airway fibrosis, independently of eosinophilic inflammation (Finotto et al, International Immunology (2005), 17(8), 993- 1007; Padilla et al, Journal of Immunology (2005), 174(12), 8097-8105).

[00351] IL-13 has been implicated as having a pivotal role in causing pathological responses associated with asthma. The development of anti-IL-13 monoclonal antibody therapy to reduce the effects of IL-13 in the lung is an exciting new approach that offers considerable promise as a novel treatment for asthma. However other mediators of differential immunological pathways are also involved in asthma pathogenesis, and blocking these mediators, in addition to IL-13, may offer additional therapeutic benefit. Such target pairs include, but are not limited to, IL-13 and a pro-inflammatory cytokine, such as tumor necrosis factor-a (TNF-a). TNF-a may amplify the inflammatory response in asthma and may be linked to disease severity (McDonnell, et al, Progress in Respiratory Research (2001), 31(New Drugs for Asthma, Allergy and COPD), 247-250.). This suggests that blocking both IL-13 and TNF-a may have beneficial effects, particularly in severe airway disease. In a preferred embodiment the FIT-Ig of the invention binds the targets IL-13 and TNFa and is used for treating asthma.

[00352] Animal models such as OVA-induced asthma mouse model, where both inflammation and AHR can be assessed, are known in the art and may be used to determine the ability of various FIT-Ig molecules to treat asthma. Animal models for studying asthma are disclosed in Coffman, et al, Journal of Experimental Medicine (2005), 201(12), 1875- 1879; Lloyd, et al, Advances in Immunology (2001), 77, 263-295; Boyce et al, Journal of Experimental Medicine (2005), 201(12), 1869-1873; and Snibson, et al, Journal of the British Society for Allergy and Clinical Immunology (2005), 35(2), 146-52. In addition to routine safety assessments of these target pairs specific tests for the degree of immunosuppression may be warranted and helpful in selecting the best target pairs (see Luster et al, Toxicology (1994), 92(1-3), 229-43; Descotes, et al, Developments in biological standardization (1992), 77 99-102; Hart et al, Journal of Allergy and Clinical Immunology (2001), 108(2), 250-257).

[00353] Based on the rationale disclosed above and using the same evaluation model for efficacy and safety other pairs of targets that FIT-Ig molecules can bind and be useful to treat asthma may be determined. Preferably such targets include, but are not limited to, IL-13 and IL-lbeta, since IL-lbeta is also implicated in inflammatory response in asthma; IL-13 and cytokines and chemokines that are involved in inflammation, such as IL-13 and IL-9; IL- 13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13 and ADAM8. The present invention also contemplates FIT-Igs capable of binding one or more targets involved in asthma selected from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG, histamine and histamine receptors, ILIA, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R1, TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24,CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GAT A3, JAK1, JAK3, STAT6, TBX21, TGFB1, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase.

[00354] Rheumatoid arthritis (RA), a systemic disease, is characterized by a chronic inflammatory reaction in the synovium of joints and is associated with degeneration of cartilage and erosion of juxta-articular bone. Many pro-inflammatory cytokines including

TNF, chemokines, and growth factors are expressed in diseased joints. Systemic administration of anti-TNF antibody or sTNFR fusion protein to mouse models of RA was shown to be anti -inflammatory and joint protective. Clinical investigations in which the activity of TNF in RA patients was blocked with intravenously administered infliximab

(Harriman G, Harper LK, Schaible TF. 1999 Summary of clinical trials in rheumatoid arthritis using infliximab, an anti-TNF alpha treatment. Ann Rheum Dis 58 Suppl 1 :161-4. ), a chimeric anti-TNF monoclonal antibody (mAB), has provided evidence that TNF regulates IL-6, IL-8, MCP-1 , and VEGF production, recruitment of immune and inflammatory cells into joints, angiogenesis, and reduction of blood levels of matrix metalloproteinases-1 and -3. A better understanding of the inflammatory pathway in rheumatoid arthritis has led to identification of other therapeutic targets involved in rheumatoid arthritis. Promising treatments such as interleukin-6 antagonists (MRA), CTLA4Ig (abatacept, Genovese Mc et al 2005 Abatacept for rheumatoid arthritis refractory to tumor necrosis factor alpha inhibition. N Engl J Med. 353 : 1 114-23.), and anti-B cell therapy (rituximab, Okamoto H, Kamatani N. 2004 Rituximab for rheumatoid arthritis. N Engl J Med. 351 : 1909) have already been tested in randomized controlled trials over the past year. Other cytokines have been identified and have been shown to be of benefit in animal models, including interleukin-15, interleukin-17, and interleukin-18, and clinical trials of these agents are currently under way. Dual-specific antibody therapy, combining anti-TNF and another mediator, has great potential in enhancing clinical efficacy and/or patient coverage. For example, blocking both TNF and VEGF can potentially eradicate inflammation and angiogenesis, both of which are involved in pathophysiology of RA. Blocking other pairs of targets involved in RA including, but not limited to, TNF and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-lbeta; TNF and MIF; TNF and IL-17; and TNF and IL-15 with specific FIT-Ig Igs is also contemplated. In addition to routine safety assessments of these target pairs, specific tests for the degree of immunosuppression may be warranted and helpful in selecting the best target pairs (see Luster et al, Toxicology (1994), 92(1 -3), 229-43; Descotes, et al, Developments in biological standardization (1992), 77 99-102; Hart et al, Journal of Allergy and Clinical Immunology (2001), 108(2), 250-257). Whether a FIT-Ig Ig molecule will be useful for the treatment of rheumatoid arthritis can be assessed using pre-clinical animal RA models such as the collagen-induced arthritis mouse model. Other useful models are also well known in the art (see Brand DD., Comp Med. (2005) 55(2): 1 14-22).

[00355] The immunopathogenic hallmark of systemic lupus erythematosus (SLE) is the polyclonal B cell activation, which leads to hyperglobulinemia, autoantibody production and immune complex formation. The fundamental abnormality appears to be the failure of T cells to suppress the forbidden B cell clones due to generalized T cell dysregulation. In addition, B and T-cell interaction is facilitated by several cytokines such as IL-10 as well as co-stimulatory molecules such as CD40 and CD40L, B7 and CD28 and CTLA-4, which initiate the second signal. These interactions together with impaired phagocytic clearance of immune complexes and apoptotic material, perpetuate the immune response with resultant tissue injury. The following targets may be involved in SLE and can potentially be used for FIT-Ig approach for therapeutic intervention: B cell targeted therapies: CD-20, CD-22, CD- 19, CD28, CD4, CD80, HLA-DRA, IL10, IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGS 1, SLA2, CD81, IFNB1, IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S- 6ST, HDAC4, HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA, and NT5E.; co-stimulatory signals: CTLA-4 or B7.1/B7.2; inhibition of B cell survival: BlyS, BAFF; Complement inactivation: C5; Cytokine modulation: the key principle is that the net biologic response in any tissue is the result of a balance between local levels of proinflammatory or antiinflammatory cytokines (see Sfikakis PP et al 2005 Curr Opin Rheumatol 17:550-7). SLE is considered to be a Th-2 driven disease with documented elevations in serum IL-4, IL-6, IL- 10. FIT-Ig Igs capable of binding one or more targets selected from the group consisting of IL-4, IL-6, IL-10, IFN-a, and TNF-a are also contemplated. Combination of targets discussed above will enhance therapeutic efficacy for SLE which can be tested in a number of lupus preclinical models (see Peng SL (2004) Methods Mol Med.; 102:227-72).

[00356] Multiple sclerosis (MS) is a complex human autoimmune-type disease with a predominantly unknown etiology. Immunologic destruction of myelin basic protein (MBP) throughout the nervous system is the major pathology of multiple sclerosis. MS is a disease of complex pathologies, which involves infiltration by CD4+ and CD8+ T cells and of response within the central nervous system. Expression in the CNS of cytokines, reactive nitrogen species and costimulator molecules have all been described in MS. Of major consideration are immunological mechanisms that contribute to the development of autoimmunity. In particular, antigen expression, cytokine and leukocyte interactions, and regulatory T-cells, which help balance/modulate other T-cells such as Thl and Th2 cells, are important areas for therapeutic target identification.

[00357] IL-12 is a proinflammatory cytokine that is produced by APC and promotes differentiation of Thl effector cells. IL-12 is produced in the developing lesions of patients with MS as well as in EAE-affected animals. Previously it was shown that interference in IL- 12 pathways effectively prevents EAE in rodents, and that in vivo neutralization of IL-12p40 using a anti-IL-12 mAb has beneficial effects in the myelin-induced EAE model in common marmosets.

[00358] TWEAK is a member of the TNF family, constitutively expressed in the central nervous system (CNS), with pro-inflammatory, proliferative or apoptotic effects depending upon cell types. Its receptor, Fnl4, is expressed in CNS by endothelial cells, reactive astrocytes and neurons. TWEAK and Fnl4 mRNA expression increased in spinal cord during experimental autoimmune encephalomyelitis (EAE). Anti-TWEAK antibody treatment in myelin oligodendrocyte glycoprotein (MOG) induced EAE in C57BL/6 mice resulted in a reduction of disease severity and leukocyte infiltration when mice were treated after the priming phase.

[00359] One aspect of the invention pertains to FIT-Ig Ig molecules capable of binding one or more, preferably two, targets selected from the group consisting of IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. A preferred embodiment includes a dual-specific anti- IL-12/TWEAK FIT-Ig Ig as a therapeutic agent beneficial for the treatment of MS. Several animal models for assessing the usefulness of the FIT-Ig molecules to treat MS are known in the art (see Steinman L, et al, (2005) Trends Immunol. 26(11):565-71; Lublin FD., et al, (1985) Springer Semin Immunopathol.8(3): 197-208; Genain CP, et al, (1997) J Mol Med. 75(3): 187-97; Tuohy VK, et al, (1999) J Exp Med. 189(7): 1033-42; Owens T, et al, (1995) Neurol Clin. l3(l):51-73; and 't Hart BA, et al, (2005) J Immunol 175(7):4761-8. In addition to routine safety assessments of these target pairs specific tests for the degree of immunosuppression may be warranted and helpful in selecting the best target pairs (see Luster et al, Toxicology (1994), 92(1-3), 229-43; Descotes, et al, Developments in biological standardization (1992), 77 99-102; Jones R. 2000 Rovelizumab (ICOS Corp). IDrugs.3(4):442-6).

[00360] The pathophysiology of sepsis is initiated by the outer membrane components of both gram-negative organisms (lipopolysaccharide [LPS], lipid A, endotoxin) and gram- positive organisms (lipoteichoic acid, peptidoglycan). These outer membrane components are able to bind to the CD 14 receptor on the surface of monocytes. By virtue of the recently described toll-like receptors, a signal is then transmitted to the cell, leading to the eventual production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-I). Overwhelming inflammatory and immune responses are essential features of septic shock and play a central part in the pathogenesis of tissue damage, multiple organ failure, and death induced by sepsis. Cytokines, especially tumor necrosis factor (TNF) and interleukin (IL)-l, have been shown to be critical mediators of septic shock. These cytokines have a direct toxic effect on tissues; they also activate phospholipase A2. These and other effects lead to increased concentrations of platelet-activating factor, promotion of nitric oxide synthase activity, promotion of tissue infiltration by neutrophils, and promotion of neutrophil activity.

[00361] The treatment of sepsis and septic shock remains a clinical conundrum, and recent prospective trials with biological response modifiers (i.e. anti-TNF, anti-MIF) aimed at the inflammatory response have shown only modest clinical benefit. Recently, interest has shifted toward therapies aimed at reversing the accompanying periods of immune suppression. Studies in experimental animals and critically ill patients have demonstrated that increased apoptosis of lymphoid organs and some parenchymal tissues contribute to this immune suppression, anergy, and organ system dysfunction. During sepsis syndromes, lymphocyte apoptosis can be triggered by the absence of IL-2 or by the release of glucocorticoids, granzymes, or the so-called 'death' cytokines: tumor necrosis factor alpha or Fas ligand. Apoptosis proceeds via auto-activation of cytosolic and/or mitochondrial caspases, which can be influenced by the pro- and anti-apoptotic members of the Bcl-2 family. In experimental animals, not only can treatment with inhibitors of apoptosis prevent lymphoid cell apoptosis; it may also improve outcome. Although clinical trials with anti- apoptotic agents remain distant due in large part to technical difficulties associated with their administration and tissue targeting, inhibition of lymphocyte apoptosis represents an attractive therapeutic target for the septic patient. Likewise, a dual-specific agent targeting both inflammatory mediator and a apoptotic mediator, may have added benefit. One aspect of the invention pertains to FIT-Ig Igs capable of binding one or more targets involved in sepsis, preferably two targets, selected from the group consisting TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4, IL10, IL1B, NFKB1, PROC, TNFRSF1A, CSF3, IL10, IL1B, IL6, ADORA2A, CCR3, IL10, IL1B, IL1RN, MIF, NFKB1, PTAFR, TLR2, TLR4, GPR44, HMOX1, midkine, IRAKI, NFKB2, SERPINA1, SERPINE1, and TREM1. The efficacy of such FIT-Ig Igs for sepsis can be assessed in preclinical animal models known in the art (see Buras JA, et al.,(2005) Nat Rev Drug Discov. 4(10):854-65 and Calandra T, et al, (2000) Nat Med. 6(2): 164-70). [00362] Chronic neurodegenerative diseases are usually age-dependent diseases characterized by progressive loss of neuronal functions (neuronal cell death, demyelination), loss of mobility and loss of memory. Emerging knowledge of the mechanisms underlying chronic neurodegenerative diseases (e.g. Alzheimer's disease) show a complex etiology and a variety of factors have been recognized to contribute to their development and progression e.g. age, glycemic status, amyloid production and multimerization, accumulation of advanced glycation-end products (AGE) which bind to their receptor RAGE (receptor for AGE), increased brain oxidative stress, decreased cerebral blood flow, neuroinflammation including release of inflammatory cytokines and chemokines, neuronal dysfunction and microglial activation. Thus these chronic neurodegenerative diseases represent a complex interaction between multiple cell types and mediators. Treatment strategies for such diseases are limited and mostly constitute either blocking inflammatory processes with non-specific antiinflammatory agents (e.g., corticosteroids, COX inhibitors) or agents to prevent neuron loss and/or synaptic functions. These treatments fail to stop disease progression. Recent studies suggest that more targeted therapies such as antibodies to soluble Α-β peptide (including the A-b oligomeric forms) can not only help stop disease progression but may help maintain memory as well. These preliminary observations suggest that specific therapies targeting more than one disease mediator (e.g. Α-β and a pro-inflammatory cytokine such as TNF) may provide even better therapeutic efficacy for chronic neurodegenerative diseases than observed with targeting a single disease mechanism (e.g. soluble A- alone) (see C.E. Shepherd, et al, Neurobiol Aging. 2005 Oct 24; Nelson RB., Curr Pharm Des. 2005;11 :3335; William L. Klein.; Neurochem Int. 2002 ;41 :345; Michelle C Janelsins, et al, J Neuroinflammation. 2005 ;2:23; Soloman B., Curr Alzheimer Res. 2004;1 : 149; Igor Klyubin, et al., Nat Med. 2005; 11 :556-61 ; Arancio O, et al, EMBO Joumal (2004) 1-10; Bomemann KD, et al, Am J Pathol. 2001; 158:63; Deane R, et al., Nat Med. 2003;9:907-13; and Eliezer Masliah, et al, Neuron. 2005;46:857).

[00363] The FIT-Ig molecules of the invention can bind one or more targets involved in Chronic neurodegenerative diseases such as Alzheimer's. Such targets include, but are not limited to, any mediator, soluble or cell surface, implicated in AD pathogenesis e.g. AGE (SI 00 A, amphoterin), pro-inflammatory cytokines (e.g. IL-1), chemokines (e.g. MCP 1), molecules that inhibit nerve regeneration (e.g. Nogo, RGM A), molecules that enhance neurite growth (neurotrophins). The efficacy of FIT-Ig molecules can be validated in pre- clinical animal models such as the transgenic mice that over-express amyloid precursor protein or RAGE and develop Alzheimer's disease-like symptoms. In addition, FIT-Ig molecules can be constructed and tested for efficacy in the animal models and the best therapeutic FIT-Ig can be selected for testing in human patients. FIT-Ig molecules can also be employed for treatment of other neurodegenerative diseases such as Parkinson's disease. Alpha-Synuclein is involved in Parkinson's pathology. A FIT-Ig capable of targeting alpha- synuclein and inflammatory mediators such as TNF, IL-1, MCP-1 can prove effective therapy for Parkinson's disease and are contemplated in the invention.

[00364] Despite an increase in knowledge of the pathologic mechanisms, spinal cord injury (SCI) is still a devastating condition and represents a medical indication characterized by a high medical need. Most spinal cord injuries are contusion or compression injuries and the primary injury is usually followed by secondary injury mechanisms (inflammatory mediators e.g. cytokines and chemokines) that worsen the initial injury and result in significant enlargement of the lesion area, sometimes more than 10-fold. These primary and secondary mechanisms in SCI are very similar to those in brain injury caused by other means e.g. stroke. No satisfying treatment exists and high dose bolus injection of methylprednisolone (MP) is the only used therapy within a narrow time window of 8 h post injury. This treatment, however, is only intended to prevent secondary injury without causing any significant functional recovery. It is heavily criticized for the lack of unequivocal efficacy and severe adverse effects, like immunosuppression with subsequent infections and severe histopathological muscle alterations. No other drugs, biologies or small molecules, stimulating the endogenous regenerative potential are approved, but promising treatment principles and drug candidates have shown efficacy in animal models of SCI in recent years. To a large extent the lack of functional recovery in human SCI is caused by factors inhibiting neurite growth, at lesion sites, in scar tissue, in myelin as well as on injury-associated cells. Such factors are the myelin-associated proteins NogoA, OMgp and MAG, RGM A, the scar- associated CSPG (Chondroitin Sulfate Proteoglycans) and inhibitory factors on reactive astrocytes (some semaphorins and ephrins). However, at the lesion site not only growth inhibitory molecules are found but also neurite growth stimulating factors like neurotrophins, laminin, LI and others. This ensemble of neurite growth inhibitory and growth promoting molecules may explain that blocking single factors, like NogoA or RGM A, resulted in significant functional recovery in rodent SCI models, because a reduction of the inhibitory influences could shift the balance from growth inhibition to growth promotion. However, recoveries observed with blocking a single neurite outgrowth inhibitory molecule were not complete. To achieve faster and more pronounced recoveries either blocking two neurite outgrowth inhibitory molecules e.g. Nogo and RGM A, or blocking an neurite outgrowth inhibitory molecule and enhancing functions of a neurite outgrowth enhancing molecule e.g. Nogo and neurotrophins, or blocking a neurite outgrowth inhibitory molecule e.g. Nogo and a pro-inflammatory molecule e.g. TNF, may be desirable (see McGee AW, et al, Trends Neurosci. 2003;26: 193; Marco Domeniconi, et al, J Neurol Sci. 2005;233:43; Milan Makwanal, et al, FEBS J. 2005;272:2628; Barry J. Dickson, Science. 2002;298: 1959; Felicia Yu Hsuan Teng, et al, J Neurosci Res. 2005;79:273; Tara Kamezis, et al., Nature Neuroscience 2004; 7, 736; Gang Xu, et al, J. Neurochem.2004; 91 ; 1018).

[00365] Other FIT-Igs contemplated are those capable of binding target pairs such as NgR and RGM A; NogoA and RGM A; MAG and RGM A; OMGp and RGM A; RGM A and RGM B; CSPGs and RGM A; aggrecan, midkine, neurocan, versican, phosphacan, Te38 and TNF-a; AB globulomer-specific antibodies combined with antibodies promoting dendrite & axon sprouting. Dendrite pathology is a very early sign of AD and it is known that NOGO A restricts dendrite growth. One can combine such type of ab with any of the SCI-candidate (myelin-proteins) Ab. Other FIT-Ig targets may include any combination of NgR-p75, NgR- Troy, NgR-Nogo66 (Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG or Omgp. Additionally, targets may also include any mediator, soluble or cell surface, implicated in inhibition of neurite e.g Nogo, Ompg, MAG, RGM A, semaphorins, ephrins, soluble A-b, pro-inflammatory cytokines (e.g. IL-1), chemokines (e.g. MIP la), molecules that inhibit nerve regeneration. The efficacy of anti-nogo / anti-RGM A or similar FIT-Ig molecules can be validated in pre-clinical animal models of spinal cord injury. In addition, these FIT-Ig molecules can be constructed and tested for efficacy in the animal models and the best therapeutic FIT-Ig can be selected for testing in human patients. In addition, FIT-Ig molecules can be constructed that target two distinct ligand binding sites on a single receptor e.g. Nogo receptor which binds three ligand Nogo, Ompg, and MAG and RAGE that binds A-b and S 100 A. Furthermore, neurite outgrowth inhibitors e.g. nogo and nogo receptor, also play a role in preventing nerve regeneration in immunological diseases like multiple sclerosis. Inhibition of nogo-nogo receptor interaction has been shown to enhance recovery in animal models of multiple sclerosis. Therefore, FIT-Ig molecules that can block the function of one immune mediator eg a cytokine like IL-12 and a neurite outgrowth inhibitor molecule eg nogo or RGM may offer faster and greater efficacy than blocking either an immune or an neurite outgrowth inhibitor molecule alone.

