ANTIBODIES

STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0001] This invention was made with government support under grant nos. R01 CA118919, R01 CA129491, R01 CA171315, and R01 CA223767 awarded by The National Institutes of Health. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62/897,598, filed on September 9, 2019. The disclosure of the above-referenced application is herein expressly incorporated by reference it its entirety, including any drawings.

INCORPORATION OF THE SEQUENCE LISTING

[0003] The material in the accompanying Sequence Listing is hereby incorporated by reference into this application. The accompanying Sequence Listing text file, named “048536- 647001WO_Sequence Listing.txt,” was created on September 7, 2020 and is 182 KB.

FIELD

[0004] The present disclosure relates generally to the field of antibody engineering and particularly relates to multispecific antibodies that specifically bind to two or more different target antigens or epitopes. The disclosure also provides compositions and methods useful for producing such multispecific antibodies and pharmaceutical compositions containing the same.

BACKGROUND

[0005] Antibody constructs having more than one binding specificities have been reported to have several advantages compared to antibodies and antibody fragments having only one single binding site, such as an improved potency, multispecificity, multifunctionality. Compared to traditional monospecific antibodies that specifically recognize one ligand, multispecific antibodies can recognize two or more ligands and thus may provide an advantage in co -engaging different cell types, creating synthetic specificity, altering internalization dynamics, synergistically neutralizing virus and toxin, and simultaneously block multiple signaling pathways to maximize therapeutic benefits.

[0006] However, the production and use of multispecific antibodies has been hindered by the difficulty of obtaining multispecific antibodies in sufficient quantity and purity for both preclinical and clinical studies. A common problem in multispecific antibody generation is how to properly pair heavy and light chains from different specificities. For example, traditional production of full-length bispecific antibodies is typically based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities. The intrinsic tendency of the Fc portion of the antibody molecule to homodimerize leads to the formation of complex mixtures of up to 10 different IgG molecules consisting of various combinations of heavy and light chains, of which only one has the correct bispecific structure. In addition, purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Thus, the production of a bispecific antibody molecule with the two Fab arms engineered to bind two different targets using traditional hybridoma techniques is challenging.

[0007] Another traditional method for multispecific antibody production is chemical conjugation of two antibodies or their fragments having different specificities. However, this method has been reported to be complex, and the chemical modification process may inactivate the antibody or promote aggregation. In addition, in the manufacture of multispecific antibodies, it is desirable to increase the yields of the desired multispecific antibodies. Because purification from undesired products remains difficult, the resulting low yield and poor quality of multispecific antibody make this process unsuitable for the large scale production required for clinical development. In addition, these molecules may not maintain the traditional antibody conformation.

[0008] In view of different problems and aspects of multispecific antibodies such as, e.g. pharmacokinetic and biological properties, stability, aggregation, expression yield, there remains a need in the art for alternative multispecific variant antibodies which have been modified to select for heterodimers with an increased stability and purity. SUMMARY

[0009] Provided herein, inter alia , are compositions and methods concerning the production of engineered antibodies having (i) multiple antigen-binding specificities and (ii) Fc regions that have been modified to promote heterodimer formation between heavy chains from two different specificities. More particularly, provided herein are engineered antibodies having (i) two singe-chain antigen binding fragments specific for two different epitopes and (ii) Fc regions associated with one another via an interface which has been modified to promote heterodimer formation. Also provided are recombinant nucleic acids encoding at least one single-chain polypeptide chain of such engineered antibodies, recombinant cells including at least one recombinant nucleic acid as disclosed herein, as well as pharmaceutical compositions containing same.

[0010] In one aspect, provided herein are various engineered antibodies including a first and a second polypeptide chain, each of the first and second polypeptide chains including: (a) a single-chain antigen-binding (scFab) fragment which includes, in N-terminal to C-terminal direction, (i) a light chain variable domain (VL); (ii) a light chain constant domain (CL); (iii) a removable linker; (iv) a heavy chain variable domain (VH); and (v)a heavy chain constant domain CHI, wherein the scFab fragments of the first and second polypeptide chains have specificity for different antigens; and (b) an antibody Fc region N-terminally linked to the scFab fragment in (a), wherein the Fc regions of the first and a second polypeptide chains are associated with one another via an interface which has been modified to promote heterodimer formation.

[0011] In another aspect, provided herein are various engineered antibodies including a first and a second polypeptide chain, each of the first and second polypeptide chains including: (a) a single-chain antigen-binding (scFab) fragment which includes, in N-terminal to C-terminal direction, (i) a light chain variable domain (VL); (ii) a light chain constant domain (CL); (iii) a removable linker; (iv) a heavy chain variable domain (VH); and (v)a heavy chain constant domain CHI, wherein the scFab fragments of the first and second polypeptide chains have specificity for different antigens, and optionally wherein the N-terminus of the first polypeptide chain and/or the second polypeptide chain is operably linked to one or more additional scFab fragments having specificity for further antigens; and (b) an antibody Fc region N-terminally linked to the scFab fragment in (a), wherein the Fc regions of the first and a second polypeptide chains are associated with one another via an interface which has been modified to promote heterodimer formation.

[0012] In another aspect, provided herein are various engineered antibodies including a first and a second polypeptide chain, each of the first and second polypeptide chains including: (a) a single-chain antigen-binding (scFab) fragment which includes, in N-terminal to C-terminal direction, (i) a light chain variable domain (VL); (ii) a light chain constant domain (CL); (iii) a removable linker; (iv) a heavy chain variable domain (VH); and (v)a heavy chain constant domain CHI, wherein the scFab fragments of the first and second polypeptide chains have specificity for different antigens, and optionally wherein the C-terminus of the first polypeptide chain and/or the second polypeptide chain is operably linked to one or more additional scFab fragments having specificity for further antigens; and (b) an antibody Fc region N-terminally linked to the scFab fragment in (a), wherein the Fc regions of the first and a second polypeptide chains are associated with one another via an interface which has been modified to promote heterodimer formation.

[0013] Non-limiting exemplary embodiments of the disclosed engineered antibodies of the disclosure include one or more of the following features. In some embodiments, each additional scFab fragment including, in N-terminal to C-terminal direction, a VL domain, a CL domain, a removable linker, a VH domain, and a CHI domain. In some embodiments, the removable linker includes one or more proteolytic cleavage sites. In some embodiments, the one or more proteolytic cleavage sites are positioned within the sequence of the removable linker and/or flanking at either end of the removable linker. In some embodiments, the one or more proteolytic cleavage sites can be cleaved by a protease or an endopeptidase. In some embodiments, at least one of the one or more proteolytic cleavage sites can be cleaved by a protease selected from the group consisting of thrombin, PreScission™ protease, and tobacco etch virus (TEV) protease. In some embodiments, the protease is thrombin. In some embodiments, the removable linker includes the polypeptide sequence of SEQ ID NO: 80. In some embodiments, at least one of the one or more proteolytic cleavage sites can be cleaved by an endopeptidase selected from the group consisting of trypsin, chymotrypsin, elastase, thermolysin, pepsin, glutamyl endopeptidase, or neprilysin. [0014] In some embodiments, the removable linker further includes one or more affinity tags. In some embodiments, the one or more affinity tags is selected from the group consisting of polyhistidine (poly-His) tags, Hemagglutinin (HA) tags, AviTag™, protein C tags, FLAG tags, Strep-tag® II, and Twin-Strep-tag®, glutathione -S-transferase (GST), C-myc tag, chitin-binding domain, Streptavidin binding proteins (SBP), maltose binding protein (MBP), cellulose-binding domains, calmodulin-binding peptides, and S-tags. In some embodiments, at least one of the one or more affinity tags includes a poly-His tag or a Twin-Strep-tag®. In some embodiments, the removable linkers of the scFab fragments of the first and second polypeptide chains includes the same affinity tags. In some embodiments, the removable linkers of the scFab fragments of the first and second polypeptide chains includes different affinity tags. In some embodiments, the removable linker further comprises one or more polypeptide dimerization motifs selected from the group consisting of homodimerization motifs, heterodimerization motifs, leucine zipper motifs, and combinations of any thereof.