[00366] Monoclonal antibody therapy has emerged as an important therapeutic modality for cancer (von Mehren M, et al 2003 Monoclonal antibody therapy for cancer.

Annu Rev Med.;54:343-69). Antibodies may exert antitumor effects by inducing apoptosis, redirected cytotoxicity, interfering with ligand-receptor interactions, or preventing the expression of proteins that are critical to the neoplastic phenotype. In addition, antibodies can target components of the tumor microenvironment, perturbing vital structures such as the formation of tumor-associated vasculature. Antibodies can also target receptors whose ligands are growth factors, such as the epidermal growth factor receptor. The antibody thus inhibits natural ligands that stimulate cell growth from binding to targeted tumor cells.

Alternatively, antibodies may induce an anti-idiotype network, complement-mediated cytotoxicity, or antibody-dependent cellular cytotoxicity (ADCC). The use of dual-specific antibody that targets two separate tumor mediators will likely give additional benefit compared to a mono-specific therapy. FIT-Ig Igs capable of binding the following pairs of targets to treat oncological disease are also contemplated: IGFl and IGF2; IGF 1/2 and

Erb2B; VEGFR and EGFR; CD20 and CD3, CD138 and CD20, CD38 and CD20, CD38 &

CD138, CD40 and CD20, CD138 and CD40, CD38 and CD40. Other target combinations include one or more members of the EGF/erb-2/erb-3 family. Other targets (one or more) involved in oncological diseases that FIT-Ig Igs may bind include, but are not limited to those selected from the group consisting of: CD52, CD20, CD19, CD3, CD4, CD8, BMP6, IL12A,

ILIA, IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11,

FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22,

FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGFl, IGF2, IL12A, ILIA,

IL1B, IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, EGF, FGF10, FGF18,

FGF2, FGF4, FGF7, IGFl, IGF1R, IL2, VEGF, BCL2, CD164, CDKN1A, CDKN1B,

CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1, IGFBP6, ILIA, IL1B,

ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9,

E2F1, EGFR, ENOl, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2, INSL4, MYC, NOX5,

NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23, FGF9, IGFBP3, IL2,

INHA, KLK6, TP53, CHGB, GNRH1, IGFl, IGF2, INHA, INSL3, INSL4, PRL, KLK6,

SHBG, NR1D1, NR1H3, NR1I3, NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2, NR1D2,

NR1H2, NR1H4, NR1I2, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1, PGR, RARB, FGFl, FGF2, FGF6,

KLK3, KRT1, APOC1, BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2,

ERK8, FGFl, FGF10, FGF11, FGFl 3, FGFl 4, FGFl 6, FGFl 7, FGFl 8, FGF2, FGF20,

FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRHl, IGF1,

IGF2, IGFBP3, IGFBP6, IL12A, ILIA, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10,

KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9,

MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3,

CD44, CDHl, CDH10, CDHl 9, CDH20, CDH7, CDH9, CDHl, CDH10, CDHl 3, CDH18,

CDHl 9, CDH20, CDH7, CDH8, CDH9, ROB02, CD44, ILK, ITGA1, APC, CD164,

COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAPl, AKAP2, CANT1, CAV1,

CDHl 2, CLDN3, CLN3, CYB5, CYC1, DAB2IP, DES, DNCL1, ELAC2, EN02, EN03,

FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A,

IL29, K6HF, KAIl, KRT2A, MIB1, PARTI, PATE, PC A3, PIAS2, PIK3CG, PPID, PR1,

PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6, ANGPT1,

ANGPT2, ANPEP, ECGF1, EREG, FGFl, FGF2, FIGF, FLT1, JAG1, KDR, LAMA5,

NRPl, NRP2, PGF, PLXDCl, STABl, VEGF, VEGFC, ANGPTL3, BAI1, COL4A3, IL8,

LAMA5, NRPl, NRP2, STABl, ANGPTL4, PEC AMI, PF4, PROK2, SERPINF1,

TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, IFNA1,

IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2, EGF, EPHB4, FGFR3,

HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5,

COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1,

CCNA2, CCNDl, CCNEl, CCNE2, CDHl (E-cadherin), CDKNIB (p27Kipl), CDKN2A

(pl6INK4a), COL6A1, CTNNB1 (b-catenin), CTSB (cathepsin B), ERBB2 (Her-2), ESR1,

ESR2, F3 (TF), FOSL1 (FRA-1), GAT A3, GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R,

IL6ST (glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun),

MKI67 (Ki-67), NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, CTLA-

4, OX40, GITR, TIM-3, Lag-3, B7-H3, B7-H4, GDF8, CGRP, Lingo-1, ICOS, GARP,

BTLA, CD160, ROR1, SERPINB5 (maspin), SERPINE1 (PAI-1), TGFA, THBS 1

(thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A (topoisomerase

Iia), TP53, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wapl/Cipl),

CLDN7 (claudin-7), CLU (clusterin), ERBB2 (Her-2), FGFl, FLRT1 (fibronectin), GABRP

(GABAa), GNAS 1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin), KLF5 (GC Box BP),

KRT19 (Keratin 19), KRTHB6 (hair-specific type II keratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Sprl), THBS1, THBS2, THBS4, and TNFAIP2 (B94).

[00367] In an embodiment, diseases that can be treated or diagnosed with the compositions and methods provided herein include, but are not limited to, primary and metastatic cancers, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract (including cervix, uterus, and ovaries as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as well as Kaposi's sarcoma), tumors of the brain, nerves, eyes, and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, and meningiomas), solid tumors arising from hematopoietic malignancies such as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin's lymphomas).

[00368] In an embodiment, the antibodies provided herein or antigen-binding portions thereof, are used to treat cancer or in the prevention of metastases from the tumors described herein either when used alone or in combination with radiotherapy and/or other chemotherapeutic agents.

[00369] According to another embodiment of the invention, the human immune effector cell is a member of the human lymphoid cell lineage. In this embodiment, the effector cell may advantageously be a human T cell, a human B cell or a human natural killer (NK) cell. Advantageously, such cells will have either a cytotoxic or an apoptotic effect on the target cell. Especially advantageously, the human lymphoid cell is a cytotoxic T cell which, when activated, exerts a cytotoxic effect on the target cell. According to this embodiment, then, the recruited activity of the human effector cell is this cell's cytotoxic activity.

[00370] According to a preferred embodiment, activation of the cytotoxic T cell may occur via binding of the CD3 antigen as effector antigen on the surface of the cytotoxic T cell by a bispecific antibody of this embodiment of the invention. The human CD3 antigen is present on both helper T cells and cytotoxic T cells. Human CD3 denotes an antigen which is expressed on T cells as part of the multimolecular T cell complex and which comprises three different chains: CD3-epsilon, CD3-delta and CD3-gamma.

[00371] The activation of the cytotoxic potential of T cells is a complex phenomenon which requires the interplay of multiple proteins. The T cell receptor ("TCR") protein is a membrane bound disulfide-linked heterodimer consisting of two different glycoprotein subunits. The TCR recognizes and binds foreign peptidic antigen which itself has been bound by a member of the highly diverse class of major histocompatibility complex ("MHC") proteins and has been presented, bound to the MHC, on the surface of antigen presenting cells ("APCs").

[00372] Although the variable TCR binds foreign antigen as outlined above, signaling to the T cell that this binding has taken place depends on the presence of other, invariant, signaling proteins associated with the TCR. These signaling proteins in associated form are collectively referred to as the CD3 complex, here collectively referred to as the CD3 antigen.

[00373] The activation of T cell cytotoxicity, then, normally depends first on the binding of the TCR with an MHC protein, itself bound to foreign antigen, located on a separate cell. Only when this initial TCR-MHC binding has taken place can the CD3- dependent signaling cascade responsible for T cell clonal expansion and, ultimately, T cell cytotoxicity ensue.

[00374] However, binding of the human CD3 antigen by the first or second portion of a bispecific antibody of the invention activates T cells to exert a cytotoxic effect on other cells in the absence of independent TCR-MHC binding. This means that T cells may be cytotoxically activated in a clonally independent fashion, i.e., in a manner which is independent of the specific TCR clone carried by the T cell. This allows an activation of the entire T cell compartment rather than only specific T cells of a certain clonal identity.

[00375] In light of the foregoing discussion, then, an especially preferred embodiment of the invention provides a bispecific antibody in which the effector antigen is the human CD3 antigen. The bispecific antibody according to this embodiment of the invention may have a total of either two or three antibody variable domains.

[00376] According to further embodiments of the invention, other lymphoid cell- associated effector antigens bound by a bispecific antibody of the invention may be the human CD 16 antigen, the human NKG2D antigen, the human NKp46 antigen, the human CD2 antigen, the human CD28 antigen or the human CD25 antigen. [00377] According to another embodiment of the invention, the human effector cell is a member of the human myeloid lineage. Advantageously, the effector cell may be a human monocyte, a human neutrophilic granulocyte or a human dendritic cell. Advantageously, such cells will have either a cytotoxic or an apoptotic effect on the target cell. Advantageous antigens within this embodiment which may be bound by a bispecific antibody of the invention may be the human CD64 antigen or the human CD89 antigen.

[00378] According to another embodiment of the invention, the target antigen is an antigen which is uniquely expressed on a target cell or effector cell in a disease condition, but which remains either non-expressed, expressed at a low level or non-accessible in a healthy condition. Examples of such target antigens which might be specifically bound by a bispecific antibody of the invention may advantageously be selected from EpCAM, CCR5, CD19, HER-2 neu, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-0-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue-1, Plasma Cell Antigen, (membrane- bound) IgE, Melanoma Chondroitin Sulfate Proteoglycan (MCSP), CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6; desmoglein 4, E-cadherin neoepitope, Fetal Acetylcholine Receptor, CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory Substance (MIS) Receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, LG, SAS and CD63.

[00379] According to a specific embodiment, the target antigen specifically bound by a bispecific antibody may be a cancer-related antigen, e.g., an antigen related to a malignant condition. Such an antigen is either expressed or accessible on a malignant cell, whereas the antigen is either not present, not significantly present, or is not accessible on a non-malignant cell. As such, a bispecific antibody according to this embodiment of the invention is a bispecific antibody which recruits the activity of a human immune effector cell against the malignant target cell bearing the target antigen, or rendering the target antigen accessible.

[00380] Gene Therapy : In a specific embodiment, nucleic acid sequences encoding a binding protein provided herein or another prophylactic or therapeutic agent provided herein are administered to treat, prevent, manage, or ameliorate a disorder or one or more symptoms thereof by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment, the nucleic acids produce their encoded antibody or prophylactic or therapeutic agent provided herein that mediates a prophylactic or therapeutic effect.

[00381] Any of the methods for gene therapy available in the art can be used in the methods provided herein. For general reviews of the methods of gene therapy, see Goldspiel et al. (1993) Clin. Pharmacy 12:488-505; Wu and Wu (1991) Biotherapy 3:87-95; Tolstoshev (1993) Ann Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993) Science 260:926-932; Morgan and Anderson (1993) Ann Rev. Biochem. 62: 191-217; and May (1993) TIBTECH 11(5): 155-215. Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). Detailed description of various methods of gene therapy is disclosed in US Patent Publication No. US20050042664.

[00382] Diagnostics : The disclosure herein also provides diagnostic applications including, but not limited to, diagnostic assay methods, diagnostic kits containing one or more binding proteins, and adaptation of the methods and kits for use in automated and/or semi-automated systems. The methods, kits, and adaptations provided may be employed in the detection, monitoring, and/or treatment of a disease or disorder in an individual. This is further elucidated below.

[00383] A. Method of Assay : The present disclosure also provides a method for determining the presence, amount or concentration of an analyte, or fragment thereof, in a test sample using at least one binding protein as described herein. Any suitable assay as is known in the art can be used in the method. Examples include, but are not limited to, immunoassays and/or methods employing mass spectrometry. Immunoassays provided by the present disclosure may include sandwich immunoassays, radioimmunoassay (RIA), enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), competitive-inhibition immunoassays, fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), bioluminescence resonance energy transfer (BRET), and homogenous chemiluminescent assays, among others. A chemiluminescent microparticle immunoassay, in particular one employing the ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park, III), is an example of an immunoassay. Methods employing mass spectrometry are provided by the present disclosure and include, but are not limited to MALDI (matrix-assisted laser desorption/ionization) or by SELDI (surface-enhanced laser desorption/ionization).

[00384] Methods for collecting, handling, processing, and analyzing biological test samples using immunoassays and mass spectrometry would be well-known to one skilled in the art, are provided for in the practice of the present disclosure (US 2009-0311253 Al).

[00385] B. Kit : A kit for assaying a test sample for the presence, amount or concentration of an analyte, or fragment thereof, in a test sample is also provided. The kit comprises at least one component for assaying the test sample for the analyte, or fragment thereof, and instructions for assaying the test sample for the analyte, or fragment thereof. The at least one component for assaying the test sample for the analyte, or fragment thereof, can include a composition comprising a binding protein, as disclosed herein, and/or an anti- analyte binding protein (or a fragment, a variant, or a fragment of a variant thereof), which is optionally immobilized on a solid phase. Optionally, the kit may comprise a calibrator or control, which may comprise isolated or purified analyte. The kit can comprise at least one component for assaying the test sample for an analyte by immunoassay and/or mass spectrometry. The kit components, including the analyte, binding protein, and/or anti-analyte binding protein, or fragments thereof, may be optionally labeled using any art-known detectable label. The materials and methods for the creation provided for in the practice of the present disclosure would be known to one skilled in the art (US 2009-0311253 Al).

[00386] C. Adaptation of Kit and Method: The kit (or components thereof), as well as the method of determining the presence, amount or concentration of an analyte in a test sample by an assay, such as an immunoassay as described herein, can be adapted for use in a variety of automated and semi-automated systems (including those wherein the solid phase comprises a microparticle), as described, for example, in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as commercially marketed, for example, by Abbott Laboratories (Abbott Park, 111.) as ARCHITECT®. Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM®, IMx® (see, for example, U.S. Pat. No. 5,294,404, PRISM®, EIA (bead), and Quantum™ II, as well as other platforms. Additionally, the assays, kits and kit components can be employed in other formats, for example, on electrochemical or other hand-held or point-of-care assay systems. The present disclosure is, for example, applicable to the commercial Abbott Point of Care (i-STAT®, Abbott Laboratories) electrochemical immunoassay system that performs sandwich immunoassays. Immunosensors and their methods of manufacture and operation in single-use test devices are described, for example in, U.S. Pat. Nos. 5,063,081, 7,419,821, and 7,682,833; and US Publication Nos. 20040018577, 20060160164 and US 20090311253. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein are obvious and may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

[00387] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.

EXAMPLES

Example 1. Construction, expression, purification, and analysis of anti-IL17/IL-20 Fabs-in-tandem immunoglobulin (FIT-Ig)

[00388] To demonstrate the FIT-Ig technology, we have generated a group of anti-IL-

17/IL-20 FIT-Ig molecules: FITl-Ig, FIT2-Ig, and FIT3-Ig, all of which contains 3 different polypeptides, as shown in Figure 1, where antigen A is IL-17 and antigen B is IL-20. The DNA construct used to generate FIT-Ig capable of binding IL-17 and IL-20 is illustrated in Figure IB. Briefly, parental mAbs included two high affinity antibodies, anti-IL-17 (clone LY) (U.S. Patent No. 7,838,638) and anti-hIL-20 (clone 15D2) (U.S. Patent Application Publication No. US2014/0194599). To generate FIT-Ig construct #1, the VL-CL of LY was directly (FITl-Ig), or through a linker of 3 amino acids (FIT2-Ig) or 7 amino acids (FIT3-Ig) fused to the N-terminus of the 15D2 heavy chain (as shown in Table 1). The construct #2 is VH-CH1 of LY, and the 3 ^rd construct is VL-CL of 15D2. The 3 constructs for each FIT-Ig were co-transfected in 293 cells, resulting in the expression and secretion of FIT-Ig protein. [00389] We also generated a group of anti-IL-17/IL-20 FIT-Ig molecules: FIT4-Ig, FIT5-Ig, and FIT6-Ig, each of which contains 2 different polypeptides, as shown in Figure 2. The DNA constructs used to generate FIT-Ig capable of binding IL-17 and IL-20 are illustrated in Figure 2B, where antigen A is IL-17 and antigen B is IL-20. Briefly, parental mAbs included two high affinity antibodies, anti-IL-17 (clone LY) and anti-hIL-20 (clone 15D2). To generate FIT-Ig construct #1, the VL-CL of LY was directly (FIT4-Ig), or through a linker of 3 amino acids (FIT5-Ig) or 7 amino acids (FIT6-Ig) fused to the N-terminus of the 15D2 heavy chain (as shown in Table 1). To generate FIT-Ig construct #4, the VH-CHl of LY was directly (FIT4-Ig), or through a linker of 3 amino acids (FIT5-Ig) or 7 amino acids (FIT6-Ig) fused to the N-terminus of the 15D2 light chain. The 2 DNA constructs (construct #1 and #4) for each FIT-Ig were co-transfected in 293 cells, resulting in the expression and secretion of FIT-Ig protein. The detailed procedures of the PCR cloning are described below.

Example 1.1: Molecular cloning of anti-IL-17/IL-20 FIT-Ig molecules:

[00390] For construct #1 cloning, LY light chain was amplified by PCR using forward primers annealing on light chain signal sequence and reverse primers annealing on C- terminus of the light chain. 15D2 heavy chain was amplified by PCR using forward primers annealing on N-terminus of 15D2 VH and reverse primers annealing on C-terminus of CH. These 2 PCR fragments were gel purified and combined by overlapping PCR using signal peptide and CH primer pair. The combined PCR product was cloned into a 293 expression vector, which already contained the human Fc sequence.

Table 1. Anti-IL-17/IL-20 FIT-Ig molecules and DNA constructs.

[00391] For construct #2 cloning, LY VH-CH1 was amplified by PCR using forward primers annealing on heavy chain signal peptide and reverse primer annealing on C-terminal of CHI. The PCR product was gel purified before cloning into 293 expression vector.

[00392] For construct #3, 15D2 light chain was amplified by PCR using forward primer annealing on N-terminal of light chain signal peptide and reverse primer annealing on the end of CL. The PCR product was gel purified before cloning into 293 expression vector.

[00393] For construct #4 cloning, LY VH-CH1 was amplified by PCR using forward primer annealing on N-terminus of heavy chain signal peptide and reverse primer annealing on the end of CHI. 15D2 VL was amplified using primers annealing on the end of 15D2 VL. Both PCR products were gel purified and combined by overlap PCR. The combined PCR product was gel purified and cloned in 293 expression vector. Table 2 shows sequences of PCR primers used for above molecular cloning.