[0015] In some embodiments, the Fc regions of the first and the second polypeptide chains are associated with one another via a modified interface within a constant domain of the Fc regions. In some embodiments, the constant domain is a CH2 domain or a CH3 domain. In some embodiments, the modified interface of the first polypeptide chain includes a protuberance which is positionable in a cavity in the modified interface of the second polypeptide chain. In some embodiments, the amino acid sequence of an original interface has been modified so as to introduce the protuberance and/or cavity into the modified interface such that a greater ratio of heterodimenhomodimer forms than that for a dimer having a non-modified interface. In some embodiments, the cavity includes an amino acid residue substituted into the interface of the second polypeptide, and wherein the substituted amino acid residue is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). In some embodiments, the protuberance includes an amino acid residue substituted into the interface of the first polypeptide, and wherein the substituted amino acid residue is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some embodiments, the amino acid residue is substituted into the interface of the first polypeptide at position 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the CH3 domain of human IgGi. In some embodiments, the protuberance includes a T366W amino acid substitution within the constant domain CH3 of the Fc region of the first polypeptide. In some embodiments, the cavity includes an amino acid substitution selected from the group consisting of S354C, T366S, L368A, and Y407V present within the constant domain CH3 of the Fc region of the second polypeptide.

[0016] In some embodiments, at least one the antigens is a cell-surface antigen. In some embodiments, the antigens are selected from the group consisting of CD3, CD4, CD8, CD25, CD28, CD27, T-cell receptors, CD 16 A, CD38, CD46, CD47, CD56, CD14, CD16b, CD71, CD79, CD68, CCR5, CCL2, SLAM, NKG2D, NKG2A, NKp46, killer-cell immunoglobulin-like receptors (KIRs), CD98, beta 2 microglobulin, CD20, CD22, CD30, CD33, CD 123, CD 137, CD133, BCMA, CD19, CDla-c, prostate-specific membrane antigen (PSMA), B7-H3 (CD276), mesothelin, prostate stem cell antigen (PSCA), CEA, CLEC12A, ALPPL2, ALPP, ALPI, GD2, TAG-72, EpCAM, GPC3, GPA33, GPRC5D, Her2, SSTR2 (somatostatin receptor 2), Mucl6, Mucl, FLT3, Mucl8, MELAN-A, DLL3, CD307, EGFRvIII, EGFR, P-cadherin, N-cadherin, ICAM-1, VLA-4, YCAM, a4/b7 integrin, an/b8 intergrins, an/b3 integrins, CD44 and CD44 splicing variants, glycoprotein llb/llla, LFA-1, CD40, 0X40, GITR, 4 IBB, c-Met, inducible T- cell costimulator (ICOS), leucine rich repeat-containing G protein-coupled receptor 5 (LGR5), VEGF, CD80, CD86, CD55, CD59, members of ErbB family, members of insulin receptor family, members of PDGF receptor family, members of VEGF receptors family, members of FGF receptor family, members of CCK receptor family, members of NGF receptor family, members of HGF receptor family, members of Eph receptor family, members of AXL receptor family, members of DDR receptor family, members of RET receptor family, members of ROS receptor family, members of LTK receptor family, members of ROR receptor family, G protein- coupled receptors (GPCRs), PD-1, PD-L1, PD-L2, CTLA-4 (CD 152), B7-H3 (CD276), B7-H4 (VTCN1), LAG3, TIM-3, VISTA, SIGLEC7 (CD328), SIGLEC9 (CD329), BTLA (CD272), A2AR, IDO (indoleamine 2,3 -dioxygenase), TΰRbϊa, TϋRbϋII, and TGFbR3.