Table 2. PCR primers used for molecular construction of anti-IL-17/anti-CD20 FIT-Igs

[00394] The final sequences of hIL-17/hIL-20 FITl-Ig, FIT2-Ig, FIT3-Ig, FIT4-Ig,

FIT5-Ig, and FIT6-Ig are listed in Table 3. Table 3. Amino acid sequences of anti-IL-17/IL-20 FIT-Ig molecules

Protein Sequence

Sequence

Protein region Identifier 12345678901234567890

DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY

Anti-IL-17/IL-20 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR FITl-Ig VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF

POLYPEPTIDE #1 SEQ ID PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS

NO.:15 QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGECQVQLVQSGAEVKRPGASVKVSCKASGY TFTND11HWVRQAPGQRLEWMGWINAGYGNTQYSQNFQDRV SITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPH DYYGMD GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNWHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK

LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY NO.:16 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR

VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW NO.:17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Linker None

15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTND11HWVRQAP

NO.:18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME

LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDWGQGTTV TVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNWHKPSNTKVDKKVEPKSC

Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.:20 VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME

Anti-IL-17/IL-20 LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTK FITl-Ig SEQ ID GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA

POLYPEPTIDE #2 NO.:21 LTSGWTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNW

HKPSNTKVDKKVEPKSC

LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.:22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME

LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNWHKPSNTKVDKKVEPKSC

SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG NO.:23 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED

Anti-IL-17/IL-20 FATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDE FITl-Ig QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVT Protein Sequence

Sequence

Protein region Identifier 12345678901234567890

POLYPEPTIDE #3 EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV

TKSFNRGEC

15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG

NO.:24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED

FATYYCQQFNSYPLTFGGGT VEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA VQW NO.:17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Anti-IL-17/IL-20 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY FIT2-Ig NO.:25 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR

POLYPEPTIDE #1 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF

PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGSGQVQLVQSGAEVKRPGASV VSCKA SGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQ DRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGES SPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSWTVPSSSLGTQTYICNVNHKPSNT VDK VEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR WSVLTVLHQDWLNGKEYKC VSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK

LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY NO.:16 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR

VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA VQW NO.:17 VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Linker SEQ ID GSG

NO.:26

15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTND11HWVRQAP

NO.:18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME

LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTV TVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNVNHKPSNTKVDKKVEPKSC

Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.:20 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQWTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTA/DKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

Anti-IL-17/IL-20 QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP FIT2-Ig GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME

POLYPEPTIDE #2 LSSLRSEDTAWYCARYDYFTGTGWWGQGTLVTVSSASTK

SEQ ID GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA NO.:21 LTSGA/HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNW

HKPSNTKVDKKVEPKSC

LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.:22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME

LSSLRSEDTAWYCARYDYFTGTGWWGQGTLVTVSS Protein Sequence

Sequence

Protein region Identifier 12345678901234567890

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNVNHKPSNTKVDKKVEPKSC

Anti-IL-17/IL-20 SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG FIT2-Ig NO.:23 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED

POLYPEPTIDE #3 FATYYCQQFNSYPLTFGGGT VEIKRTVAAPSVFIFPPSDE

QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC

15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG

NO.:24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED

FATYYCQQFNSYPLTFGGGT VEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA VQW NO.:17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Anti-IL-17/IL-20 SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY FIT3-Ig NO.:27 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR

POLYPEPTIDE #1 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF

PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGGGGSGSQVQLVQSGAEVKRPGASV V SCKASGYTFTND11HWVRQAPGQRLEWMGWINAGYGNTQYS QNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLW FGESSPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY NO.:16 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR

VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW NO.:17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Linker SEQ ID GGGGSGS

NO.:28

15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTND11HWVRQAP

NO.:18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME

LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTV TVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNVNHKPSNTKVDKKVEPKSC

Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.:20 WVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

Anti-IL-17/IL- SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP 20 FIT3-Ig NO.:21 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME

LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTK Protein Sequence

Sequence

Protein region Identifier 12345678901234567890

POLYPEPTIDE GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA

#2 LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN

HKPSNTKVDKKVEPKSC

LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.:22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME

LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNWHKPSNTKVDKKVEPKSC

Anti-IL-17/IL- SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG 20 FIT3-Ig NO.:23 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED POLYPEPTIDE FATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDE

#3 QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVT

EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC

15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG

NO.:24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED

FATYYCQQFNSYPLTFGGGTKVEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW NO.:17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Anti-IL-17/IL-20 FIT SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLWSRGNTYLHWY 4-Ig POLYPEPTIDE NO.:15 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR

#1 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF

PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECQVQLVQSGAEVKRPGASVKVSCKASGY TFTND11HWVRQAPGQRLEWMGWINAGYGNTQYSQNFQDRV SITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGESSPH DYYGMD GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNWHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK

LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY NO.:16 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR

VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW NO.:17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Linker None

15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTND11HWVRQAP

NO.:18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME

LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDWGQGTTV TVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNWHKPSNTKVDKKVEPKSC

Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.:20 VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP Protein Sequence

Sequence

Protein region Identifier 12345678901234567890

REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

Anti-IL-17/IL-20 SEQ ID QVQLVQSGAEVKKPGSSV VSCKASGYSFTDYHIHWVRQAP FIT4-Ig NO.:29 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME POLYPEPTIDE #4 LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTK

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNT VDK VEPKSCAIQLTQSPSSLSASVGDRVTITCR ASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGT VEI KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC

LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.:22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME

LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNVNHKPSNTKVDKKVEPKSC

Linker none

15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG

NO.:24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED

FATYYCQQFNSYPLTFGGGTKVEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW NO.:17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Anti-IL-17/IL-20 FIT SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY 5-Ig POLYPEPTIDE NO.:25 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR

#1 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF

PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGSGQVQLVQSGAEVKRPGASVKVSCKA SGYTFTNDIIHWVRQAPGQRLEWMGWINAGYGNTQYSQNFQ DRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLWFGES SPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK

LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY NO.:16 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR

VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW NO.:17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Linker SEQ ID GSG

NO.:26

15D2 VH SEQ ID QVQLVQSGAEVKRPGASVKVSCKASGYTFTND11HWVRQAP

NO.:18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME Protein Sequence

Sequence

Protein region Identifier 12345678901234567890

LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTV TVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNVNHKPSNTKVDKKVEPKSC

Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.:20 VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

Anti-IL-17/IL-20 SEQ ID QVQLVQSGAEVKKPGSSV VSCKASGYSFTDYHIHWVRQAP FIT5-Ig NO.:30 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME POLYPEPTIDE #4 LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTK

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNT VDK VEPKSCGSGAIQLTQSPSSLSASVGDRVTI TCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA KVQW VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKH VYACEVTHQGLSSPVTKSFNRGEC

LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.:22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME

LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNVNHKPSNTKVDKKVEPKSC

Linker SEQ ID GSG

NO.:26

15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG

NO.:24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED

FATYYCQQFNSYPLTFGGGTKVEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW NO.:17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Anti-IL-17/IL-20 FIT SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY 6-Ig POLYPEPTIDE NO.:27 LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR

#1 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIKRTVAAPSVFIF

PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGGGGSGSQVQLVQSGAEVKRPGASVKV SCKASGYTFTND11HWVRQAPGQRLEWMGWINAGYGNTQYS QNFQDRVSITRDTSASTAYMELISLRSEDTAVYYCAREPLW FGESSPHDYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

LY VL SEQ ID DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWY LQKPGQSPQLLIYKVSNRFIGVPDRFSGSGSGTDFTLKISR Protein Sequence

Sequence

Protein region Identifier 12345678901234567890

NO.:16 VEAEDVGVYYCSQSTHLPFTFGQGTKLEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA VQW NO.:17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Linker GGGGSGS

15D2 VH SEQ ID QVQLVQSGAEVKRPGASV VSCKASGYTFTND11HWVRQAP

NO.:18 GQRLEWMGWINAGYGNTQYSQNFQDRVSITRDTSASTAYME

LISLRSEDTAVYYCAREPLWFGESSPHDYYGMDVWGQGTTV TVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNVNHKPSNTKVDKKVEPKSC

Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC NO.:20 VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

Anti-IL-17/IL-20 SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP FIT6-Ig NO.:31 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME POLYPEPTIDE #4 LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSASTK

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGGGGSGSAIQLTQSPSSLSASVGD RVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFG GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

LY VH SEQ ID QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAP NO.:22 GQGLEWMGVINPMYGTTDYNQRFKGRVTITADESTSTAYME

LSSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NO.:19 NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYI

CNVNHKPSNTKVDKKVEPKSC

Linker SEQ ID GGGGSGS

NO.:28

15D2 VL SEQ ID AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPG

NO.:24 KAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPED

FATYYCQQFNSYPLTFGGGTKVEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW NO.:17 KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Example 1.2: Expression, purification, and analysis of anti-IL-17/IL-20 FIT-Ig proteins:

[00395] All DNA constructs of each FIT-Ig were subcloned into pBOS based vectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 of each FIT-Ig (1, 2, and 3), or construct #1 and #4 of each FIT-Ig (4, 5, and 6) were transiently co-expressed using Polyethyleneimine (PEI) in 293E cells. Briefly, DNA in FreeStyle™ 293 Expression Medium was mixed with the PEI with the final concentration of DNA to PEI ratio of 1:2, incubated for 15min (no more than 20min) at room temperature, and then added to the 293E cells (1.0-1.2 ^χ lOVml, cell viability > 95%) at 60μg DNA/120ml culture. After 6-24 hours culture in shaker, peptone was added to the transfected cells at a final concentration of 5%, with shaking at 125 rpm/min., at 37°C, 8% CC . On the 6th - 7th day, supernatant was harvested by centrifugation and filtration, and FIT-Ig protein was purified using protein A chromatography (Pierce, Rockford, IL) according to the manufacturer's instructions. The proteins were analyzed by SDS-PAGE and their concentrations determined by A280 and BCA (Pierce, Rockford, IL).

[00396] For the expression of FITl-Ig, FIT2-Ig, and FIT3-Ig, different DNA molar ratios of the 3 constructs were used, including construct #1 :#2:#3 = 1 : 1: 1, construct #1:#2:#3 = 1: 1.5: 1.5, and construct #1 :#2:#3 = 1 :3:3 (Table 4). FIT-Ig proteins were purified by protein A chromatography. The purification yield (7-16 mg/L) was consistent with hlgG quantification of the expression medium for each protein. The composition and purity of the purified FIT-Igs were analyzed by SDS-PAGE in both reduced and non-reduced conditions. In non-reduced conditions, FIT-Ig migrated as a single band of approximately 250 KDa. In reducing conditions, each of the FIT-Ig proteins yielded two bands, one higher MW band is construct #1 of approximately 75 KDa, and one lower MW band corresponds to both construct#2 and #3 overlapped at approximately 25 KDa. The SDS-PAGE showed that each FIT-Ig is expressed as a single species, and the 3 polypeptide chains are efficiently paired to form an IgG-like molecule. The sizes of the chains as well as the full-length protein of FIT- Ig molecules are consistent with their calculated molecular mass based on amino acid sequences.

Table 4. Expression and SEC analysis of WL-17/IL-20 FIT-Ig proteins

[00397] To further study the physical properties of FIT-Ig in solution, size exclusion chromatography (SEC) was used to analyze each protein. For SEC analysis of the FIT-Ig, purified FIT-Ig, in PBS, was applied on a TSKgel SuperSW3000, 300 x 4.6 mm column (TOSOH). An HPLC instrument, Model U3000 (DIONEX) was used for SEC. All proteins were determined using UV detection at 280 nm and 214 nm. The elution was isocratic at a flow rate of 0.25 mL/min. All 3 FIT-Ig proteins exhibited a single major peak, demonstrating physical homogeneity as monomeric proteins (Table 4). The ratio of construct #1 :#2:#3 = 1 :3:3 showed a better monomeric profile by SEC for all 3 FIT-Ig proteins (Table 4).

[00398] Table 4 also shows that the expression levels of all the FIT-Ig proteins are comparable to that of the regular mAbs, indicating that the FIT-Ig can be expressed efficiently in mammalian cells. For the expression of FIT4-Ig, FIT5-Ig, and FIT6-Ig, the DNA ration of construct #1 :#4 = 1 : 1, and the expression level were in the range of 1-10 mg/L, and the % Peak monomeric fraction as determined by SEC was in the range of 58-76%. Based on this particular mAb combination (LY and 15D2), the 3-polypepide FIT-Ig constructs (FITl-Ig, FIT2-Ig, and FIT3-Ig) showed better expression profile than that of the 2-polypeptide FIT-Ig constructs (FIT4-Ig, FIT5-Ig, and FIT6-Ig), therefore FITl-Ig, FIT2-Ig, and FIT3-Ig were further analyzed for functional properties

Example 1.3 Determination of antigen binding affinity of anti-IL-17/IL-20 FIT-Igs

[00399] The kinetics of FIT-Ig binding to rhIL-17 and rhIL-20 was determined by surface plasmon resonance (Table 5) with a Biacore XI 00 instrument (Biacore AB, Uppsala, Sweden) using HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25°C. Briefly, goat anti-human IgG Fey fragment specific polyclonal antibody (Pierce Biotechnology Inc, Rockford, IL) was directly immobilized across a CM5 research grade biosensor chip using a standard amine coupling kit according to manufacturer's instructions. Purified FIT-Ig samples were diluted in HEPES-buffered saline for capture across goat anti -human IgG Fc specific reaction surfaces and injected over reaction matrices at a flow rate of 5 μΐ/min. The association and dissociation rate constants, kon (M-ls-1) and koff (s-1) were determined under a continuous flow rate of 30 μΕ/ιηίη. Rate constants were derived by making kinetic binding measurements at ten different antigen concentrations ranging from 1.25 to 1000 nM. The equilibrium dissociation constant (M) of the reaction between FIT-Ig and the target proteins was then calculated from the kinetic rate constants by the following formula: KD = koff/kon. Aliquots of antigen samples were also simultaneously injected over a blank reference and reaction CM surface to record and subtract any nonspecific binding background to eliminate the majority of the refractive index change and injection noise. Surfaces were regenerated with two subsequent 25 ml injections of 10 mM Glycine (pH 1.5) at a flow rate of 10 μί/πϋη. The anti-Fc antibody immobilized surfaces were completely regenerated and retained their full capture capacity over twelve cycles.

Table 5. Functional characterizations of anti-IL-17/IL-20 FIT-Ig molecules

[00400] The Biacore analysis indicated the overall binding parameters of the three FIT-Igs to hIL-17 and hIL-20 were similar, with the affinities of the FIT-Igs being very close to that of the parental mAb LY and 15D2, and there was no lose of binding affinities for either antigen binding domains (Table 5).

[00401] In addition, tetravalent dual-specific antigen binding of FIT-Ig was also analyzed by Biacore. FITl-Ig was first captured via a goat anti-human Fc antibody on the Biacore sensor chip, and the first antigen was injected and a binding signal observed. As the FITl-Ig was saturated by the first antigen, the second antigen was then injected and the second signal observed. This was done either by first injecting IL-17 then IL-20 or by first injecting IL-20 followed by IL-17 for FIT2-Ig (Figure 3). In either sequence, a dual-binding activity was detected, and both antigen binding was saturated at 25-30 RU. Similar results were obtained for FIT2-Ig and FIT3-Ig. Thus each FIT-Ig was able to bind both antigens simultaneously as a dual-specific tetravalent molecule. [00402] The expression profile and dual-binding properties of FIT-Ig clearly demonstrated that, within the FIT-Ig molecule, both VL-CL paired correctly with their corresponding VH-CH1 to form 2 functional binding domains, and expressed as a single monomeric, tetravalent, and bispecific full length FIT-Ig protein. This is in contrast to the multivalent antibody type of molecules (Miller and Presta, US Patent 8722859), which displayed tetravalent but mono-specific binding activities to one target antigen.

Example 1.4 Determination of biological activity of anti-IL-17/IL-20 FIT-Ig

[00403] The biological activity of FIT-Ig to neutralize IL-17 function was measured using GROoc bioassay. Briefly, Hs27 cells were seeded at 10000 into 96 well plates. FIT-Ig or anti-IL-17 control antibody (25 μί) were added in duplicate wells, with starting concentration at 2.5 nM followed by 1 :2 serial dilutions until 5 pM. IL-17A (25 μί) was then added to each well. The final concentration of IL-17A was 0.3 nM. Cells were incubated at 37°C for 17h before cell culture supernatant were collected. Concentrations of GRO-oc in cell culture supernatants were measured by human CACL1/GRO alpha Quantikine kit according to the manufacturer's protocol (R&D systems).

[00404] The biological activity of FIT-Ig to neutralize IL-20 function was measured using IL-20R ⁺ BAF3 cell proliferation assay. Briefly, 25μΙ _^ of recombinant human IL-20 at 0.8 nM was added to each well of 96-well plates (the final concentration of IL-20 is 0.2 nM). Anti-IL20 antibody or FIT-Ig or other control antibody were diluted to 400 nM (working concentration was 100 nM) followed by 5-fold serial dilutions and were added to 96-well assay plates (25μί per well). BaF3 cells stably transfected with IL-20 receptor were then added to each well at concentration of 10000 cell/well in volume of 50 μί RPMI 1640 plus 10% FBS, Hygromycin B at the concentration of 800μg/ml, G418 at the concentration of 800μg/ml. After 48-hr incubation, ΙΟΟμί CellTiter-Glo Luminescent buffer were added to each well. Contents were mixed for 2 minutes on an orbital shaker to induce cell lysis and plates were incubated at room temperature for 10 minutes to stabilize luminescent signal. Luminescence was recorded by SpectraMax M5.

[00405] As shown in Table 5, all FIT-Igs were able to neutralize both hIL-20 and hlL-

17, with affinities similar to that of the paternal antibodies. Based on functional analysis using both Biacore and cell-based neutralization assays, it appears that all 3 FIT-Igs fully retain the activities of the parental mAbs. There was no significant functional difference among the three FIT-Igs, indicating that the linker was optional, and that FIT-Ig construct provided sufficient flexibility and special dimension to allow dual binding in the absence of a peptide spacer between the 2 Fab binding regions. This is in contrast to DVD-Ig type of molecules, where a linker between the 2 variable domains on each of the 2 polypeptide chain is required for retaining activities of the lower (2 ^nd ) variable domain.

Example 1.5 Stability study of anti-IL-17/IL-20 FIT-Ig

[00406] FITl-Ig protein samples in citrate buffer (pH=6.0) were individually incubated at constant 4 °C, 25 °C and 40 °C for 1 day, 3 days or 7 days; Similarly, FITl-Ig protein samples were freeze-thawed once, twice or three times. The fractions of intact full monomeric protein of all samples was detected by SEC-HPLC, with 10 μg of each protein sample injected into Utimate 3000 HPLC equipping Superdex200 5/150 GL at flow rate 0.3 mL/min for 15 min, and data was recorded and analyzed using Chromeleon software supplied by the manufacturer. Table 6 shows that FITl-Ig and FIT3-Ig remained full intact monomeric molecule under these thermo-challenged conditions.

Table 6. Stability analysis of FIT-Ig by measuring % full monomeric fractions by SEC

Example 1.6 Solubility study of anti-IL-17/IL-20 FIT-Ig [00407] The solubility of FITl-Ig was analyzed by measuring sign of precipitation in the presence of increasing concentration of PEG6000 (PEG6000 was purchased from Shanghai lingfeng chemical reagent co. Ltd). Briefly, solubility of protein in the presence of PEG6000 was obtained as a function of PEG6000 concentration (0, 5%, 10%, 15%, 20%, 25% and 30%). The solubility studies were conducted at a temperature of 25 °C at a solution pH of 6.0. Briefly, protein was precipitated by mixing appropriate quantities of buffered stock solutions of the protein, PEG and the buffer to get the desired concentration of the components. The final volume was made up to 200 μΐ and the concentration of protein was set at 1.0 mg/mL. The final solutions were mixed well and equilibrated for 16 h. After equilibration, the solutions were centrifuged at 13000 rpm for 10 min to separate the protein precipitate. Protein solubility was measured at 280 nm using Spectra Max Plus384 (Molecular Device) and obtained from the absorbance of the supernatant, and calculating the concentration based on standard curve of protein concentration (Figure 4A). We also analyzed a commercial antibody Rituxan using the same experimental method under 3 different pH conditions (Figure 4B). It appears that the protein solubility is dependent on the pH conditions, and that the predicted solubility of FIT-Ig would be in the range of monoclonal antibodies.

Example 1.7 Pharmacokinetic study of anti-IL-17/IL-20 FIT-Ig

[00408] Pharmacokinetic properties of FITl-Ig were assessed in male Sprague-Dawley

(SD) rats. FIT-Ig proteins were administered to male SD rats at a single intravenous dose of 5 mg/kg via a jugular cannula or subcutaneously under the dorsal skin. Serum samples were collected at different time points over a period of 28 days with sampling at 0, 5, 15, and

30min; 1, 2, 4, 8, and 24hr; and 2, 4, 7, 10, 14, 21, and 28 day serial bleeding via tail vein, and analyzed by human IL-17 capture and/or human IL-20 capture ELISAs. Briefly, ELISA plates were coated with goat anti-biotin antibody (5 μg/ml, 4° C, overnight), blocked with

Superblock (Pierce), and incubated with biotinylated human IL-17 (IL-17 capture ELISA) or

IL-20 (IL-20 capture ELISA) at 50 ng/ml in 10% Superblock TTBS at room temperature for

2 h. Serum samples were serially diluted (0.5% serum, 10% Superblock in TTBS) and incubated on the plate for 30 min at room temperature. Detection was carried out with HRP- labeled goat anti human antibody and concentrations were determined with the help of standard curves using the four parameter logistic fit. Several animals, especially in the subcutaneous group, showed a sudden drop in FIT-Ig concentrations following day 10, probably due to developing an anti-human response. These animals were eliminated from the final calculations. Values for the pharmacokinetic parameters were determined by non- compartmental model using WinNonlin software (Pharsight Corporation, Mountain View, Calif).

[00409] The rat PK study, FITl-Ig serum concentrations were very similar when determined by the two different ELISA methods, indicating that the molecule was intact, and capable of binding both antigens in vivo. Upon IV dosing, FITl-Ig exhibited a bi-phasic pharmacokinetic profile, consisting of a distribution phase followed by an elimination phase, similar to the PK profile of conventional IgG molecules. The pharmacokinetic parameters calculated based on the two different analytical methods were very similar and are shown in Table 7. Clearance of FIT-Ig was low (~12 mL/day/kg), with low volumes of distribution (Vss~130 mL/kg) resulting in a long half-life (Tl/2>10 days). Following subcutaneous administration, FIT-Ig absorbed slowly, with maximum serum concentrations of approximately 26.9 μg/ml reached at 4 days post-dose. The terminal half-life was about 11 days and the subcutaneous bioavailability was close to 100%. As demonstrated by these results, the properties of FITl-Ig are very similar to a conventional IgG molecule in vivo, indicating a potential for therapeutic applications using comparable dosing regimens.

[00410] The pharmacokinetics study of FIT-Ig has demonstrated a surprising breakthrough in the field of multi-specific Ig-like biologies development. The rat pharmacokinetic system is commonly used in the pharmaceutical industry for preclinical evaluation of therapeutic mAbs, and it well predicts the pharmacokinetic profile of mAbs in humans. The long half-life and low clearance of FIT-Ig will enable its therapeutic utility for chronic indications with less frequent dosing, similar to a therapeutic mAb. In addition, FIT- Ig, being 100-kDa larger than an IgG, seemed to penetrate efficiently into the tissues based on its IgG-like volume of distribution parameter from the PK study.