[0017] In some embodiments, the scFab fragment of the first and/or second polypeptide chains includes the VL, CL, VH, and CHI domains derived from abciximab, abciximab, adalimumab, aducanumab, alacizumab, alemtuzumab, alirocumab, alirocumab, ascrinvacumab, atezolizumab, atinumab, bapineuzumab, basiliximab, basiliximab, belimumab, bevacizumab, blinatumomab, blosozumab, bococizumab, brentuximab, canakinumab, caplacizumab, capromab, certolizumab, cetuximab, crenezumab, daclizumab, daratumumab, demcizumab, denosumab, denosumab, dinutuximab, ecukinumab, eculizumab, eculizumab, efalizumab, elotuzumab, enoticumab, etaracizumab, evinacumab, evolocumab, evolocumab, fasinumab, fulranumab, gantenerumab, golimumab, ibritumomab, icrucumab, idarucizumab, idarucizumab, inciacumab, infliximab, ipilimumab, mepolizumab, natalizumab, necitumumab, nesvacumab, nivolumab, obinutuzumab, ofatumumab, omalizumab, opicinumab, orticumab, ozanezumab, palivizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, ponezumab, ralpancizumab, ramucirumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, rinucumab, rituximab, romosozumab, siltuximab, solanezumab, stamulumab, tadocizumab, tanezumab, tocilizumab, trastuzumab, ustekinumab, vedolizumab, or vesencumab . In some embodiments, the scFab fragment of the first polypeptide chain is derived from ipilimumab and the scFab fragment of the second polypeptide chain is derived from daratumumab. In some embodiments, the scFab fragment of the first polypeptide chain is derived from ipilimumab and the scFab fragment of the second polypeptide chain is derived from trastuzumab.

[0018] In some embodiments, the one or more additional scFab fragments are operably linked to the first polypeptide chain and/or the second polypeptide chain by a connector. In some embodiments, the connector is a peptide connector. In some embodiments, the additional antigens are selected from the group consisting of CD3, CD4, CD8, CD25, CD28, CD27, T-cell receptors, CD 16 A, CD38, CD46, CD47, CD56, CD14, CD16b, CD71, CD79, CD68, CCR5, CCL2, SLAM, NKG2D, NKG2A, NKp46, killer-cell immunoglobulin-like receptors (KIRs), CD98, beta 2 microglobulin, CD20, CD22, CD30, CD33, CD 123, CD 137, CD 133, BCMA, CD19, CDla-c, prostate-specific membrane antigen (PSMA), B7-H3 (CD276), mesothelin, prostate stem cell antigen (PSCA), CEA, CLEC12A, ALPPL2, ALPP, ALPI, GD2, TAG-72, EpCAM, GPC3, GPA33, GPRC5D, Her2, SSTR2 (somatostatin receptor 2), Mucl6, Mucl, FLT3, Mucl 8, MELAN-A, DLL3, CD307, EGFRvIII, EGFR, P-cadherin, N-cadherin, ICAM-1, VLA-4, YCAM, a4/b7 integrin, an/b8 intergrins, an/b3 integrins, CD44 and CD44 splicing variants, glycoprotein llb/llla, LFA-1, CD40, 0X40, GITR, 4 IBB, c-Met, inducible T-cell costimulator (ICOS), leucine rich repeat-containing G protein-coupled receptor 5 (LGR5), VEGF, CD80, CD86, CD55, CD59, members of ErbB family, members of insulin receptor family, members of PDGF receptor family, members of VEGF receptors family, members of FGF receptor family, members of CCK receptor family, members of NGF receptor family, members of HGF receptor family, members of Eph receptor family, members of AXL receptor family, members of DDR receptor family, members of RET receptor family, members of ROS receptor family, members of LTK receptor family, members of ROR receptor family, G protein- coupled receptors (GPCRs), PD-1, PD-L1, PD-L2, CTLA-4 (CD 152), B7-H3 (CD276), B7-H4 (VTCN1), LAG3, TIM-3, VISTA, SIGLEC7 (CD328), SIGLEC9 (CD329), BTLA (CD272), A2AR, IDO (indoleamine 2,3 -dioxygenase), TGFpRI, TGFpRII, and TGF[SR3.

[0019] In some embodiments, the one or more additional scFab fragment includes the VL, CL, Vn, and CHI domains derived from abciximab, abciximab, adalimumab, aducanumab, alacizumab, alemtuzumab, alirocumab, alirocumab, ascrinvacumab, atezolizumab, atinumab, bapineuzumab, basiliximab, basiliximab, belimumab, bevacizumab, blinatumomab, blosozumab, bococizumab, brentuximab, canakinumab, caplacizumab, capromab, certolizumab, cetuximab, crenezumab, daclizumab, daratumumab, demcizumab, denosumab, denosumab, dinutuximab, ecukinumab, eculizumab, eculizumab, efalizumab, elotuzumab, enoticumab, etaracizumab, evinacumab, evolocumab, evolocumab, fasinumab, fulranumab, gantenerumab, golimumab, ibritumomab, icrucumab, idarucizumab, idarucizumab, inciacumab, infliximab, ipilimumab, mepolizumab, natalizumab, necitumumab, nesvacumab, nivolumab, obinutuzumab, ofatumumab, omalizumab, opicinumab, orticumab, ozanezumab, palivizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, ponezumab, ralpancizumab, ramucirumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, rinucumab, rituximab, romosozumab, siltuximab, solanezumab, stamulumab, tadocizumab, tanezumab, tocilizumab, trastuzumab, ustekinumab, vedolizumab, or vesencumab. In some embodiments, at least one of the one or more additional scFab fragments includes the VL, CL, VH, and CHI domains derived from atezolizumab. In some embodiments, at least one of the first and second polypeptide chains comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to any one of SEQ ID NOS: 1-12.

[0020] In another aspect, provided herein are various recombinant nucleic acids including a nucleotide sequence that encodes (a) the first polypeptide chain of an engineered antibody as disclosed herein, or a scFab fragment thereof; (b) the second polypeptide chain of an engineered antibody as disclosed herein, or a scFab fragment thereof; or (c) both (a) and (b) above. In some embodiments, the nucleic acid sequence is incorporated into an expression cassette or a vector.

[0021] In another aspect, provided herein are various recombinant cells including the first polypeptide chain of an engineered antibody as disclosed herein, or a scFab fragment thereof; (b) the second polypeptide chain of an engineered antibody as disclosed herein, or a scFab fragment thereof; (c) both (a) and (b) above; (d) an engineered antibody as disclosed herein; and/or (e) a recombinant nucleic acid as disclosed herein. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is Human Embryonic Kidney 293 A (HEK293A) cell, a HEK293 cell, a HEK293T cell, a HEK293F cell, a Chinese Hamster Ovary (CHO) cell, a CHO K1 cells, or a CHO-S cell.

[0022] In yet another aspect, provided herein are various methods for preparing an engineered antibody, including: (a) providing an engineered antibody as disclosed herein; and (b) removing the removable linker to produce an antibody that does not contain the removable linker.