Table 7. Pharmacokinetics analysis of FITl-Ig in SD Rats

sc

PK parameters Tmax Cmax t½ AUClNF CL/F F

Unit Day ug/mL Day Day x ug/mL mL/day/kg %

IL-17 ELISA 4.00 26.9 11.0 406 12.4 103.5

IL-20 ELISA 4.00 23.1 10.4 350 14.3 86.4

Example 1.8 Stable CHO cell line development studies of FIT-Ig

[00411] It has been observed that FIT-Ig was efficiently expressed in transiently- transfected 293E cells. In order to further determine the manufacturing feasibility of FIT-Ig, stable transfections were carried out in both CHO-DG44 and CHO-S cell lines, and subsequent clone selections as well as productivity analysis were performed. Briefly, CHO cells were transfected by electroporation with 8xl0 ⁶ cells in 400 μΐ transfection solution plus 20ug DNA (for CHO DG44 cells) or 25 μg DNA (for CHO-S cells) subcloned in Freedom pCHO vector (Life Technologies). The stable cell line selection was done using routine procedures. Briefly, for CHO-DG44 selection, upon transfection, stable pool was selected (- HT/2P/400G, where P is μg/mL Puromycin, G is μg/mL G418), and protein production was analyzed by IgG ELISA. Top pools were selected and proceed to amplification for several rounds with increasing concentration of MTX (50, 100, 200 and 500 nM), followed by analysis of protein production by IgG ELISA. The top pools were then selected for subcloning. For CHO-S cell selection, the first phase selection was performed in medium containing 10P/400G/100M (M is nM MTX), followed by analysis of protein production. Then the top pools were selected and proceed to 2 ^nd phase selection in either 30P/400G/500M or 50P/400G/1000M, followed by protein production measurement by ELISA. The top pools were then selected for subcloning. For protein productivity analysis, fully recovered cell pools (viability >90%) were seeded at 5 ^χ 10 ⁵ viable cells/mL (CHO DG44) or 3 ^χ 10 ⁵ viable cells/mL (CHO-S) using 30 mL fresh medium (CD FortiCHO™ medium supplemented with 6 mM L-glutamine) in 125-mL shake flasks. The cells were incubated on a shaking platform at 37°C, 80% relative humidity, 8% C02, and 130 rpm. Sample cultures daily or at regular intervals (e.g., on day 0, 3, 5, 7, 10, 12, and 14) to determine the cell density, viability, and productivity until culture viability drops below 50% or day 14 of culture is reached. After sampling, feed the cultures with glucose as needed.

[00412] The overall process of FITl-Ig CHO stable cell line development showed features similar to that of a monoclonal antibody development in CHO cells. For example, during DG44 pool analysis under 2P/400G, the VCD continued to increase until day 10-12 up to about 1.3E7, whereas cell viability remained above 80% up to day 13-14, and the productivity reached almost 40 mg/mL on day 14. Upon amplification at 5P/400G/50M, productivity reached above 50 mg/mL on day 14. For CHO-S cell selection, the titer reached above 200 mg/mL during the phase 1 selection, and above 370 mg/mL at the phase 2 selection. These levels of productivity are similar to what have been previously observed for regular human mAb development is our laboratory, suggesting that FIT-Ig display mAb-like manufacturing feasibility for commercial applications.

Example 2; Construction, expression, and purification of anti-CD3/CD20 Fabs-in- tandem immunoglobulin (FIT-Ig)

[00413] To demonstrate if a FIT-Ig can bind to cell surface antigens, we have generated an anti-CD3/CD20 FIT-Ig molecule FIT7-Ig and FIT8-Ig, which is the 3- polypeptide construct, as shown in Figure 1. The construct used to generate FIT-Ig capable of binding cell surface CD3 and CD20 is illustrated in FigurelB. Briefly, parental mAbs include two high affinity antibodies, anti-CD3 (OKT3) and anti-CD20 (Ofatumumab). To generate FIT7-Ig construct #1, the VL-CL of OKT3 was fused directly (FIT7-Ig) or through a linker of 7 amino acids linker (FIT8-Ig) to the N-terminus of the Ofatumumab heavy chain (as shown in Table 8). The construct #2 is VH-CH1 of OKT3 and the 3 ^rd construct is VL-CL of Ofatumumab. The 3 constructs for FIT-Ig were co-transfected in 293 cells, resulting in the expression and secretion of FIT-Ig proteins. The detailed procedures of the PCR cloning are described below:

Example 2.1 Molecular cloning of anti-CD3/CD20 FIT-Ig:

[00414] The molecular cloning method is similar as that for anti-hIL-17/hIL-20 FIT-Ig.

Table 8. Anti-CD3/CD20 FIT-Ig molecules and constructs.

[00415] Table 9 shows sequences of PCR primers used for molecular construction above. Table 9. PCR primers used for molecular construction of anti-IL-17/IL-20 FIT-Igs

SEQ ID NO.

P4 : GTCTGCGGCCGCTCATTTACCCGGAGACAGGGAGAG 32

P12 TCGAGCGGCCGCTCAACAAGATTTGGGCTCAACTTTCTTG 33

P20 CAGGTCCAGCTGCAGCAGTCTG 34

P22 GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAG 35

GGG

P23 TACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAG 36

P24 TGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA 37

P25 CTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGAAGTGCAGCTGGTGGAGT 38

CTG

P28 GCTGCTGCTGTGGTTCCCCGGCTCGCGATGCGAAATTGTGTTGACACAGTC 39

P29 AAGATGAAGACAGATGGTGCAGCCACCGTACGTTTAATCTCCAGTCGTGTCC 40

[00416] The final sequences of anti-CD3/CD20 FIT-Ig are described in Table 10.

Table 10. Amino acid sequences of anti-CD3/CD20 FIT-Ig

Protein Sequence

Sequence

Protein region Identifier 12345678901234567890

OKT3/Ofatumuniab SEQ ID QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKS FIT7-Ig POLYPEPTIDE NO.:41 GTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGME

#1 AEDAATYYCQQWSSNPFTFGSGTKLEINRTVAAPSVFIF

PPSDEQLKSGTASWCLLNNFYPREA VQW VDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKH VYACEV THQGLSSPVTKSFNRGECEVQLVESGGGLVQPGRSLRLS CAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGY ADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKD IQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT V DK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK

OKT3 VL SEQ ID QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKS

NO.:42 GTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGME

AEDAATYYCQQWSSNPFTFGSGTKLEIN

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKV NO.: 17 QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD

YEKHKVYACEVTHQGLSSPVTKSFNRGEC

Linker none

Ofatumuniab VH SEQ ID EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQ

NO.:43 APGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKS

LYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTT VTVSS Protein Sequence

Sequence

Protein region Identifier 12345678901234567890

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV NO.: 19 SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT

QTYICNVNHKPSNT VDK VEPKSC

Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV NO.:20 TCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

OKT3/Ofatumuniab SEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ FIT7-Ig POLYPEPTIDE NO.:44 RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSST

#2 AYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNT VDK VEPKSC

OKT3 VH SEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ

NO.:45 RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSST

AYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTV SS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV NO.: 19 SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT

QTYICNVNHKPSNTKVDKKVEPKSC

OKT3/Ofatumuniab SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK FIT7-Ig POLYPEPTIDE NO.:46 PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL

#3 EPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFI

FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

Ofatumuniab VL SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK

NO.:47 PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL

EPEDFAVYYCQQRSNWPITFGQGTRLEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKV NO: 17 QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD

YEKHKVYACEVTHQGLSSPVTKSFNRGEC

OKT3/Ofatumuniab SEQ ID QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKS FIT8-Ig POLYPEPTIDE NO.:48 GTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGME

#1 AEDAATYYCQQWSSNPFTFGSGTKLEINRTVAAPSVFIF

PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECGGGGSGSEVQLVESGGGLVQP GRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISW NSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTA LYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK

OKT3 VL SEQ ID QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKS

NO.:42 GTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGME

AEDAATYYCQQWSSNPFTFGSGTKLEIN

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKV Protein Sequence

Sequence

Protein region Identifier 12345678901234567890

NO.: 17 QW VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD

YEKH VYACEVTHQGLSSPVTKSFNRGEC

Linker SEQ ID GGGGSGS

NO.:28

Ofatumuniab VH EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYA HWVRQ

APGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKS LYLQMNSLRAEDTALYYCAKDIQYGNYYYGMD GQGTT VTVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV NO.: 19 SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT

QTYICNVNHKPSNT VDK VEPKSC

Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV NO.:20 TCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS

TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

OKT3/Ofatumuniab SEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ FIT8-Ig POLYPEPTIDE NO.:44 RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSST

#2 AYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNT VDK VEPKSC

OKT3 VH SEQ ID QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQ

NO.:45 RPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSST

AYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTV SS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV NO: 19 SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT

QTYICNVNHKPSNTKVDKKVEPKSC

OKT3/Ofatumuniab SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK FIT8-Ig POLYPEPTIDE NO.:46 PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL

#3 EPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFI

FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

Ofatumuniab VL SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK

NO.:47 PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL

EPEDFAVYYCQQRSNWPITFGQGTRLEIK

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKV NO: 17 QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD

YEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 2.2 Expression and purification of anti-CD3/CD20 FIT-Ig:

[00417] All DNA constructs of each FIT-Ig were subcloned into pBOS based vectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 of each FIT-Ig were transiently co-expressed using Polyethyleneimine (PEI) in 293E cells. Briefly, DNA in FreeStyle™ 293 Expression Medium was mixed with the PEI with the final concentration of DNA to PEI ratio of 1 :2, incubated for 15min (no more than 20min) at room temperature, and then added to the 293E cells (1.0-1.2 χ lOVml, cell viability > 95%) at 6(^g DNA/120ml culture. After 6-24 hours culture in shaker, add peptone to the transfected cells at a final concentration of 5%, with shaking at 125 rpm/mia, at 37°C, 8% CC . On the 6th - 7th day, supernatant was harvested by centrifugation and filtration, and FIT-Ig protein purified using protein A chromatography (Pierce, Rockford, IL) according to manufacturer's instructions. The proteins were analyzed by SDS-PAGE and their concentration determined by A280 and BCA (Pierce, Rockford, IL) (Table 11).

Table 11. Expression and SEC analysis of anti-CD3/CD20 FIT-Ig proteins

Example 2.3 Binding activities of anti-CD3/CD20 FIT-Ig molecules:

[00418] Binding of anti-CD3/CD20 FIT-Igs to both targets were analyzed by FACS, using Jurkat cells that express CD3 on the cell surface, as well as Raji cells that express CD20 on the cell surface. Briefly, 5 * 10 ⁵ cells were washed in ice-cold PBS and blocked with 2% FBS on ice for 1 hr. Cells were incubated with antibody, FIT-Ig (100 nM), or isotype control on ice for lhr and washed 3 times with PBS. Secondary antibody (goat anti-human IgG labeled with Alexa Fluor 488, Invitrogen) were added and incubated with cells on ice for 1 hr in dark followed by three times wash with PBS. Samples were analyzed in FACs calibur. The cell surface binding shows that both FIT7-Ig and FIT8-Ig were able to binding to both cell surface antigens CD3 and CD20 in a concentration dependent manner. Compared to the binding activities of the parental mAbs, FIT-Ig showed a reduced binding intensity to CD3 on Jurkat cells, but an enhanced binding intensity to CD20 on Raji cells. In all binding studies, FIT7-Ig and FIT8-Ig showed similar binding activities to both antigens, indicating the linker did not make a significant impact on its binding ability for FIT8-Ig (Table 12).

Table 12. Cell surface antigen binding studies of anti-CD3/CD20 FIT-Ig proteins

FIT8-Ig CD3 (Jurkat) 211

Ofatumumab 181

FIT7-Ig

CD20 (Raji) 291

FIT8-Ig 274

Example 3; Construction, expression, and purification of anti-TNF/IL-17 Fabs-in- tandem immunoglobulin (FIT-Ig)

[00419] Another FIT-Ig that can bind to human IL-17 and human TNFa (FIT9-Ig) was also generated using anti-IL-17 mAb clone LY, and anti-TNF mAb Golimumab, in the 3- poly peptide construct, as shown in Figure 1. To generate FIT9-Ig construct #1, the VL-CL of Golimumab was fused directly to the N-terminus of LY heavy chain (as shown in Table 13). The construct #2 is VH-CH1 of Golimumab and the 3 ^rd construct is VL-CL of LY. The 3 constructs for FIT9-Ig were co-transfected in 293 cells, resulting in the expression and secretion of FIT9-Ig proteins. The final sequences of anti-TNF/IL-17 FIT-Ig are described in Table 14

Example 3.1 Molecular cloning of anti-TNF/IL-17 FIT-Ig:

[00420] The molecular cloning method is similar as that for anti-hIL-17/hIL-20 FIT-Ig.

Table 13. Anti-TNF/IL-17 FIT-Ig molecule and constructs.

Table 14: Amino acid sequences of anti-TNF/IL-17 FIT-Ig molecules

Protein Sequence

Protein region SEQ ID 12345678901234567890

NO:

EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL

Anti-IL-TNF/IL-17 EPEDFAWYCQQRSNWPPFTFGPGTKVDIKRTVAAPSVF FIT9-Ig SEQ ID IFPPSDEQLKSGTASWCLLNNFYPREA VQWKA/DNALQ

POLYPEPTIDE #1 NO.:87 SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKS FNRGECQVQLVQSGAEVKKPGSSVK VSCKASGYSFTDYHIHWVRQAPGQGLEWMGVINPMYGTT DYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCA RYDYFTGTGVYWGQGTLVTVS S AS TKGP SVF PLAP S S KS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSWTVPSSSLGTQTYI CNWHKPSNTKVD Table 14: Amino acid se uences of anti-TNF/IL-17 FIT-I molecules

Example 3.2 Expression, purification, and analysis of anti-TNF/IL-17 FIT-Ig proteins:

[00421] All DNA constructs of each FIT-Ig were subcloned into pBOS based vectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 of FIT9-Ig were transiently co- expressed using Polyethyleneimine (PEI) in 293E cells as described previously and FIT9-Ig proteins were purified by protein A chromatography. The expression level was 10-23 mg/L. The purified protein was subjected to functional analysis using cell-based assays for IL-17 (production of GROa by Hs27 cells) and TNF (production of IL-8 by L929 cells). The neutralization potency of FIT9-Ig against human TNF was 11.6 pM (compared to 15.9 pM by Golimumab in the same experiment), as against human IL-17 was 122 pM (compared to 51.5 pM by LY in the same experiment). Overall FIT9-Ig maintained the biological activities of the parental mAbs.

Example 4: Construction, expression, and purification of anti-CTLA-4/PD-l Fabs-in- tandem immunoglobulin (FIT-Ig)

[00422] Another FIT-Ig that can bind to human CTLA-4 and human PD-1 (FITIO-Ig) was generated using anti-CTLA-4 mAb Ipilimumab, and anti-PD-1 mAb Nivolumab, in the 3-polypeptide construct, as shown in Figure 1. To generate FITIO-Ig construct #1, the VL- CL of Ipilimumab was fused directly to the N-terminus of Nivolumab heavy chain (as shown in Table 15). The construct #2 is VH-CH1 of Ipilimumab and the 3 ^rd construct is VL-CL of Nivolumab. The 3 constructs for FITIO-Ig were co-transfected in 293 cells, resulting in the expression and secretion of FITIO-Ig proteins.

Example 4.1 Molecular cloning of anti-CTLA-4/PD-l FIT-Ig:

[00423] The molecular cloning method is similar as that for anti-hIL-17/hIL-20 FIT-Ig.

The final sequences of anti- CTLA-4/PD-1 FIT-Ig are described in Table 16.

Table 15. Anti-CTLA-4/PD-l FIT-Ig molecule and constructs.

Table 16. Amino acid sequences of anti-CTLA-4/PD-l FIT-Ig molecules Protein Sequence

Protein region SEQ ID 12345678901234567890

NO:

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQ APRLLIYGAFSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAV

Anti-CTLA-4 PD-l YYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSG FITIO-Ig SEQ ID TASWCLLNNFYPREA VQW VDNALQSGNSQESVTEQDSKDS

POLYPEPTIDE #1 NO.:92 TYSLSSTLTLSKADYEKH VYACEVTHQGLSSPVTKSFNRGEC

QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQAPGK GLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSL RAEDTAVYYCATNDDYWGQGTLVTVS SASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKE YKC VSNKALPAP I EKT I SKAKGQPRE PQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK

IPILIIY1UIY1AB VL SEQ ID EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQ

APRLLIYGAFSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAV NO.:93 YYCQQYGSS PWTFGQGTKVE I K

CL SEQ ID RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW V DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA NO.:17 CEVTHQGLS SPVTKS FNRGE C

Linker None

NIVOLUIY1AB VH SEQ ID QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQAPGK

NO.:94 GLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSL

RAEDTAVYYCATNDDYWGQGTLVTVS S

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS NO.:19 GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN

HKP SNTKVDKKVE PKS C

Fc SEQ ID DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCW NO.:20 VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK

QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQAPGK GLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSL

Anti-CTLA-4 PD-l RAEDTAI YYCARTGWLGP FD YWGQGTLVTVS S AS TKGP SVF PL FITIO-Ig SEQ ID APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP

POLYPEPTIDE #2 NO.:95 AVLQSSGLYSLSSWTVPSSSLGTQTYI CNVNHKPSNTKVDKK

VEPKSC

D7ILIMUMAB VH SEQ ID QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQAPGK

NO.:96 GLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSL

RAEDTAI YYCARTGWLGPFDYWGQGTLVTVSS

CHI SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS NO.:19 GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN

HKP SNTKVDKKVE PKS C

SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA NO.:97 PRLLIYDASNRATGI PARFSGSGSGTDFTLTI SSLEPEDFAVY

Anti-CTLA-4 PD-l YCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGT FITIO-Ig ASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST

POLYPEPTIDE #3 YSLSSTLTLSKAD YE KHKVYACEVTHQGLS SPVTKS FNRGE C

Nivolumab VL SEQ ID EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQA

NO.:98 PRLLIYDASNRATGI PARFSGSGSGTDFTLTI SSLEPEDFAVY Table 16. Amino acid se uences of anti-CTLA-4/PD-l FIT-I molecules

Example 4.2 Expression, purification, and functional analysis of anti-CTLA-4/PD-l FIT-Ig proteins:

[00424] All DNA constructs of each FIT-Ig were subcloned into pBOS based vectors, and sequenced to ensure accuracy. Construct #1, #2, and #3 of FITIO-Ig were transiently co- expressed using Polyethyleneimine (PEI) in 293E cells as described previously and FIT9-Ig proteins were purified by protein A chromatography to 98% monomeric full protein. The expression levels were up to 43 mg/L. The purified protein was subjected to binding analysis using ELISA against recombinant CTLA-4Ig and PD-1. Briefly, for binding to CTLA-4, human CTLA-4Ig (R&D Systems) was immobilized on 96-well plates, followed by routine wash and blocking procedures. Then FIT-10-Ig or Ipilimumab at various concentrations were added to the plate, followed by incubation and multiple wash steps, and detected with anti- human Fab -HRP. For binding to PD-1, human PD-1 (with a his tag) (R&D Systems) was immobilized on 96-well plates, followed by routine wash and blocking procedures. Then FIT- 10-Ig or Nivolumab at various concentrations were added to the plate, followed by incubation and multiple wash steps, and detected with anti-human Fc -HRP (Figure 5). It appears that FITIO-Ig was able to bind both CTLA-4 (A) and PD-1 (B) with similar activities as the parental mAbs Ipilimumab and Nivolumab, respectively.

[00425] In addition, multiple-antigen binding study was done using OctetRed to determine if FITIO-Ig was able to bind recombinant CTLA-4 and PD-1 simultaneously. Briefly, FITIO-Ig was immobilize on AR2G sensor at concentration of 10 μg/ml, followed by binding of CTLA-4Ig and then PD-1 (or PD-1 first, then CTLA-4Ig) in assay buffer (PBS pH 7.4, 0.1% BSA, 0.02%) Tween), with concentration at 80 nM. At the end of the experiment, the surface was regenerated with 10 mM glycine at pH1.5 five times (Figure 6). This experiment shows that FITIO-Ig was able to bind PD-1 when it had already bound to CTLA- 4, and vice versa, indicating that FITIO-Ig was able to bind both CTLA-4Ig and PD-1 simultaneously. Example 5; Construction, expression, and purification of additional Fabs-in-tandem immunoglobulin (FIT-Ig)

[00426] FIT-Ig having specificity for EGFR and PD-Ll; cMet and EGFR; Factor IXa and Factor X; Her3 and IGF-IR; DLL-4 and VEGF; CD20 and CD3; Her3 and EGFR; PD-1 and PD-Ll; and Her3 and PD-1 were constructed as in the foregoing Examples. These exemplary FIT-Ig and their corresponding sequences are provided below in Table 17. Table 18 provides the expression level in 293E cells and the SEC profile for each of the FIT-Ig.

Table 17. Amino acid se uences of additional exemplary FIT-Ig

Table 18. Expression level in 293E cells and SEC profile for FIT-Igs

[00427] The SEC profiles for each of the FIT-Ig of Tables 17 and 18 are also provided in Figure 7a-7i.

[00428] Functional binding data for FIT13a-Ig are provided below in Table 19. In addition, a multiple-antigen binding study was performed to determine if FIT13a-Ig was able to bind cMet and EGFR. The results of the study are shown in Figure 8, and show that FIT13a-Ig was able to bind both cMet and EGFR simultaneously.

Table 19. Functional binding data for FIT13a-Ig

[00429] Functional binding data for the Factor IXa binding activity of FIT14-Ig are provided below in Table 20. Table 20. Functional binding data for FIT14-Ig

[00430] The results of the study show that additional FIT-Ig can be constructed, expressed, and purified and will exhibit functional binding to the target proteins.

Example 6; Construction, expression, and purification of new anti-CTLA-4/PD-l Fabs- in-tandem immunoglobulin (FIT-Ig)

[00431] New FIT-Ig having specificity for CTLA4 and PDl were constructed as in the foregoing Examples. These exemplary FIT-Ig and their corresponding sequences are provided below in Table 21. Table 21 provides the expression level in 293E cells and the SEC profile for each of the FIT-Ig.

Table 21. Amino acid sequences of additional exemplary FIT-Ig for CTLA4 and PDl

Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

NBS3-Ig Long chain 126 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGER

(IpiliVL-hCk- ATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATG

CTLA4 NivoluVH- IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT

(ipilimumab)/ hCglmut) FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN

PD-l(nivolumab) NF YPRE AKVQ WKVDNALQ SGNS QE SVTE QD S KD ST YS LS S

TLTL SKAD YE KHKVYACE VTHQGL S S PVTKS FNRGE CQVQ LVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQAPGK GLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQM NSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKS CDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Ipili VL 127 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQK

PGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSPWTFGQGTKVEIK Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

Ipili VL - CDR1 128 RASQSVGSSYLA

Ipili VL - CDR2 129 GAFSRAT

Ipili VL - CDR3 130 QQYGSSPWT

NivoluVH 131 QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQA

PGKGLEWVAVIWYDGSKRYYADSVKGRFTI SRDNSKNTLF LQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

Nivolu VH - CDRl 132 NSGMH

Nivolu VH - CDR2 133 VIWYDGSKRYYADSVKG

Nivolu VH - CDR3 134 NDDY

Short Chain #1 (Ipili 135 MEFGLSWLFLVAILKGVQCQVQLVESGGGWQPGRSLRLS VH-CH1) CAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGN YYA

DSV GRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGW LGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK VEPKSC

Ipili VH 136 QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQA

PGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS

Ipili VH - CDR1 137 SYTMH

Ipili VH - CDR2 138 FI SYDGNNKYYADSVKG

Ipili VH - CDR3 139 TGWLGPFDY

Short Chain #2 140 VIDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGERA

(Nivolu VL-hCK) rLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA

RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQG

rKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Nivolu VL 141 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP

GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQSSNWPRTFGQGTKVEIK

Nivolu VL - CDR1 142 RASQSVSSYLA

Nivolu VL - CDR2 143 DASNRAT

Nivolu VL - CDR3 144 QQSSNWPRT

NBS3R - Ig Long Chain (Nivolu 145 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGER

VL-hCk-Ipili VH- ATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI

PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSN PRTF

PD- hCglmut)

GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN

l(nivolumab)/

FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST

CTLA4

LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECQVQL

(ipilimumab) VESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG

LEWVTFISYDGN YYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

SPGK

Nivolu VL 146 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP

GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQSSNWPRTFGQGT VEIK

Nivolu VL - CDR1 147 RASQSVSSYLA

Nivolu VL - CDR2 148 DASNRAT

Nivolu VL - CDR3 149 QQSSNWPRT

Ipili VH 150 QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQA

PGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS

Ipili VH - CDR1 151 SYTMH

Ipili VH - CDR2 152 FI SYDGNNKYYADSVKG

Ipili VH - CDR3 153 TGWLGPFDY

Short Chain #1 154 MEFGLSWLFLVAILKGVQCQVQLVESGGGWQPGRSLRLD (Nivolu VH-CH1) CKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYA

DSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAT DD

YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNWHKPSNT VDK VEPKSC

Nivolu VH 155 QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQA

PGKGLEWVAVIWYDGSKRYYADSVKGRFTI SRDNSKNTLF LQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

Nivolu VH - CDR1 156 GMH

Nivolu VH - CDR2 157 VIWYDGSKRYYADSVKG

Nivolu VH - CDR3 158 NDDY

Short Chain #2 (Ipili 159 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGER

VL-hCK) ATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATG

IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT

FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKH VYACEVTHQGLSSPVTKSFNRGEC

Ipili VL 160 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQK

PGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSPWTFGQGTKVEIK

Ipili VL - CDR1 161 RASQSVGSSYLA

Ipili VL - CDR2 162 GAFSRAT

Ipili VL - CDR3 163 YGSSPWT

NBS3-C - Ig Long Chain 164 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGER

(IpiliVL-hCk- ATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATG

CTLA4 NivoluVH-hCg4): IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT

FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN

(ipilimumab)/

NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS

PD-l(nivolumab)

TLTLSKADYEKH VYACEVTHQGLSSPVTKSFNRGECQVQ LVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQAPGK GLEWVAVIWYDGSKRYYADSV GRFTISRDNSKNTLFLQM NSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFP LAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPS NT VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

LMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRWSVLTVLHQDWLNGKEYKC VSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGOTFSCSVMHEALHNHYTQKSLSLSLGK

IpiliVL 165 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQK

PGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSPWTFGQGTKVEIK

IpiliVL - CDR1 166 RASQSVGSSYLA

IpiliVL - CDR2 167 GAFSRAT

IpiliVL - CDR3 168 QQYGSS

NivoluVH 169 QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQA

PGKGLEWVAVIWYDGSKRYYADSVKGRFTI SRDNSKNTLF LQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

NivoluVH - CDR1 170 NSGMH

NivoluVH - CDR2 171 VIWYDGSKRYYADSVKG

NivoluVH - CDR3 172 NDDY

Short Chain #1 (Ipili 173 MEFGLSWLFLVAILKGVQCQVQLVESGGGWQPGRSLRLS VH-CH1) CAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGN YYA

DSV GRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGW LGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK VEPKSC

Ipili VH 174 QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQA

PGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS

Ipili VH - CDR1 175 SYTMH

Ipili VH - CDR2 176 FI SYDGNNKYYADSVKG

Ipili VH - CDR3 177 TGWLGPFDY

Short Chain #2 178 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGER (Nivolu VL-hCK) ATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI

PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSN PRTF

GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Nivolu VL 179 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP

GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQSSNWPRTFGQGTKVEIK

Nivolu VL - CDR1 180 RASQSVSSYLA

Nivolu VL - CDR2 181 DASNRAT

Nivolu VL - CDR3 182 QQSSNWPRT

NBS3R-C - Long Chain (Nivolu 183 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGER

VL-hCk-Ipili VH- ATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI Ig

hCglmut) PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSN PRTF

GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN

PD- FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST l(nivolumab)/ LTLSKADYEKHKWACEVTHQGLSSPVTKSFNRGECQVQL

CTLA4 VESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKG

(ipilimumab) LEWVTFISYDGN YYADSVKGRFTISRDNSKNTLYLQMN

SLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGP Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNT VDK VEPKSCDKTHTCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

Nivolu VL 184 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP

GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQSSNWPRTFGQGT VEIK

Nivolu VL - CDR1 185 RASQSVSSYLA

Nivolu VL - CDR2 186 DASNRAT

Nivolu VL - CDR3 187 QQSSNWPRT

Ipili VH 188 QVQLVESGGGWQPGRSLRLSCAASGFTFSSYTMHWVRQA

PGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS

Ipili VH - CDR1 189 SYTMH

Ipili VH - CDR2 190 FI SYDGNNKYYADSVKG

Ipili VH - CDR3 191 TGWLGPFDY

Short Chain #1 192 MEFGLSWLFLVAILKGVQCQVQLVESGGGWQPGRSLRLD (Nivolu VH-IgG4- CKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYA CH1) DSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAT DD

YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTKTYTCNA/DHKPSNT VDKRVES

Nivolu VH 193 QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQA

PGKGLEWVAVIWYDGSKRYYADSVKGRFTI SRDNSKNTLF LQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

Nivolu VH - CDR1 194 NSGMH

Nivolu VH - CDR2 195 VIWYDGSKRYYADSVKG

Nivolu VH - CDR3 196 NDDY

Short Chain #2 (Ipili 197 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGER

VL-hCK) ATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATG

IPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWT

FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKH VYACEVTHQGLSSPVTKSFNRGEC

Ipili VL 198 EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQK

PGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYGSSPWTFGQGTKVEIK

Ipili VL - CDR1 199 RASQSVGSSYLA

Ipili VL - CDR2 200 GAFSRAT

Ipili VL - CDR3 201 QQYGSSPWT

[00432] The long chain and short chain comprise a leader sequence, which either MDMRVPAQLLGLLLLWFPGSRC (SEQ ID NO: 487),

MEFGLSWLFLVAILKGVQC (SEQ ID NO: 488). Table 22. SEC Profile/Expression level in 293E cells;

[00433] Expression level was evaluated in small scale production without any optimization, except NBS3. All SEC samples are under one step Protein A purification. FIT- Ig in both IgGl and IgG4 format can be produced as homogeneous protein from 293 cells.

Functional Studies

[00434] Functional binding data for these antibodies is provided below in Table 23.

The data suggests that the affinity was not affected by changing IgG constant sequences, but can be improved by place certain Fab in upper domain.

Table 23. Functional binding data

[00435] ELISA binding study: ELISA binding of NBS3R was tested. Briefly, for binding to CTLA-4, human CTLA-4Ig (R&D Systems) was immobilized on 96-well plates, followed by routine wash and blocking procedures. Then FIT-Ig or related monoclonal antibodies at various concentrations were added to the plate, followed by incubation and multiple wash steps, and detected with anti -human Fab -HRP. For binding to PD-1, human PD-1 (with a his tag) (R&D Systems) was immobilized on 96-well plates, followed by routine wash and blocking procedures. Then NBS3R or Nivolumab at various concentrations was added to the plate, followed by incubation and multiple wash steps, and detected with anti-human Fc -HRP. It appears that NBS3R was able to bind both CTLA-4 (A) and PD-1 (B), respectively. The result is shown in Figure 9A and Figure. 9B. [00436] Multiple binding study: A multiple binding study of NBS3 was also carried out. The result is shown in Figure. 10.

[00437] Thermo stability study: Thermo stability test was also performed on these antibodies, the result of which is shown in Table 24. The melting temperature was measured by DSC.

Table 24. Thermo stability test

[00438] Storage stability study: The storage stability of NBS3 was assessed by SEC-

HPLC method, and result is shown in Table 25. Samples were treated by freeze/thaw cycle for one time, two times or three times, no aggregation or degradation was observed by SEC- HPLC profile. Samples was treated at 4°C, 25°C or 40°C for 1 day, 3 days or 7 days, no aggregation or degradation was observed by SEC-HPLC profile.

Table 25. Storage stability of NBS3

[00439] NBS3 was further tested in rat PK study, and the result is shown in Figure

11A and Figure 11B. The purpose of this study was to evaluate the pharmacokinetic of NBS3 following single intravenous (IV) or subcutaneous (SC) administrations in SD rats. The IV dose was administered via foot dorsal vein injection and SC dose was administered via subcutaneous injection. At the designated time-points, the animals were restrained manually, and approximately 240 blood/time point was collected via tail vein puncture or cardiac puncture into tubes. The blood samples were placed at room temperature for 0.5 hr. Then blood samples were centrifuged (10000 g, 5 min under 4°C) to obtain the serum samples. The serum samples were immediately stored at -80°C until analysis. Samples were analyzed together with dosing solution via ELISA. The measured dosing concentration was used for the PK parameter calculation.

[00440] NBS3, NBS3-C, and NBS3R-C were tested in cell-based receptor blocking assay. Briefly, PDl-Fc was immobilized on 96-well plates, followed by routine wash and blocking procedures. Then diluted FIT-Ig and biotinylated PD-Ll-Fc was added to each well, followed by incubation and multiple wash steps, and detected with Streptavidin-HRP. The result is shown in Figure 12.

[00441] NBS3, NBS3-C, and NBS3R-C were further tested in MLR (Mixed

Lymphocyte Reaction) assays and PBMC SEB-stimulation assay for their functional activity.

[00442] In the MLR assays, mixed lymphocyte reaction was performed using monocyte-derived dendritic cells from one donor and allogeneic CD4 T cells from another donor. The whole blood samples were collected from healthy donors, and PBMC were isolated from whole blood using Ficoll-Pague gradient centrifugation. On day 1, PBMC from one donor was isolated and diluted with serum-free RPMI 1640 at lXlOVml. The diluted

PBMC was seeded into 6-well tissue culture plate at 3ml/well and incubated for 3h.

Supernatant was removed and unattached cells were washed off. The attached monocyte were polarized into dendritic cells with 250 U/ml IL-4 and 500 U/ml GM-CSF in RPMI1640 with

10 %FBS. The medium was replaced with fresh IL-4 and GM-CSF at day 4. At day 7, immature DC was collected and treated with ^g/ml LPS in RPMI 1640 with 10% FBS for additional 24h for maturation. At Day 8, CD4 T cells were isolated from another donor

PBMC by negative selection and adjusted to final concentration at 2X10 ⁶ cells/ml. Mature

DC were treated with mitomycin C at 37°C for 1.5 hr. Then DC were washed with PBS and adjusted to final concentration at 1X10 ⁶ cells/ml. CD4 T cells (Responder cells) were added into 96 well plate at ΙΟΟμΙ/well and pre-treated with test antibody at diluted concentration for

30 minutes. Then mature DC (Stimulator cells) were added into the well at ΙΟΟμΙ/well. The final volume of each well is 200μ1. The MLR were incubated at 37 degree for 72 hr for IL-2 test and 120 hr for IFN-gamma test respectively using ELISA. The result is shown in Figure 13A and Figure 13B.

[00443] In the PBMC SEB-stimulation assays, PBMC were isolated from healthy donor blood by Ficoll-Pague gradient centrifugation. The isolated PBMC were seeded into 96-well tissue culture plate at 1X10 ⁵ cells/well. Then the PBMC were pre-treated with diluted test antibodies for 30min. Staphylococcus enterotoxin B (SEB) was added into cell culture medium with final concentration at lOOng/ml. The final assay volume was 200μ1Λνβ11. The cells were cultured for 96 hr and the culture supernatant was collected. IL-2 cytokine production in the supernatant was detected by ELISA. The result is shown in Figure 14.

Example 7; Construction, expression, and purification of additional anti-EGFR/PD- Ll Fabs-in-tandem immunoglobulin (FIT-Ig)

[00444] New FIT-Igs having specificity for EGFR and PD-L1 were constructed as in the foregoing Examples. These exemplary FIT-Igs and their corresponding sequences are provided below in Table 26. Table 27 provides the expression level in 293E cells and the SEC profile for each of the FIT-Ig.

Table 26. Amino acid sequences of additional exemplary FIT-Ig for EGFR and PD-L1

Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

Pani VL 203 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKP

GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP EDIATYFCQHFDHLPLAFGGGT VEIK

Pani VL - CDR1 204 QASQDISNYLN

Pani VL - CDR2 205 DASNLET

Pani VL - CDR3 206 QHFDHLPLA

1B 12VH 207 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSSYAISWVRQA

PGQGLEWMGGI IPIFGRAHYAQKFQGRVTITADESTSTAY MELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVT VSS

1B 12VH - CDR1 208 SYAIS

1B 12VH - CDR2 209 GI I PI FGRAHYAQKFQG

1B 12VH - CDR3 210 KFHFVSGSPFGMDV

Short Chain #1 211 MEFGLSWLFLVAILKGVQCQVQLQESGPGLV PSETLSLT (Pani VH-CHlh) CTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNY

NPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDR

VTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGT

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNT VDK VEPKS C

Pani VH 212 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIR

QSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQF SLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS

Pani VH - CDR1 213 SGDYYWT

Pani VH - CDR2 214 HIYYSGNTNYNPSLKS

Pani VH - CDR3 215 DRVTGAFDI

Short Chain #2 216 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGER (1B12 VL-hCk) ATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI

PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFG

QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNF YPREA VQW VDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

IB 12 VL 217 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP

GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPTFGQGTKVEIK

IB 12 VL - CDR1 218 RASQSVSSYLA

IB 12 VL - CDR2 219 DASNRAT

IB 12 VL - CDR3 220 QQRSNWPT

Long Chain (Pani 221 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDR

VL-hCk-10A5VH- VTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGV hCgl) PSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAF

GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKH VYACEVTHQGLSSPVTKSFNRGECQVQL VQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQR LEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELS SLRSEDTAVYYCARERIQLWFDYWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC V SNKALPAPIEKTISKAKGQPREPQWTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

Pani VL 222 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKP

GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP EDIATYFCQHFDHLPLAFGGGT VEIK

Pani VL - CDR1 223 QASQDISNYLN

Pani VL - CDR2 224 DASNLET

Pani VL - CDR3 225 QHFDHLPLA

10A5VH 226 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQA

PGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAY MELSSLRSEDTAWYCARERIQLWFDYWGQGTLVTVSS

10A5VH - CDR1 227 SYDVH

10A5VH - CDR2 228 WLHADTGITKFSQKFQG

10A5VH - CDR3 229 ERIQLWFDY

Short Chain #1 230 MEFGLSWLFLVAILKGVQCQVQLQESGPGLV PSETLSLT (Pani VH-CHlh) CTVSGGSVSSGDYY TWIRQSPGKGLEWIGHIYYSGNTNY

NPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCVRDR

VTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGT

AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNT VDK VEPKS C

Pani VH 231 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIR

QSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQF SLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS

Pani VH - CDR1 232 SGDYYWT

Pani VH - CDR2 233 HIYYSGNTNYNPSLKS

Pani VH - CDR3 234 DRVTGAFDI

Short Chain #2 235 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDR (10A5 VL-hCk) VTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGV

PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTF

GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKH VYACEVTHQGLSSPVTKSFNRGEC

10A5 VL 236 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKP

EKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYNSYPYTFGQGTKLEIK

10A5 VL - CDR1 237 RASQGISSWLA

10A5 VL - CDR2 238 AASSLQS

10A5 VL - CDR3 239 QQYNSYPYT

Table 27. SEC Profile/Expression level in 293E cells:

Expression

FIT-Ig Monomer% in SEC

level(mg/L) FIT012b - Ig 74.72% 5.3

FIT012d - Ig > 87% 1.05

[00445] The SEC profile and expression data suggest that FIT012d exhibited better purity than FIT012b by changing PD-L1 antibody sequences.

[00446] Functional binding data for these two antibodies is provided below in Table

28 and Table 29. The data suggests that the affinity was not affected by changing IgG constant sequences, but can be improved by place certain Fab in upper domain.

Table 28. Functional binding data of FIT012b

[00447] A multiple binding study of FIT012b and FIT012d was also carried out. The result is shown in Figure 15 (FIT012b) and Figure 16A to Figure 16B (FIT012d).

Example 8; Construction, expression, and purification of anti-cMet/EGFR Fabs-in- tandem immunoglobulin (FIT-Ig)

[00448] New FIT-Ig having specificity for cMet and EGFR was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 30. Table 31 provides the expression level in 293E cells and the SEC profile for each of the FIT-Ig.

Table 30. Amino acid sequences of additional exemplary FIT-Ig for cMet and EGFR

Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

FIT013a Long Chain (hi 332 240 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDR

VL-hCk-Pani VH- VTITCRASQGINTWLAWYQQKPGKAPKLLIYAASSLKSGV cMet (hi 332 hCgl) PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTF (13.3.2L- GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

A91T,H- FYPREA VQW VDNALQSGNSQESVTEQDSKDSTYSLSST 42K,S97T)) LTLSKADYEKHKWACEVTHQGLSSPVTKSFNRGECQVQL

QESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPG KGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKL SSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

hl332 VL 241 DIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQKP

GKAPKLLIYAASSLKSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQANSFPLTFGGGTKVEIK

hl332 VL - CDR1 242 RASQGINTWLA

hl332 VL - CDR2 243 AASSLKS

hl332 VL - CDR3 244 QQANSFPLT

Pani VH 245 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIR

QSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQF SLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS

Pani VH - CDR1 246 SGDYYWT

Pani VH - CDR2 247 HIYYSGNTNYNPSLKS

Pani VH - CDR3 248 DRVTGAFDI

Short Chain #1 249 MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASVKVS (hl332 VH-CH1) CKASGYTFTSYGFSWVRQAPGQGLEWMGWISASNGNTYYA

QKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVYA

DYADYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

M332 VH 250 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGFSWVRQA

PGQGLEWMGWISASNGNTYYAQKLQGRVTMTTDTSTSTAY MELRSLRSDDTAVYYCARVYADYADYWGQGTLVTVSS

M332 VH - CDR1 251 SYGFS

M332 VH - CDR2 252 WI SASNGNTYYAQKLQG

M332 VH - CDR3 253 VYADYADY

Short Chain #2 254 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGDR

(Pani VL-hCk) VTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGV

PSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAF

GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Pani VL 255 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKP

GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP EDIATYFCQHFDHLPLAFGGGTKVEIK

Pani VL - CDR1 256 QASQDISNYLN

Pani VL - CDR2 257 DASNLET Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

Pani VL - CDR3 258 QHFDHLPLA

Table 31. SEC Profile/Expression level in 293E cells:

Stability data

[00449] The storage stability of FIT013a was assessed by SEC-HPLC method, and result is shown in Table 32. Samples were treated by freeze/thaw cycle for one time, two times or three times, no aggregation or degradation was observed by SEC-HPLC profile. Samples was treated at 4°C, 25°C or 40°C for 1 day, 3 days or 7 days, no aggregation or degradation was observed by SEC-HPLC profile.