[0023] Non-limiting exemplary embodiments of the disclosed methods for preparing an engineered antibody include one or more of the following features. In some embodiments, the providing the engineered antibody includes co-expressing the first and the second polypeptide chains in the same recombinant cell. In some embodiments, the providing the engineered antibody includes culturing a host cell that co-expresses the first and the second polypeptide chains. In some embodiments, the methods further include a process of purifying the engineered antibody prior to and/or after the removal of the linker(s). In some embodiments, the purifying process includes one or more techniques selected from the group consisting of affinity chromatography, ion-exchange chromatography (IEC), anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydroxyapatite chromatography, hydrophobic interaction chromatography (HIC), size-exclusion chromatography (SEC), metal affinity chromatography, and mixed mode chromatography (MMC). In some embodiments, the purifying process includes affinity chromatography. In some embodiments, the affinity chromatography includes protein A affinity chromatography. In some embodiments, the purifying process includes ion-exchange chromatography (IEC). In some embodiments, the produced antibody includes the engineered antibody lacking the removable linker with a purity of greater than 70%, 80%, 90%, or 95%. [0024] In yet another aspect, provided herein are various engineered antibodies produced by a method disclosed herein. In a related aspect, provided herein are various pharmaceutical compositions, including an engineered antibody as disclosed herein, and a pharmaceutically acceptable carrier.

[0025] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0026] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGS. 1A-1D graphically summarize the results of experiments performed to illustrate that monoclonal antibodies processed from single-chain IgGs show similar biophysical and binding properties as the original antibodies. FIG. 1A shows an exemplary scheme to produce monoclonal IgG from single-chain IgG with a thrombin cleavable linker. FIG. IB: After thrombin cleavage, the IgG molecules produced with the thrombin-cleavable linker (Ipili- sc36TMB and Dara-sc36TMB) show molecular weights of approximately 150 kDa and can be separated into heavy chains (about 50 kDa) and light chains (about 25 kDa) with a reducing agent, b-mercapto ethanol (b-ME), a virtually identical pattern compared with the original monoclonal antibody. FIGS. 1C-1D: Ipili-sc36TMB (FIG. 1C) and Dara-sc36TMB (FIG. ID) bind to their respective ligands in ELISA assays, showing similar binding profiles compared with the original monoclonal antibodies.

[0028] FIGS. 2A-2L graphically summarize the results of experiments performed to illustrate the production of bispecific antibodies with thrombin-cleavable linkers and “knobs- into-holes” (KIH) Fc domains. FIG. 2A shows an exemplary scheme to produce bispecific antibodies. FIG. 2B: Two bispecific antibodies, Ipili-Dara-KIH and Ipili-Her-KIH, were generated, both of which show a predominant component with a molecular weight of 150 kDa. A small fraction of contamination, likely caused by incorrectly paired homodimers, can be observed at the half size of an antibody (labeled with *). Under reducing condition, separated heavy and light chains, 50 kDa and 25 kDa respectively, were observed. FIGS. 2C-2D: Bispecific antibody Ipili-Dara-KIH binding to CTLA4-Fc (FIG. 2C) and CD38 (FIG. 2D), in comparison to the original monoclonal antibodies. FIGS. 2E-2F: Bispecific antibody Ipili-Her- KIH binding to CTLA4-Fc (FIG. 2E) and ErbB2-Fc (FIG. 2F), in comparison to the original monoclonal antibodies. FIGS: 2G-2L summarize the results of a purity assessment of the bi specific antibodies Ipili-Dara-KIH and Ipili-Her-KIH by analytical hydrophobic interaction chromatography. The main peak of Ipili Dara KIH (FIG. 2G) shows an elution time in-between of Ipilimumab (FIG. 2H) and Daratumumab (FIG. 21), representing the desired bispecific product that is estimated by area integration (OpenLab CDS, Agilent) to be 71% of total protein obtained from one-step protein A purification. For the Ipili-Her-KIH antibody, the main peak of Ipili Her KIH (FIG. 2J) shows an elution time in-between of Ipilimumab (FIG. 2K) and Herceptin (FIG. 2L), representing the desired bispecific product that is estimated to be 73% of total protein obtained from one-step protein A purification.