Table 32. Storage stability of FIT013a

[00450] Functional Study: Protein based binding data for FIT013a is provided below in

Table 33 and Table 34

Table 33. Functional binding data of FIT013a (human cMet/human EGFR) ig Target Kon Koff KD IC50

H1332 4.14E+05 5.27E-04 1.27E-09

1 (human cMet)

FIT013a 4.75E+05 5.02E-04 1.06E-09

Panitumumab 2 (human 8.43E+04 5.10E-05 6.05E-10

FIT013a EGFR) 1.09E+05 5.84E-05 5.34E-10

Table 34. Functional binding data of FIT013a (Cyno cMEt/Cyno EGFR) ig Target Kon Koff KD IC50

H1332 2.21E+05 1.00E-03 4.53E-09

l(Cyno cMet)

FIT013a 2.86E+05 9.74E-04 3.40E-09

Panitumumab 5.87E+05 2.12E-04 3.62E-10

2(Cyno EGFR)

FIT013a 2.66E+05 1.54E-04 5.77E-10

[00451] A multiple binding study of FIT013a was also carried out. The result is shown in Figure 17.

[00452] Further, the binding activity of FIT013a in cancer cell lines was determined by flow cytometry using BD FACSVerse. Cells grown in culture were detached from flask with trypsin-free medium and collected. The collected cells were washed in PBS buffer containing 2% FBS. Cells were then aliquot and incubated with 1 :5 serially diluted FIT013a on ice for lhr. The starting working concentration of FTI013a was 2C^g/ml. Cells were washed, resuspended and incubated with 1: 100 diluted Alexa Fluor® 488 labeled mouse anti-human IgGl (Invitrogen, Cat.No.A- 10631) on ice protected from light for lhr. Cells were washed and signal was detected with a BD FACSVerse flow cytometer according to manufacture's protocols.

[00453] These experiments demonstrate that FIT013a can bind to many cancer cell lines, like NCI-H1993, HCC827, MK -45, SGC-7901, EBC-1, A549, KP4, NCI-H292, NCI- HI 975, etc. Geometric mean fluorescence intensity (MFI) and EC50 for each cell lines are listed in the Table 35.

Table 35. Cell based binding data for FIT013a

[00454] In addition, FACS assays were conducted to measure FIT013a's dual binding to membrane c-Met and EGFR to show its multiple binding activity.

[00455] Several cell lines were used n this assay. MK -45 (human gastric adenocarcinoma cells) expressed high level of c-Met and low level of EGFR. SGC-7901 (human gastric cancer) expressed high level of EGFR and low level of c-Met. NCI-H1975 (human non-small cell lung cancer) expressed equal level of c-Met and EGFR.

[00456] Cells grown in culture were detached from flask with trypsin-free medium and collected. The collected cells were washed in PBS buffer containing 2% FBS. Cells were aliquot and incubated with serially diluted FIT013a or FIT013a-Fab on ice for lhr. Cells were then washed and incubated with ^g/ml biotinylated human c-MET or biotinylated EGFR on ice for lhr. Cells were resuspended and incubated with 4μg/ml Alexa Fluor® 488 labeled Streptavidin (Invitrogen, Cat.No S32354) on ice protected from light for lhr. Cells were washed and signal was detected with a BD FACSVerse flow cytometer according to manufacture's protocols.

[00457] When cell membrane expression level c-Met is much higher than EGFR (e.g., on MKN-45 cell), c-Met binding cite of FIT013a and FIT013a-Fab can be occupied by membrane c-Met, the free EGFR binding cite of FIT013a and FIT013a-Fab can be detected by biotinylated EGFR. When cell membrane expression level EGFR is much higher than c- Met (e.g., on SGC-7901), EGFR binding cite of FIT013a and FIT013a-Fab can be occupied by membrane EGFR, the free c-Met binding cite of FIT013a and FIT013a-Fab can be detected by biotinylated c-Met. When cell membrane expression level c-Met is equal to EGFR (e.g., on NCI-H1975 cell), c-Met and EGFR binding cites of FIT013a and FIT013a- Fab are occupied simultaneously, no free EGFR or c-Met binding cite of FIT013a and FIT013a-Fab can be detected. As demonstrated in Figure 18A to Figure 18C, FIT013a and FIT013a-Fab can simultaneously bind to two receptors c-Met and EGFR on the cell surface.

[00458] Signaling assay: Next, FIT013a was used to inhibit HGF induced AKT phosphorylation in cells. NCI-H292 cells were plated at 2* 10 ⁵ per well in 96-well plate and serum starved overnight. Serially diluted FIT013a or other related Abs were added to plate and incubate for 30mins and then 40ng/ml HGF was added to the assay plate for 5mins. The cells were lysed and AKT phosphorylation was detected by ERK phospho-T202/Y204 kit (Cisbio, Cat: 64AKSPEG). The experiment demonstrates that FIT013a shows superior activity than mAbs combo in neutralizing HGF induced AKT phosphorylation in NCI-H292 cell. FIT013a inhibited 80% of AKT phosphorylation while EGFR and c-Met antibody combo inhibit 60% of AKT phosphorylation, see Figure 19.

[00459] Agonist assay: Agonist effect of c-Met, EGFR antibody in FIT013a was tested by AKT phosphorylation. NCI-H441 cells were plated at 2 X 10 ⁵ per well in 96-well plate and serum starved overnight. Serially diluted FIT013a or other related Abs were added to plate and incubate for 30mins. The cells were lysed and AKT phosphorylation was detected by ERK phospho-T202/Y204 kit (Cisbio, Cat: 64AKSPEG). As shown in Figure 20, the experiment demonstrates that FIT013a showed weak agonist effect in absence of HGF.

[00460] Receptor depletion study: FIT013a was tested to see if it can deplete both c- Met and EGFR on cell membrane. The c-Met antibody, EGFR antibody and FIT013a were incubated with H441 cell for more than 16 hrs at 37°C and then EGFR and c-Met remaining on the cell surface was detected by FACS. The result indicates that FIT013a can deplete near 70% of cell membrane c-Met and EGFR, higher than the c-Met antibody or the EGFR antibody, see Table 36.

Table 36. Receptor depletion by FIT013a

[00461] Rat PK study: The purpose of this study was to evaluate the pharmacokinetic of FIT013a (c-met/EGFR) following single intravenous (IV) or subcutaneous (SC) administrations in SD rats. For IV and SC dosing, the test article FIT013a (c-met/EGFR) was dissolved (in lOmM sodium citrate, 50mM NaCl, pH 6.0) at 2.3 mg/mL (lot: 160408001). A total of 8 male SD rats, approximately 195-208g of body weight, purchased from SLAC Laboratory Animal Co. LTD. with Qualification No.: SCXK (SH) 2008001659659, were used in this study. The dosing and sampling is designed as Table 37.

Table 37. Dosing and Sampling Design

Route Dose Solution Dose

Treatment No. of

Treatment of Level Cone. Volume Time points Group animals

admin. (mg/kg) (mg/mL) (niL/kg)

Sampling at 0,

FIT013a

lOmin, 1, 4, 8, 24hr,

1 (c- 4 IV 5 2 2.5

2, 4, 7, 10, 14, 21, met/EGFR)

28d, serial bleeding via tail vein for

serum only. -240 uL blood per time point.

Sampling at 0, 30min, 1, 4, 8, 12, 24hr, 2, 4, 7, 10, 14,

FIT013a

21, 28d, serial

(c- SC 2.5

bleeding via tail vein met/EGFR)

for serum only.

-240 uL blood per time point.

[00462] The IV dose was administered via foot dorsal vein injection and SC dose was administered via subcutaneous injection. At the designated time-points, the animals were restrained manually, and approximately 240 blood/time point was collected via tail vein puncture or cardiac puncture into tubes. The blood samples were placed at room temperature for 0.5 hr. Then blood samples were centrifuged (10000 g, 5 min under 4°C) to obtain the serum samples. The serum samples were immediately stored at -80°C until analysis. The samples were detected by ELISA using c-Met and EGFR protein respectively. The concentration-time data of FIT013a (c-met/EGFR) in rat serum for IV and SC studies are listed in Table 38 to Table 41, and are illustrated in Figure 21A to Figure 21D.

Table 38. Serum concentration-time data and pharmacokinetic parameters of FIT013a (c-met/EGFR) after an IV dose at 5 mg/kg in male SD rats (c-met plate)

Dose Dose Sampling Concentration Mean

(mg/kg) route time ^g/mL) ^g/mL) SD CV (%)

(Day) Rat #l Rat #2 Rat #3 Rat #4

5 IV 0 BQL BQL BQL BQL BQL NA NA c-met plate 0.00694 138 118 82.0 108 112 23.3 20.9

0.0417 114 105 74.3 99.3 98.2 17.0 17.4

0.167 103 97.8 71.7 83.8 89.0 14.0 15.8

0.333 79.1 87.9 62.8 70.9 75.2 10.8 14.4

1 60.8 62.8 56.3 53.6 58.4 4.17 7.14

2 51.0 54.2 45.4 42.6 48.3 5.27 10.9

4 44.0 45.5 37.7 39.0 41.5 3.81 9.17

7 31.9 33.1 29.6 28.6 30.8 2.06 6.68

10 24.7 22.7 24.4 21.8 23.4 1.40 5.97

14 20.0 19.7 18.5 18.0 19.1 0.946 4.96

21 12.3 11.9 12.1 11.5 12.0 0.319 2.67

28 7.68 8.12 9.53 7.43 8.19 0.938 11.5

PK parameters Unit Rat #l Rat #2 Rat #3 Rat #4 Mean SD CV (%)

CL mL/day/kg 6.24 6.14 6.21 6.86 6.36 0.335 5.27

Vss mL/kg 82.5 82.7 105 97.6 91.8 11.0 12.0

VI mL/kg 38.2 44.4 65.3 47.6 48.9 11.6 23.8

Alpha tie day 0.235 0.491 0.879 0.309 0.478 0.288 60.2

Beta tie day 9.46 9.80 12.3 10.21 10.4 1.26 12.1

AUC Day x μg/mL 802 815 805 729 788 39.5 5.02 MRT day 13.2 13.5 16.8 14.2 14.4 1.65 11.4

Table 39. Serum concentration-time data and pharmacokinetic parameters of FIT013a (c-met/EGFR) after a SC dose of 5 mg kg in male SD rats (c-met plate)

Dose Dose Sampling Concentration Mean SD CV(%)

(mg kg) route time ^g mL) ^g/mL)

(Day) Rat #5 Rat #6 Rat #7 Rat #8

5 SC 0 BQL BQL BQL BQL BQL NA NA c-Met plate 0.00694 0.0780 BQL 0.0500 BQL 0.0640 NA NA

0.0417 0.167 0.150 0.172 0.228 0.179 0.0338 18.9

0.167 0.865 2.38 1.70 3.28 2.06 1.03 49.9

0.333 2.44 4.89 3.66 6.17 4.29 1.60 37.4

0.5 5.69 8.74 7.37 10.8 8.16 2.18 26.7

1 21.3 29.5 23.6 28.3 25.7 3.85 15.0

2 39.4 47.9 40.9 43.7 43.0 3.73 8.69

4 47.4 36.8 42.3 47.2 43.4 5.01 11.5

7 36.8 38.5 34.4 37.7 36.9 1.76 4.78

10 28.8 25.7 27.0 29.9 27.9 1.85 6.64

14 19.8 *0.753 18.5 18.6 19.0 0.748 3.94

21 14.2 BQL 12.8 16.2 14.4 1.67 11.6

28 11.6 BQL 9.52 10.5 10.5 1.03 9.74

PK parameters Unit Rat #5 Rat #6 Rat #7 Rat #8 Mean SD CV(%)

Tmax day 4.00 2.00 4.00 4.00 3.50 1.00 28.6

Cmax ^πιΐ 47.4 47.9 42.3 47.2 46.2 2.63 5.70

Terminal tin day 11.6 11.6 11.4 12.0 11.7 0.262 2.24

AUO _ast Day x g/ml 656 344 611 680 573 155 27.1

AUClNF Day x g/ml 850 775 767 863 814 49.8 6.12

CL F mL/day kg 5.88 6.45 6.52 5.79 6.16 0.376 6.11

F % 108 98.4 97.4 110 103 6.32 6.12

*:The serum concentration of these time point were excluded from mean value and PK parameters calculation due to the posibility of anti-drug antibody.

Table 40. Serum concentration-time data and pharmacokinetic parameters of FIT013a (c- met/EGFR) after an IV dose at 5 mg/kg in male SD rats (EGFR plate)

Dose Dose Sampling Concentration Mean

(mg/kg) route time ^g/mL) ^g mL) SD CV(%)

(Day) Rat #l Rat #2 Rat #3 Rat #4

5 IV 0 BQL BQL BQL BQL BQL NA NA EGFR plate 0.00694 130 115 80.0 105 107 21.0 19.6

0.0417 111 103 75.8 97.3 96.9 15.2 15.7

0.167 98.0 95.5 72.3 83.4 87.3 11.9 13.6

0.333 76.2 83.6 62.1 70.8 73.2 9.06 12.4

1 57.0 59.3 56.6 53.1 56.5 2.59 4.58

2 47.6 50.9 45.6 44.6 47.2 2.79 5.92

4 45.6 45.3 39.9 39.8 42.6 3.27 7.67

7 32.7 33.8 31.1 29.5 31.8 1.88 5.90

10 25.1 23.6 25.2 22.3 24.0 1.37 5.69 14 19.2 19.7 19.1 18.4 19.1 0.524 2.75

21 12.8 12.2 12.8 12.1 12.5 0.365 2.92

28 8.38 8.62 10.0 7.71 8.68 0.973 11.2

PK parameters Unit Rat #l Rat #2 Rat #3 Rat #4 Mean SD CV(%)

CL mL/day kg 6.14 6.09 5.98 6.66 6.22 0.302 4.86

Vss mL/kg 86.3 85.2 102 96.0 92.3 7.94 8.61

VI mL/kg 39.7 44.8 65.3 48.7 49.6 11.1 22.3

Alpha ti day 0.222 0.368 0.795 0.306 0.423 0.255 60.3

Beta ti day 10.0 10.0 12.3 10.3 10.7 1.09 10.2

AUC Day x g/mL 815 821 836 751 806 37.6 4.67

MRT day 14.1 14.0 17.0 14.4 14.9 1.44 9.70

Table 41. Serum concentration-time data and pharmacokinetic parameters of FIT013a (c- met/EGFR) after a SC dose at 5 mg/kg in male SD rats (EGFRplate)

Dose Dose Sampling Concentration Mean

(mg/kg) route time ^g/mL) ^g/mL) SD CV(%)

(Day) Rat #5 Rat #6 Rat #7 Rat #8

5 SC 0 BQL BQL BQL BQL BQL NA NA

EGFR plate 0.00694 0.0760 BQL 0.0490 BQL 0.0625 NA NA

0.0417 0.164 0.144 0.167 0.231 0.177 0.0377 21.4

0.167 0.836 2.32 1.66 3.30 2.03 1.04 51.4

0.333 2.45 4.45 3.74 6.37 4.25 1.64 38.5

0.5 5.85 8.61 7.69 11.6 8.45 2.42 28.6

1 21.6 27.7 24.5 29.8 25.9 3.61 14.0

2 41.5 45.3 40.0 43.8 42.6 2.39 5.60

4 46.2 38.9 41.9 46.2 43.3 3.56 8.22

7 35.9 37.3 34.7 39.6 36.9 2.13 5.77

10 28.5 25.0 26.8 30.6 27.7 2.40 8.65

14 19.3 *0.768 19.4 19.0 19.2 0.183 0.950

21 14.7 BQL 13.9 16.5 15.0 1.33 8.84

28 11.7 BQL 10.5 11.4 11.2 0.601 5.37

PK parameters Unit Rat #5 Rat #6 Rat #7 Rat #8 Mean SD CV(%)

Tmax day 4.00 2.00 4.00 4.00 3.50 1.00 28.6

Cmax ^ ^η ιΐ 46.2 45.3 41.9 46.2 44.9 2.03 4.51

Terminal ti day 13.1 9.38 12.4 12.3 11.8 1.66 14.0

AUClast Day x g/ml 653 339 625 694 578 162 28.0

AUClNF Day x g/ml 875 677 813 896 815 98.8 12.1

CL/F mL/day/kg 5.72 7.39 6.15 5.58 6.21 0.823 13.3

F % 109 84.0 101 111 101 12.3 12.1

*:The serum concentration of these time point were excluded from mean value and PK parameters calculation due to the posibility of anti-drug antibody.

NCI-H1975-HGF xenograft model tumor distribution study

[00463] In this study, serum and tumor concentration of FIT013a was measured 24 hrs after single IP dose in NCI-H1975-HGF tumor bearing nude BALB/c Mice. [00464] NCI-H1975-HGF cells were subcutaneously inoculated to nude BALB/c mice. When the average tumor volume reached to 200-250 mm ³ , the mice were randomly allocated to four groups, FIT013a, H1332, Panitumumab and vehicle group. FIT013a group was single IP dose, 16mg/kg; HI 332 or Panitumumab was single IP dose, lOmg/kg. Vehicle group was dosed with formulation buffer lOmM sodium citrate, 50mM NaCl, pH 6.0. 24hrs after dosing, tumor and serum were collect. Tumors were homogenized and the supernatant was collected for the following ELISA study. FIT013a, Panitumumab, and HI 332 were quantified by using generic hlgG ELISA method for both serum and tumor.

[00465] The experiment demonstrates that FIT013a showed comparable distribution activity in serum and tumor with monoclonal antibodies, see Figure 22.

NCI-H1975-HGF xenograft model efficacy study

[00466] In this study, the efficacy of FIT013a was measured in NCI-H1975-HGF xenograft model.

[00467] NCI-H1975-HGF cells were subcutaneously inoculated to nude BALB/c mice. When the average tumor volume reached to 100-130 mm ³ , and the largest tumor volume was less than 140 mm ³ . The mice were randomly allocated to four groups, FIT013a, H1332, Panitumumab and vehicle group. The antibodies were dosed two times/week i.p. for three weeks. The dosing for FIT013a was 16mg/kg, for H1332 or Panitumumab was lOmg/kg. Vehicle group was dosed with formulation buffer, lOmM sodium citrate, 50mM NaCl, pH 6.0. The tumor volume and mouse body weight was measured twice/week. Percentage tumor growth inhibition (% TGI) was defined as the difference between the control-treated group mean tumor volume (MTV) and the test antibody-treated group MTV.

[00468] The experiment demonstrates that FIT013a showed FIT013a showed better efficacy than EGFR or c-Met monoclonal Ab, see Figure 23.

Example 9; Study of anti-Factor IXa/F actor X Fabs-in- tandem immunoglobulin (FIT- Ϊ21

[00469] FIT-Ig having specificity for Factor IXa and Factor X was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 42. Table 43 provides the expression level in 293E cells and the SEC profile for each of the FIT-Ig.

Table 42. Amino acid sequences of additional exemplary FIT-Ig for cMEt and EGFR

Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

VL-hCk) VTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGV

PDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTF

GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKWACEVTHQGLSSPVTKSFNRGEC

F-X VL 274 DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKP

GQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQP EDIATYYCQQYSDPPLTFGGGT VEIK

F-X VL - CDR1 275 KASRNIERQLA

F-X VL - CDR2 276 QASRKES

F-X VL - CDR3 277 QQYSDPPLT

Table 43. SEC Profile/Expression level in 293E cells:

Functional Study

[00470] Affinity measurement by surface plasmon resonance: The kinetics of FIT-Ig binding to hFactor IX and hFactor X (Enzyme Research Laboratory) was determined by surface plasmon resonance with a Biacore XI 00 instrument (Biacore AB, Uppsala, Sweden) using HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25°C. Briefly, goat anti-human IgG Fc fragment specific polyclonal antibody (Pierce Biotechnology Inc, Rockford, IL) was directly immobilized across a CM5 research grade biosensor chip using a standard amine coupling kit according to manufacturer's instructions. Purified FIT-Ig samples were diluted in HEPES -buffered saline for capture across goat anti- human IgG Fc specific reaction surfaces and injected over reaction matrices at a flow rate of 5 μΐ/min. The association and dissociation rate constants, kon (M-ls-1) and koff (s-1) were determined under a continuous flow rate of 30 μί/ιηίη. Rate constants were derived by making kinetic binding measurements at 500 nM antigen concentrations. The equilibrium dissociation constant (M) of the reaction between FIT-Ig and the target proteins was then calculated from the kinetic rate constants by the following formula: KD = koff/kon. Aliquots of antigen samples were also simultaneously injected over a blank reference and reaction CM surface to record and subtract any nonspecific binding background to eliminate the majority of the refractive index change and injection noise. Surfaces were regenerated with two subsequent 25 ml injections of 10 mM Glycine (pH 1.5) at a flow rate of 10 μί/ιηίη. The anti-Fc antibody immobilized surfaces were completely regenerated and retained their full capture capacity over twelve cycles. Protein based binding data for FIT014a is provided below in Table 44.

Table 44. Functional binding data of FIT014

[00471] Factor Villa-like activity assay: FVIIIa-like activity of the FIT-Ig was evaluated by an enzyme assay according to manufacturer's instructions of BIOPHEN FVIIFC kit(Hyphen-Biomed, 221402-RUO). The result indicates that FIT014a has comparable FVIIIa like activity with Emicizumab and purified FVIIIa, while monoclonal antibody of Factor IX and Factor X, as well as a combination of them, has no activity, see Figure 24.

[00472] Multiple-antigen binding study: this study was done using OctetRed to determine if FIT014a is able to bind hFactor IX and hFactor X simultaneously. Briefly, FIT014a was immobilize on AR2G sensor at concentration of 10 μg/ml, followed by binding of hFactor IX and then hFactor X(Enzyme Research Laboratory) in assay buffer (PBS pH 7.4, 0.1% BSA, 0.02% Tween), with concentration at 500 nM. At the end of the experiment, the surface was regenerated with 10 mM glycine at pH1.5 five times. This experiment shows that FIT014a is able to bind hFactor X when it had already bound to hFactor IX, indicating that FIT014a is able to bind both hFactor IX and hFactor X simultaneously, see Figure 25.

[00473] Stability study: FIT014a protein samples in citrate buffer (pH=6.0) were individually incubated at constant 4 °C, 25 °C and 40 °C for 1 day, 3 days or 7 days. Similarly, FIT014a protein samples were freeze-thawed once, twice or three times. The fractions of intact full monomeric protein of all samples was detected by SEC-HPLC, with 10 μg of each protein sample injected into Utimate 3000 HPLC equipping Superdex200 5/150 GL at flow rate 0.3 mL/min for 15 min, and data was recorded and analyzed using Chromeleon software supplied by the manufacturer. Table 45 shows that FIT014a remained full intact monomeric molecule under these thermo-challenged conditions.

Table 45. Storage stability of FIT014

[00474] Rat PK study: FIT014a was subjected to PK study in rat, and the result is shown in Figure 26 and Table 46. Antibody concentrations in rat serum samples were detected by ELISA with LLOQ of 62.5 ng/mL. On hlgG plate, the coating protein is anti- hlgG Fc, and the detection antibody is anti-hlgG Fab. On Factor X plate, the coating protein is hF actor X, while the detection antibody is anti-human-IgG Fc.

Table 46. Rat PK data for FIT014a

Example 10; Study of anti-HER3/IGF-lR Fabs-in-tandem immunoglobulin (FIT-Ig)

[00475] FIT-Ig having specificity for HER3 and IGF-IR was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 47. Table 48 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.

Table 47. Amino acid sequences of additional exemplary FIT-Ig for HER3 and IGF-IR

Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

FigituVH - CDR1 284 SYAMN

FigituVH - CDR2 285 AISGSGGTTFYADSVKG

FigituVH - CDR3 286 DLGWSDSYYYYYGMDV

Short Chain #1 287 MEFGLSWLFLVAILKGVQCQVQLQQWGAGLLKPSETL (PatritumabVH -CHI) SLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSG

STNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAV YYCARDKWTWYFDLWGRGTLVTVSSASTKGPSVFPLA

PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSC

PatritumabVH 288 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWI

RQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETS KNQFSLKLSSVTAADTAWYCARDKWTWYFDLWGRGT LVTVSS

PatritumabVH - CDR1 289 GYYWS

PatritumabVH - CDR2 290 EINHSGSTNYNPSLKS

PatritumabVH - CDR3 291 DKWTWYFDL

Short Chain #2 (Figitu 292 MDMRVPAQLLGLLLLWFPGSRCDIQMTQFPSSLSASV VL-hCk) GDRVTITCRASQGIR DLGWYQQKPGKAPKRLIYAAS

RLHRGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCL

QHNSYPCSFGQGTKLEIKRTVAAPSVFIFPPSDEQLK

SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Figitu VL 293 DIQMTQFPSSLSASVGDRVTITCRASQGIRNDLGWYQ

QKPGKAPKRLIYAASRLHRGVPSRFSGSGSGTEFTLT ISSLQPEDFATYYCLQHNSYPCSFGQGTKLEIK

Figitu VL - CDR1 294 RASQGIRNDLG

Figitu VL - CDR2 295 AASRLHR

Figitu VL - CDR3 296 LQHNSYPCS

Table 48. SEC Profile/Expression level in 293E cells;

Functional Studies

[00476] Functional binding study: Functional binding data for FIT016a is provided below in Table 49.

Table 49. Functional binding data

ig Target Kon Koff KD IC50

Patritumab 3.17E+05 2.85E-04 9.00E-10

1(HER3)

FIT016a 3.19E+05 3.19E-04 1.00E-09

Figitumumab 2(IGF1R) 1.30E+05 9.48E-05 7.29E-10 FIT016a 3.38E+04 9.19E-05 2.72E-09

[00477] Multiple-antigen binding study: this study was done using OctetRed to determine if FIT016a is able to bind Her3 and IGFIR simultaneously. This experiment shows that FIT016a is able to bind Her3 when it had already bound to IGFIR, indicating that FIT016a is able to bind both Her3 and IGF-1R simultaneously, see Figure 27.

Example 11; Study of anti-DLL4/VEGF Fabs-in- tandem immunoglobulin (FIT-Ig)

[00478] FIT-Ig having specificity for DLL4 and IGFIR was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 50. Table 51 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.

Table 50. Amino acid sequences of additional exemplary FIT-Ig for HER3 and IGF-IR

Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

FIT017a Long Chain (Demci 297 MDMRVPAQLLGLLLLWFPGSRCDIVMTQSPDSLAVSLGE

VL-hCk-Bevci VH- RATISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAAS

DLL4(demcizum hCgl) NQGSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQS ab)/VEGF(bevciz KEVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA umab) SWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK

DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGECEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYG MNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSL DTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFD VWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SWTVPSSSLGTQTYICNVNHKPSNT VDK VEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RWSVLTVLHQDWLNGKEYKC VSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGOTFSCSVMHEALHNHYTQKSLSLSPGK

Demci VL 298 DIVMTQSPDSLAVSLGERATI SCRASESVDNYGI SFMKW

FQQKPGQPPKLLIYAASNQGSGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQSKEVPWTFGGGT VEIK

Demci VL - CDR1 299 RASESVDNYGI SFMK

Demci VL - CDR2 300 AASNQGS

Demci VL - CDR3 301 QQSKEVPWT

Bevci VH 302 EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQ

APGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKST AYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGT LVTVSS

Bevci VH - CDR1 303 NYGMN Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

Bevci VH - CDR2 304 WINTYTGEPTYAADFKR

Bevci VH - CDR3 305 YPHYYGS SHWYFDV

Short Chain #1 306 MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASV I (Demci VH -CHlh) SCKASGYSFTAYYIHWV QAPGQGLEWIGYISSYNGATN

YNQKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCAR DYDYDVGMDYWGQGTLVTVS SASTKGPS VF PLAP S S KS T SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTYICOTNHKPSNTKVDK KVEPKSC

Demci VH 307 QVQLVQSGAEVKKPGASVKI S CKASGYS FTAYYI HWVKQ

APGQGLEWIGYI SSYNGATNYNQKFKGRVTFTTDTSTST AYMELRSLRSDDTAVYY CARD YD YDVGMD YWGQGTLVTV SS

Demci VH - CDR1 308 AYYIH

Demci VH - CDR2 309 YISSYNGATNYNQKFKG

Demci VH - CDR3 310 DYDYDVGMDY

Short Chain #2 311 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGD (Bevci VL-hCk)) RVTITCSASQDISNYLN YQQKPGKAPKVLIYFTSSLHS

GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVP

WTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWC

LLNNFYPREAKVQW VDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKWACEVTHQGLSSPVTKSFNRG EC

Bevci VL 312 D I QMTQS PS SLSASVGDRVTI TCSASQD I SNYLNWYQQK

PGKAP VLIYFTSSLHSGVPSRFSGSGSGTDFTLTI SSL QPEDFATYYCQQYSTVPWTFGQGT VEIK

Bevci VL - CDR1 313 SASQDISNYLN

Bevci VL - CDR2 314 FTSSLHS

Bevci VL - CDR3 315 QQYSTVPWT

Table 51. SEC Profile/Expression level in 293E cells;

Functional Studies

[00479] Functional binding study: Functional binding data for FIT017a is provided below in Table 52.

Table 52. Functional binding data

Target Kon Koff KD IC50 demcizumab

1(DLL4) 1.74E+05 1.28E-04 7.36E-09

FIT017a 1.88E+05 1.28E-04 6.81E-09

bevcizumab 3.46E+05 2.43E-06 7.04E-12

2(VEGF)

FIT017a 3.50E+05 1.28E-05 3.65E-11 [00480] Multiple-antigen binding study: this study was done using OctetRed to determine if FIT017a is able to bind DLL4 and VEGF simultaneously. This experiment shows that FIT017a is able to bind DLL4 when it had already bound to VEGF, indicating that FIT017a is able to bind both DLL4 and VEGF simultaneously, see Figure 28.

Example 12; Study of anti-CD20/CD3 Fabs-in-tandem immunoglobulin (FIT-Ig)

[00481] FIT-Ig having specificity for CD20 and CD3 was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 53. Table 54 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.

Table 53. Amino acid sequences of additional exemplary FIT-Ig for CD20 and CD3

Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

FIT018a Long Chain 316 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPG

(OfatuVL-hCk- ERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNR

CD20(Ofatumum CD3mAb VH- ATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS ab)/CD3 mAb hCglmut) NWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTA (described in SWCLLNNFYPREA VQW VDNALQSGNSQESVTEQDS US2009/0252683 KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT , incorporated by KSFNRGECEVQLLESGGGLVQPGGSLKLSCAASGFTFN reference) TYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD

RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGN SYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT VD KKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT LM I S RT PE VT CVWDVSHED PE VKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

OfatuVL 317 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ

KPGQAPRLLI YDASNRATGI PARFSGSGSGTDFTLTI S SLEPEDFAVYYCQQRSNWPITFGQGTRLEIK

OfatuVL - CDR1 318 RASQSVSSYLA

OfatuVL- CDR2 319 DASNRAT

OfatuVL - CDR3 320 QQRSNWPIT

CD3mAb VH 321 EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVR

QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDD SKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAY WGQGTLVTVSS Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

CD3mAb VH- CDR1 322 TYAMN

CD3mAb VH- CDR2 323 R I R S KYNNYAT Y YAD S VKD

CD3mAb VH- CDR3 324 HGNFGNSYVSWFAY

Short Chain #1 (Ofatu 325 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGRSLR

VH-CH1) LSCAASGFTF DYAMHWVRQAPGKGLEWVSTISWNSGS

IGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYY CAKDIQYGNYYYGMDV GQGTTVTVSSASTKGPSVFPL

APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSC

Ofatu VH 326 EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVR

QAPGKGLEWVSTISWNSGSIGYADSVKGRFTI SRDNAK KSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQ GTTVTVSS

Ofatu VH - CDR1 327 DYAMH

Ofatu VH - CDR2 328 TISWNSGSIGYADSVKG

Ofatu VH - CDR3 329 D I Q YGNYYYGMDV

Short Chain #2 330 MTWTPLLFLTLLLHCTGSLSELWTQEPSLTVSPGGTV

(CD3mAb VL-hCL) TLTCRSSTGAVTTSNYA VQQKPGQAPRGLIGGT R

AP GT PARF S G S L LGGKAALT L S GVQP ED EAE Y YC LWY

SNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANK

ATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQ SNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS

CD3mAb VL 331 ELWTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWV

QQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

CD3mAb VL - CDR1 332 RSSTGAVTTSNYAN

CD3mAb VL - CDR2 333 GTNKRAP

CD3mAb VL - CDR3 334 ALWYSNLWV

Table 54. SEC Profile/Expression level in 293E cells;

Functional Studies

[00482] Functional binding study: Functional binding data for FIT018a is provided below in Table 55.

Table 55. Functional binding data

[00483] Cell based binding study: The binding activity of FIT018a to human or cynomolgus B cell and T cell were determined by flow cytometry using BD FACSVerse. Raji cell was used for detecting human B cell binding. Jurakt cell was used for detecting human T cell binding. Primary cynomolgus T cell was used for detecting cynomolgus T cell binding. HEK 293 cell transit transfected with cynoCD20 was used for detecting cynomolgus B cell binding. Cells were washed in PBS buffer containing 2% FBS. Cells were then aliquot and incubated with 1:5 serially diluted FIT018a on ice for lhr. The starting working concentration of FIT018a was 2C^g/ml. Cells were washed, resuspended and incubated with 1: 100 diluted Alexa Fluor® 488 labeled mouse anti-human IgGl (Invitrogen, Cat.No.A- 10631) on ice protected from light for lhr. Cells were washed and signal was detected with a BD FACSVerse flow cytometer according to manufacture's protocols. These experiments demonstrate that FIT018a can bind to human B cell and T cell (Raji is human B cell line and Jurkat is human T cell line), see Figure 29 A and Figure 29B. FIT018a can also bind to cynomolgus CD20 and CD3, see Figure 30A and Figure 30B.

[00484] B-cell depletion assay: The in vitro activity of FIT018a was measured by B- cell depletion assay. Human PBMCs were isolated by Ficoll Paque Plus (GE HEALTHCARE, cat: GE17144002) according to manufacture's instruction. Target cell Raji was harvested and seeded to assay plate at 5*10 ⁴ per well. Antibodies were serially diluted and added to assay plate. 2.5 x l O ⁵ per well PBMCs were added to assay plate and incubate for 2 days or 3 days. After incubation, B cell was detected by anti-CD 19 antibody using FACS machine. The experiments demonstrate that FIT018a can induce B cell apoptosis, see Figure 31A and Figure 3 IB.

Example 13; Study of anti-HER3/EGFR Fabs-in-tandem immunoglobulin (FIT-Ig)

[00485] FIT-Ig having specificity for HER3 and EGFR was constructed as in the foregoing Examples. This exemplary FIT-Ig and corresponding sequences are provided below in Table 56. Table 57 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.

Table 56 Amino acid sequences of additional exemplary FIT-Ig for HER3 and EGFR Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

Long Chain (patritu 335 MDMRVPAQLLGLLLLWFPGSRCDIEMTQSPDSLAVSLGE

FIT019a -Ig VL-hCk-PaniVH- RATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYW hCgl) ASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQ

HER3(patrituma QYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG b)/EGFR(Panitu TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD mumab) SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT

KSFNRGECQVQLQESGPGLVKPSETLSLTCTVSGGSVSS GDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLT ISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIW GQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNT VDK VEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKC VSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GOTFSCSVMHEALHNHYTQKSLSLSPGK

patritu VL 336 DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYL

AWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFT LTISSLQAEDVAVYYCQQYYSTPRTFGQGT VEIK

patritu VL - CDR1 337 RSSQSVLYSSSNRNYLA

patritu VL- CDR2 338 WASTRES

patritu VL - CDR3 339 QYYSTPRT

PaniVH 340 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWI

RQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKT QFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTV SS

Pani VH - CDR1 341 SGDYYWT

Pani VH - CDR2 342 HIYYSGNTNYNPSLKS

Pani VH - CDR3 343 DRVTGAFDI

Short Chain #1 344 MEFGLSWLFLVAILKGVQCQVQLQQWGAGLLKPSETLSL (Patritumab VH - TCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY CHI) NPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARD

KWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTQTYICOTNHKPSNTKVDKKV EPKSC

Patritumab VH 345 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQ

PPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQF SLKLSSVTAADTAWYCARDKWTWYFDLWGRGTLVTVSS

Patritumab VH - 346 GYYWS

CDR1

Patritumab VH - 347 EINHSGSTNYNPSLKS

CDR2

Patritumab VH - 348 DKWTWYFDL

CDR3

Short Chain #2 349 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGD (Pani VL-hCk) RVTITCQASQDISNYLN YQQKPGKAPKLLIYDASNLET

GVPSPRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHL

PLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASW

CLLNNFYPREA VQW VDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKH VYACEVTHQGLSSPVTKSFNR Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

GEC

Pani VL 350 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQK

PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSL QPEDIATYFCQHFDHLPLAFGGGTKVEIK

Pani VL - CDR1 351 QASQDISNYLN

Pani VL - CDR2 352 DASNLET

Pani VL - CDR3 353 QHFDHLPLA

FIT019b - Ig Long Chain (Panitu 354 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSLSASVGD

VL-hCk-Patritu- RVTITCQASQDISNYLN YQQKPGKAPKLLIYDASNLET

EGFR hCgl) GVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLP

(Panitumumab)/ LAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWC HER3 LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY

(patritumab) SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG

ECQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWI RQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKN QFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK

Panitu VL 355 DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQK

PGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSL QPEDIATYFCQHFDHLPLAFGGGTKVEIK

Panitu VL - CDR1 356 QASQDISNYLN

Panitu VL- CDR2 357 DASNLET

Panitu VL - CDR3 358 QHFDHLPLA

PatrituVH 359 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQ

PPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQF SLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS

Patritu VH - CDR1 360 GYYWS

Patritu VH - CDR2 361 EINHSGSTNYNPSLKS

Patritu VH - CDR3 362 DKWTWYFDL

Short Chain #1 363 MEFGLSWLFLVAILKGVQCQVQLQESGPGLVKPSETLSL (Panitu VH -CHI) TCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNT

NYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYCV RDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSC

Panitu VH 364 QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWI

RQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKT QFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTV SS

Panitu VH - CDR1 365 DYYWT

Panitu VH - CDR2 366 HIYYSGNTNYNPSLKS

Panitu VH - CDR3 367 DRVTGAFDI

Short Chain #2 368 MDMRVPAQLLGLLLLWFPGSRCDIEMTQSPDSLAVSLGE Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

(Patritu VL-hCk) RATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYW

ASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQ

QYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG

TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC

Patritu VL 369 DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYL

AWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFT LTI SSLQAEDVAVYYCQQYYSTPRTFGQGT VEIK

Patritu VL - CDR1 370 RSSQSVLYSSSNRNYLA

Patritu VL - CDR2 371 WASTRES

Patritu VL - CDR3 372 QQYYSTPRT

Table 57. SEC Profile/Expression level in 293E cells:

Functional Studies

[00486] Functional binding study: Functional binding data for FIT019a and FIT019b is provided below in Table 58 and Table 59, respectively.

Table 58. Functional binding data of FIT019a

[00487] Multiple binding study: A multiple binding study of FIT019a and FIT019b was carried out. The result is shown in Figure 32 (FIT019a) and Figure 33A to Figure 33B (FIT019b), respectively. The result indicates that both FIT019a and FIT019b can bind to HER3 and EGFR simultaneously. Example 14; Study of anti-PD-Ll/PD-1 Fabs-in-tandem immunoglobulin (FIT-Ig)

[00488] FIT-Ig having specificity for PD-Ll and PD-1 was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 60. Table 61 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.

Table 60. Amino acid sequences of additional exemplary FIT-Ig for PD-Ll and PD-1

Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

FIT020b Long Chain (1B12 373 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE

VL-hCk-Nivolu RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT

PD-Ll VH-hCglMut) GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP

(1B12)/PD-1 TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCL

(nivolumab) LNNFYPREA VQW VDNALQSGNSQESVTEQDSKDSTYS

LSSTLTLSKADYEKH VYACEVTHQGLSSPVTKS FNRGE CQVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVR QAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKN TLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY I CN VNHKP S NT VDK VE PKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

I1B12 VL 374 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK

PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI SSL EPEDFAVYYCQQRSNWPTFGQGTKVEIK

IB 12 VL - CDRl 375 RASQSVSSYLA

IB 12 VL - CDR2 376 DASNRAT

IB 12 VL - CDR3 377 QQRSNWPT

NivoluVH 378 QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQ

APGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNT LFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

NivoluVH - CDR1 379 NSGMH

NivoluVH - CDR2 380 VI WYDGS KRYYAD SVKG

NivoluVH - CDR3 381 NDDY

Short Chain #1 382 MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGSSVKV (1B12 VH-CH1) SCKTSGDTFSSYAISWVRQAPGQGLEWMGGIIPIFGRAH

YAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCAR KFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPS

SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVE PKSC

1B12 VH 383 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSSYAI SWVRQ Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

APGQGLEWMGG11PIFGRAHYAQKFQGRVTITADESTST AYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGT TVTVSS

IB 12 VH - CDR1 384 SYAIS

IB 12 VH - CDR2 385 GI I PI FGRAHYAQKFQG

IB 12 VH - CDR3 386 KFHFVSGSPFGMDV

Short Chain #2 387 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE (Nivolu VL-hCK) RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT

GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP

RTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWC

LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKWACEVTHQGLSSPVTKSFNRG EC

Nivolu VL 388 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK

PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQSSNWPRTFGQGT VEIK

Nivolu VL - CDR1 389 RASQSVSSYLA

Nivolu VL - CDR2 390 DASNRAT

Nivolu VL - CDR3 391 QQSSNWPRT

Table 61. SEC Profile/Expression level in 293E cells:

Functional Studies

[00489] Affinity measurement by surface plasmon resonance: The kinetics of FIT-Ig binding to rhPD-Ll and rhPD-1 was determined by surface plasmon resonance with a Biacore X100 instrument (Biacore AB, Uppsala, Sweden) using HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25°C. Briefly, for rhPD-Ll and rhPD-1 was directly immobilized at 40 RU across a CM5 research grade biosensor chip using a standard amine coupling kit according to manufacturer's instructions. Rate constants were derived by making kinetic binding measurements at seven different antigen concentrations ranging from 1 to 40 nM. The equilibrium dissociation constant (M) of the reaction between FIT-Ig and the target proteins was then calculated from the kinetic rate constants by the following formula: KD = koff/kon. Aliquots of FIT-Ig/ Ab samples were also simultaneously injected over a blank reference and reaction CM surface to record and subtract any nonspecific binding background to eliminate the majority of the refractive index change and injection noise. Surfaces were regenerated with two subsequent 25 ml injections of 10 mM Glycine (pH 1.5) at a flow rate of 10 μί/ηϋη. Functional binding data for FIT020b is provided below in Table 62 .