[0029] FIGS. 3A-3E graphically summarize the results of experiments performed to illustrate the production of bispecific antibody with high purity using a pair of thrombin- cleavable dual-tagged affinity linkers. FIG. 3A shows an exemplary scheme to produce bispecific antibodies with built-in affinity tags, e.g ., 10-His tag and Twin-Strep-Tag® that enables sequential purification of the desired heterodimer by Ni-NTA resin and Strep- Tactin®XT resin. As shown in FIG. 3B, the bispecific product of Ipili-Dara-KIH purified via the dual tags (Ni-NTA and Strep-Tactin®XT) was compared with the same product purified by protein A agarose. The contamination in the bispecific products that could be observed at the half-antibody size (the * band) was eliminated when purified with dual tags. In FIGS. 3C-3D, the purity of bispecific products was assessed by analytical hydrophobic interaction chromatography (HIC). As shown in FIG. 3E, flow cytometry analysis of Jurkat-cell binding by each of the antibodies Ipili-Dara-KIH, Daratumumab, and Ipilimumab at various concentrations (0-200 nM). FIG. 3F shows flow cytometry analysis of MCF7-cell binding by each of the antibodies Ipili-Her-KIH, Herceptin, and Ipilimumab at various concentrations (0-200 nM).

[0030] FIGS. 4A-4E graphically summarize the results of experiments illustrating the design of Tri-N-Fabs as a new format for tri-specific antibodies produced with thrombin- removable linkers and KIH Fc heterodimers. An exemplary production scheme and final structure of the Tri-N-Fabs is presented in FIG. 4A. The extra Fab domain (III) is appended to the N-terminus of the bispecific antibody. For the Tri-N-Fabs produced, Fabs of Ipilimumab were placed at position I, Daratumumab at position II, and Herceptin at position III. FIG. 4B pictorially summarizes the results of an SDS-PAGE analysis where the Tri-N-Fabs showed an approximate molecular weight of 200 kDa and can be separated into polypeptide chains of 50 kDa and 25 kDa under reducing conditions. Small fractions of contamination were observed, marked as (*) and (†), likely caused by homodimers. As shown in FIG. 4C, the purity of the Tri- N-Fabs is assessed by analytical hydrophobic interaction chromatography. As shown in FIGS. 4D, 4E, and 4F, Tri-N-Fabs interacts with the three intended ligands in an ELISA assay. Parental antibodies were included as references.

[0031] FIGS. 5A-5G graphically summarize the results of experiments performed to illustrate the production of tetra-N-Fabs, a new format for tetra-specific antibodies, with thrombin-removable linkers and KIH Fc heterodimers. FIG. 5 A shows an exemplary design of Tetra-N-Fabs. Additional Fab domains (III and IV) were appended to the N-terminus of a bispecific antibody described above. For the Tetra-N-Fabs produced, Fabs of Ipilimumab were placed at position I, Daratumumab at position II, Herceptin at position III, and Atezolizumab at position IV. As shown in FIG. 5B, the Tetra-N-Fabs displayed a molecular weight of 250 kDa by non-reducing SDS-PAGE analysis and could be separated into two polypeptide chains of 50 kDa and 25 kDa under reducing conditions. As shown in FIG. 5A, the purity of the Tetra-N- Fabs was estimated to be 95% by analytical HIC. FIGS. 5D, 5E, 5F, and 5G show the binding of the Tetra-N-Fabs to all four ligands with parental antibodies included as references.