Table 62. Functional binding data

[00490] Functional activity test by MLR assay: Mixed lymphocyte reaction was performed using monocyte-derived dendritic cells from one donor and allogeneic CD4 T cells from another donor. The whole blood samples were collected from healthy donors, and PBMC were isolated from whole blood using Ficoll-Pague gradient centrifugation. On day 1, PBMC from one donor was isolated and diluted with serum-free RPMI 1640 at lX10 ⁶ /ml. The diluted PBMC was seeded into 6-well tissue culture plate at 3ml/well and incubated for 3h. Supernatant was removed and unattached cells were washed off. The attached monocyte were polarized into dendritic cells with 250 U/ml IL-4 and 500 U/ml GM-CSF in RPMI1640 with 10 %FBS. The medium was replaced with fresh IL-4 and GM-CSF at day 4. At day 7, immature DC was collected and treated with ^g/ml LPS in RPMI 1640 with 10% FBS for additional 24h for maturation. At Day 8, CD4 T cells were isolated from another donor PBMC by negative selection and adjusted to final concentration at 2X10 ⁶ cells/ml. Mature DC were treated with mitomycin C at 37°C for 1.5 hr. Then DC were washed with PBS and adjusted to final concentration at 1X10 ⁶ cells/ml. CD4 T cells (Responder cells) were added into 96 well plate at ΙΟΟμΙ/well and pre-treated with test antibody at diluted concentration for 30 minutes. Then mature DC (Stimulator cells) were added into the well at ΙΟΟμΙ/well. The final volume of each well is 200μ1. The MLR were incubated at 37 degree for 72 hr for IL-2 test. The result is shown in Figure 34A and Figure 34B.

[00491] Multiple binding study: A multiple binding study of FIT020b was carried out.

The result is shown in Figure 35. The result indicates that FIT020b can bind to PD-L1 and PD-1 simultaneously.

Example 15; Study of anti-CD20/CD-22 Fabs-in-tandem immunoglobulin (FIT-Ig) [00492] FIT-Ig having specificity for CD20 and CD22 was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 63. Table 64 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.

Table 63. Amino acid sequences of additional exemplary FIT-Ig for CD20 and CD22

Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

FIT021b Long Chain (Ofatu 392 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE

VL-hCk-Epratu VH- RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT

CD20 hCgl) GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP

(Ofatumumab) / ITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASWC CD22 LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY

(Epratuzumab) SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG

ECQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWV RQAPGQGLEWIGYINPRNDYTEYNQNFKDKATITADEST NTAYMELSSLRSEDTAFYFCARRDITTFYWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQTYICNWHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPE VKFNWWDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

Ofatu VL 393 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK

PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWPITFGQGTRLEIK

Ofatu VL - CDR1 394 RASQSVSSYLA

Ofatu VL - CDR2 395 DASNRAT

Ofatu VL - CDR3 396 QQRSNWPIT

Epratu VH 397 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQ

APGQGLEWIGYINPRNDYTEYNQNFKDKATITADESTNT AYMELSSLRSEDTAFYFCARRDITTFYWGQGTTVTVSS

Epratu VH - CDR1 398 SYWLH

Epratu VH - CDR2 399 YINPRNDYTEYNQNFKD

Epratu VH - CDR3 400 RDITTFY

Short Chain #1 401 MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGRSLRL (Ofatu VH-CH1) SCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIG

YADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAK DIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSC

Ofatu VH 402 EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQ

APGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKS LYLQMNSLRAEDTALYYCAKDIQYGNYYYGMD GQGTT VTVSS Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

Ofatu VH - CDR1 403 DYAMH

Ofatu VH - CDR2 404 T I S WNSGS I GYADSVKG

Ofatu VH - CDR3 405 D I Q YGNYYYGMDV

Short Chain #2 406 MDMRVPAQLLGLLLLWFPGSRCDIQLTQSPSSLSASVGD (Epratu VL-hCk) RVTMSCKSSQSVLYSANHKNYLAWYQQKPGKAPKLLIYW

ASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCH

QYLSSWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGT

AS WCLLNNFYPREA VQW VDNALQ SGNSQE SVTE QD S KDSTYSLSSTLTLSKADYEKH VYACEVTHQGLSSPVTK SFNRGEC

Epratu VL 407 DIQLTQSPSSLSASVGDRVTMSCKSSQSVLYSANHKNYL

AWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFT FTI SSLQPEDIATYYCHQYLSSWTFGGGTKLEIK

Epratu VL - CDR1 408 KS S QS VL YS ANHKNYLA

Epratu VL - CDR2 409 WASTRES

Epratu VL - CDR3 410 HQYLSSWT

Table 64. SEC Profile/Expression level in 293E cells:

Functional Studies

[00493] Cell based binding study: The binding activity of FIT021b to B lymphoma cell lines Raji or Daudi were determined by flow cytometry using BD FACSVerse. Cells were washed in PBS buffer containing 2% FBS. Cells were then aliquot and incubated with 1:5 serially diluted FIT021b on ice for lhr. The starting working concentration of FIT021b was 2C^g/ml. Cells were washed, resuspended and incubated with 1 : 100 diluted Alexa Fluor® 488 labeled mouse anti-human IgGl (Invitrogen, Cat.No.A-10631) on ice protected from light for lhr. Cells were washed and signal was detected with a BD FACSVerse flow cytometer according to manufacture's protocols. These experiments demonstrate that FIT021b can bind to B lymphoma cell lines Raji or Daudi, see Figure 36A and Figure 36B.

Example 16: Study of anti-HER3/PD-l Fabs-in-tandem immunoglobulin (FIT-Ig)

[00494] FIT-Ig having specificity for HER3 and PD1 was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 65. Table 66 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.

Table 65. Amino acid sequences of additional exemplary FIT-Ig for HER3 and PD-1

Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

FIT022a Long Chain (patritu 411 MDMRVPAQLLGLLLLWFPGSRCDIEMTQSPDSLAVSLGE

VL-hCk-Nivolu RATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYW

HER3 VH-hCglmut) ASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQ

(patritumab) / QYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG PD-1 TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD

(nivolumab) SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT

KSFNRGECQVQLVESGGGWQPGRSLRLDCKASGITFSN SGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTI SRDNSKNTLFLQMNSLRAEDTAVYYCAT DDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

patritu VL 412 DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYL

AWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFT LTISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIK

patritu VL - CDR1 413 RSSQSVLYSSSNRNYLA

patritu VL - CDR2 414 WASTRES

patritu VL - CDR3 415 QQYYSTPRT

Nivolu VH 416 QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQ

APGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNT LFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

Nivolu VH - CDR1 417 NSGMH

Nivolu VH - CDR2 418 VIWYDGSKRYYADSVKG

Nivolu VH - CDR3 419 NDDY

Short Chain #1 420 MEFGLSWLFLVAILKGVQCQVQLQQWGAGLLKPSETLSL (Patritumab -CHI) TCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY

NPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARD KWTWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSC

Patritumab VH 421 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQ

PPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQF SLKLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS

Patritumab VH - 422 GYYWS

CDR1

Patritumab VH - 423 EINHSGSTNYNPSLKS

CDR2 Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

Patritumab VH - 424 DKWTWYFDL

CDR3

Short Chain #2 425 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE (NivoVL-hCK) RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT

GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP

RTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWC

LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKH VYACEVTHQGLSSPVTKSFNRG EC

Nivo VL 426 EIVLTQSPATLSLSPGERATLSCRASQSVSSYL

AWYQQKPGQAPRLLIYDASNRATGIPARFSGSG SGTDFTLTI SSLEPEDFAVYYCQQSSNWPRTFG QGT VEIK

Nivo VL - CDR1 427 RASQSVSSYLA

Nivo VL - CDR2 428 DASNRAT

Nivo VL - CDR3 429 QQSSNWPRT

Table 66. SEC Profile/Expression level in 293E cells:

Functional Studies

[00495] Affinity measurement by surface plasmon resonance: The kinetics of

FIT022a-Ig binding to Her3-his and hPD-l-Fc was determined by surface plasmon resonance. The result is shown in Table 67.

Table 67. Functional binding data for FIT022a

[00496] Multiple binding study: A multiple binding study of FIT022a was carried out.

The result is shown in Figure 37. The result indicates that FIT022a can bind to PD-Ll and PD-1 simultaneously.

[00497] MLR functional assay: Mixed lymphocyte reaction was performed using monocyte-derived dendritic cells from one donor and allogeneic CD4 T cells from another donor. The whole blood samples were collected from healthy donors, and PBMC were isolated from whole blood using Ficoll-Pague gradient centrifugation. On day 1, PBMC from one donor was isolated and diluted with serum-free RPMI 1640 at lXlOVml. The diluted PBMC was seeded into 6-well tissue culture plate at 3ml/well and incubated for 3h. Supernatant was removed and unattached cells were washed off. The attached monocyte were polarized into dendritic cells with 250 U/ml IL-4 and 500 U/ml GM-CSF in RPMI1640 with 10 %FBS. The medium was replaced with fresh IL-4 and GM-CSF at day 4. At day 7, immature DC was collected and treated with ^g/ml LPS in RPMI 1640 with 10% FBS for additional 24h for maturation. At Day 8, CD4 T cells were isolated from another donor PBMC by negative selection and adjusted to final concentration at 2X10e6 cells/ml. Mature DC were treated with mitomycin C at 37 ^° C for 1.5 hr. Then DC were washed with PBS and adjusted to final concentration at 1X10 ⁶ cells/ml. CD4 T cells (Responder cells) were added into 96 well plate at ΙΟΟμΙ/well and pre-treated with test antibody at diluted concentration for 30 minutes. Then mature DC (Stimulator cells) were added into the well at ΙΟΟμΙ/well. The final volume of each well is 200μ1. The MLR were incubated at 37 degree for 72 hr for IL-2 test and 120 hr for IFN-gamma test respectively using ELISA. The result is shown in Figure 38A and Figure 38B

Example 17; Study of anti-cMet/PD-Ll Fabs-in-tandem immunoglobulin (FIT-Ig)

[00498] FIT-Ig having specificity for cMet and PD-L1 was constructed as in the foregoing Examples. This exemplary FIT-Ig and its corresponding sequences are provided below in Table 68. Table 69 provides the expression level in 293E cells and the SEC profile for the FIT-Ig.

Table 68. Amino acid sequences of additional exemplary FIT-Ig for cMet and PD-L1

Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

FIT023a Long Chain (hi 332 430 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGD

VL-hCk-lB12 VH- RVTITCRASQGINTWLAWYQQKPGKAPKLLIYAASSLKS cMet (hl332)/ hCgl) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFP PD-L1 (1B12) LTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWC

LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG ECQVQLVQSGAEV KPGSSVKVSCKTSGDTFSSYAISWV Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

RQAPGQGLEWMGG11PIFGRAHYAQKFQGRVTITADEST STAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDV GQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT VPSSSLGTQTYICNVNHKPSNTKVDK VEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS VLTVLHQDWLNGKEYKC VSNKALPAPIEKTI SKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK

hl332 VL 431 DIQMTQSPSSVSASVGDRVTITCRASQGINTWLAWYQQK

PGKAPKLLIYAASSLKSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQANSFPLTFGGGT VEIK

hl332 VL - CDR1 432 RASQGINTWLA

hl332 VL - CDR2 433 AASSLKS

hl332 VL - CDR3 434 QQANSFPLT

1B12 VH 435 QVQLVQSGAEVKKPGSSV VSCKTSGDTFSSYAISWVRQ

APGQGLEWMGG11PIFGRAHYAQKFQGRVTITADESTST AYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGT TVTVSS

IB 12 VH - CDR1 436 SYAIS

IB 12 VH - CDR2 437 GIIPIFGRAHY

IB 12 VH - CDR3 438 KFHFVSGSPFGMDV

Short Chain #1 439 MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASVKV (hl332 VH-CH1) SCKASGYTFTSYGFSWVRQAPGQGLEWMGWISASNGNTY

YAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR VYADYADYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSWTVPSSSLGTQTYICOTNHKPSNTKVDKKV EPKSC

M332 VH 440 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGFSWVRQ

APGQGLEWMGWISASNGNTYYAQKLQGRVTMTTDTSTST AYMELRSLRSDDTAVYYCARVYADYADYWGQGTLVTVSS

M332 VH - CDR1 441 SYGFS

M332 VH - CDR2 442 WISASNGNTYYAQKLQG

M332 VH - CDR3 443 VYADYADY

Short Chain #2 444 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE (1B12 VL-hCK) RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT

GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP

TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCL

LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C

IB 12 VL 445 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK

PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWPTFGQGTKVEIK

IB 12 VL - CDR1 446 RASQSVSSYLA

IB 12 VL - CDR2 447 DASNRAT Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

IB 12 VL - CDR3 448 QQRSNWPT

Table 69. SEC Profile/Expression level in 293E cells:

Functional Studies

[00499] Affinity measurement by surface plasmon resonance: The kinetics

FIT023a-Ig binding to cMet and PD-Ll was determined by surface plasmon resonance. ^' result is shown in Table 70.

Table 70. Functional binding data for FIT023a

[00500] Multiple binding study: A multiple binding study of FIT023a was carried out.

The result is shown in Figure 39A and Figure 39B. The result indicates that FIT023a can bind to cMet and PD-Ll simultaneously.

Example 18: Study of anti-BTLA/PD- 1 Fabs-in-tandem immunoglobulin (FIT-Ig)

[00501] FIT-Ig having specificity for BTLA and PD-Ll were constructed as in the foregoing Examples. This exemplary FIT-Ig and their corresponding sequences are provided below in Table 71. Table 72 provides the expression level in 293E cells and the SEC profile for the FIT-Ig. Both FIT024a and FIT024b have good purity after one-step Protein A purification.

Table 71. Amino acid sequences of additional exemplary FIT-Ig for BTLA and PD-1 Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

FIT024a - Ig Long Chain (6A5 449 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGE

VL-hCk-Nivolu RATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRA

BTLA (6A5)/ VH-hCglmut) TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSS PD-1 PPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASV

(Nivolumab) VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS

TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGECQVQLVESGGGWQPGRSLRLDCKASGITFSNSGMH WVRQAPGKGLEWVAVIWYDGSKRYYADSV GRFTISRDN SKNTLFLQMNSLRAEDTAVYYCAT DDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICOTNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA AGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD WLNGKEYKC VSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK

6A5 VL 450 EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQ

KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR LEPEDFAVYYCQQYGSSPPITFGQGTRLEIK

6A5 VL - CDR1 451 RASQSVSSTYLA

6A5 VL - CDR2 452 GASSRAT

6A5 VL - CDR3 453 QQYGSSPPIT

Nivolu VH 454 QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQ

APGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNT LFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

Nivolu VH - CDR1 455 NSGMH

Nivolu VH - CDR2 456 VIWYDGSKRYYADSVKG

Nivolu VH - CDR3 457 NDDY

Short Chain #1 (6A5 458 MEFGLSWLFLVAILKGVQCQITLKESGPTLVKPTQTLTL VH-CH1) TCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIYWDDDK

RYSPSLKSRLTITKDTSKNQWLTMA MDPVDTATYYCA HIRITEVRGVI ISYYGMDVWGQGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSC

6A5 VH 459 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWI

RQPPGKALEWLALIYWDDDKRYSPSLKSRLTITKDTSKN QWLTMANMDPVDTATYYCAHIRITEVRGVI I SYYGMDV WGQGTTVTVSS

6A5 VH - CDR1 460 TSGVGVG

6A5 VH - CDR2 461 LIYWDDDKRYSPSLKS

6A5 VH - CDR3 462 IRITEVRGVI I SYYGMDV

Short Chain #2 463 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE (Nivolu VL-hCK) RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT

GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP

RTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWC

LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

Nivolu VL 464 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQSSNWPRTFGQGTKVEIK

Nivolu VL - CDR1 465 RASQSVSSYLA

Nivolu VL - CDR2 466 DASNRAT

Nivolu VL - CDR3 467 QQSSNWPRT

FIT024b - Ig Long Chain (Nivolu 468 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPATLSLSPGE

VL-hCk-6A5 VH- RATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRAT PD-1 hCglmut) GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWP

(Nivolumab)/ RTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWC

LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY BTLA (6A5) SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG

ECQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVG WIRQPPGKALEWLALIYWDDDKRYSPSLKSRLTITKDTS KNQWLTMANMDPVDTATYYCAHIRITEVRGVIISYYGM DVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQWTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Nivolu VL 469 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK

PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQSSNWPRTFGQGTKVEIK

Nivolu VL - CDR1 470 RASQSVSSYLA

Nivolu VL - CDR2 471 DASNRAT

Nivolu VL - CDR3 472 QQSSNWPRT

6A5 VH 473 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWI

RQPPGKALEWLALIYWDDDKRYSPSLKSRLTITKDTSKN QWLTMANMDPVDTATYYCAHIRITEVRGVI I SYYGMDV WGQGTTVTVSS

6A5 VH - CDR1 474 TSGVGVG

6A5 VH - CDR2 475 LIYWDDDKRYSPSLKS

6A5 VH - CDR3 476 IRITEVRGVI I SYYGMDV

Short Chain #1 477 MEFGLSWLFLVAILKGVQCQVQLVESGGGWQPGRSLRL (Nivolu VH-CH1) DCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRY

YADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAT NDDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C

Nivolu VH 478 QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQ

APGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNT LFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS

Nivolu VH - CDR1 479 NSGMH

Nivolu VH - CDR2 480 VIWYDGSKRYYADSVKG

Nivolu VH - CDR3 481 NDDY

Short Chain #2 (6A5 482 MDMRVPAQLLGLLLLWFPGSRCEIVLTQSPGTLSLSPGE VL-hCK) RATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRA

TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSS Name Protein region SEQ Sequences

Target (mAb) ID

NO

mAbl (upper

domain) / mAb2

(lower domain)

PPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASV

VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

6A5 VL 483 EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQ

KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISR LEPEDFAVYYCQQYGSSPPITFGQGTRLEIK

6A5 VL - CDR1 484 RASQSVSSTYLA

6A5 VL - CDR2 485 GASSRAT

6A5 VL - CDR3 486 QQYGSSPPIT

Table 72. SEC Profile/Expression level in 293E cells:

Functional Studies

[00502] Affinity measurement by surface plasmon resonance: The kinetics of

FIT024a-Ig and FIT024b-Ig binding to BTLA4 and PD-1 was determined by surface plasmon resonance. The result is shown in Table 73.

Table 73. Functional binding data for FIT024a and FIT024b

[00503] Multiple binding study: A multiple binding study of FIT024a and FIT024b was carried out. The result is shown in Figure 40A to Figure 40B and Figure 41A to Figure 41B. The result indicates that both FIT024a and FIT024b can bind to BTLA4 and PD-1 simultaneously.

[00504] MLR functional assay: Mixed lymphocyte reaction was performed using monocyte-derived dendritic cells from one donor and allogeneic CD4 T cells from another donor. The whole blood samples were collected from healthy donors, and PBMC were isolated from whole blood using Ficoll-Pague gradient centrifugation. On day 1, PBMC from one donor was isolated and diluted with serum-free RPMI 1640 at lXlOVml. The diluted PBMC was seeded into 6-well tissue culture plate at 3ml/well and incubated for 3h. Supernatant was removed and unattached cells were washed off. The attached monocyte were polarized into dendritic cells with 250 U/ml IL-4 and 500 U/ml GM-CSF in RPMI1640 with 10 %FBS. The medium was replaced with fresh IL-4 and GM-CSF at day 4. At day 7, immature DC was collected and treated with ^g/ml LPS in RPMI 1640 with 10% FBS for additional 24h for maturation. At Day 8, CD4 T cells were isolated from another donor PBMC by negative selection and adjusted to final concentration at 2X10e6 cells/ml. Mature DC were treated with mitomycin C at 37 ^° C for 1.5 hr. Then DC were washed with PBS and adjusted to final concentration at 1X10 ⁶ cells/ml. CD4 T cells (Responder cells) were added into 96 well plate at ΙΟΟμΙ/well and pre-treated with test antibody at diluted concentration for 30 minutes. Then mature DC (Stimulator cells) were added into the well at ΙΟΟμΙ/well. The final volume of each well is 200μ1. The MLR were incubated at 37 degree for 72 hr for IL-2 test. The result is shown in Figure 42A and Figure 42B. Both FIT024a and FIT024b have higher activity to enhance T cell activation in MLR study compared to Nivolumab.

[00505] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference in its entirety.

[00506] Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[00507] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[00508] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.