[0032] FIGS. 6A-6L graphically summarize the results of experiments performed to illustrate the production of Tri-C-Fabs and Tetra-C-Fabs as alternative formats for tri- and tetra- specific antibodies using the thrombin-removable linker and KIH Fc heterodimer. FIGS. 6 A and 6B are schematic presentations of two exemplary antibodies: Tri-C-Fabs (FIG. 6 A; SEQ ID NOS: 4 and 11) and Tetra-C-Fabs (FIG. 6B; SEQ ID NOS: 11 and 12). In these antibodies, scFab domains representing the 3 ^rd and 4 ^th specificity were appended to the C-terminus of the bispecific antibody. Fabs of Ipilimumab were placed at position I, Daratumumab at position II, Herceptin at position III, and Atezolizumab at position IV (in the case of Tetra-C-Fabs). FIG. 6C pictorially summarizes the results of an SDS-PAGE analysis of Tri-C-Fabs and Tetra-C-Fabs, where the Tri-C-Fabs displayed an approximate molecular weight of 200 kDa and can be separated into polypeptide chains of 75 kDa, 50 kDa, and 25 kDa under reducing conditions. The Tetra-C-Fabs shows an approximate molecular weight of 250 kDa and can be separated into polypeptide chains of 75 kDa and 25 kDa under reducing conditions. FIGS. 6D, 6E, and 6F show that the Tri-C-Fabs binds to all three intended ligands by ELISA, with the parental antibodies included as references. FIGS. 6G, 6H, 61, and 6J graphically summarize the binding of the Tetra-C-Fabs to all four intended ligands by ELISA, with parental antibodies included as references. FIGS: 6K-6L summarize the results of a purity assessment of tri-specific and tetra- specific antibodies by analytical hydrophobic interaction chromatography. Purities of Tri-C-Fabs (FIG: 6K) and Tetra-CFabs (FIG: 6L) are estimated to be 93% and 79%, respectively.

[0033] FIG. 7 depicts the amino acid sequence of the sc36TMB linker (SEQ ID NO: 80). The thrombin cleavage site are indicated with arrows.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0034] The present disclosure relates generally to the field of antibody engineering and particular concerns multispecific antibodies that specifically bind to two or more different target antigens or epitopes. In particular, provided herein, inter alia , are compositions and methods concerning the production of engineered antibodies having (i) multiple antigen-binding specificities and (ii) Fc regions that have been modified to promote heterodimer formation between heavy chains from two different specificities. More particularly, provided herein are engineered antibodies having (i) two singe-chain antigen binding (scFab) fragments specific for two different epitopes and (ii) Fc regions associated with one another via an interface which has been modified to promote heterodimer formation.

[0035] A fundamental problem in multispecific antibody generation is how to properly pair heavy and light chains from antibodies with different specificities. For example, in bispecific antibody construction, although the “knobs-into-holes” (KIH) approach has been used to promote heterodimer formation between heavy chains from two different specificities, the efficiency is less than desired, and homodimer formations are frequently observed. In addition, the light chain pairing problem cannot be solved by the original KIH design. As a variation of the original strategy, KIH technique has been used also for light chain pairing but again the proper pairing efficiency remains an issue. As such, the overall efficiency of bispecific antibody generation is less than desired even with the dual knob-into-hole strategy.

[0036] For multispecific antibody generation, there are only a small number of studies that have ventured into this area, due to complexity associated with higher order of specificity. Most of the prior approaches are based on simple joining of multiple scFvs in tandem or appendixing antibody fragments (often scFvs) to the IgG framework. However, the resulting multispecific molecules often have production and stability problems, limiting their use in therapeutic development. As described in greater detail below, various compositions and methods disclosed herein enable facile generation of multispecific antibody and furthermore the resulting multispecific antibodies can use Fab as the basic binding module with robust binding affinity and specificity.

[0037] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.

DEFINITIONS

[0038] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. [0039] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising mixtures thereof “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0040] Certain values and ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. Generally, the term “about” has its ordinary meaning of approximately. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. Where ranges are provided, they are inclusive of the boundary values.

[0041] The terms, “cell”, “cell culture”, “cell line”, “recombinant cell”, “recipient cell” and “host cell” as used herein, include the primary subject cells and any progeny thereof, without regard to the number of transfers. It should be understood that not all progeny are exactly identical to the parental cell (due to deliberate or inadvertent mutations or differences in environment); however, such altered progeny are included in these terms, so long as the progeny retain the same functionality as that of the originally transfected cell.

[0042] As used herein, the term “construct” is intended to mean any recombinant nucleic acid molecule such as an expression cassette, plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular, single-stranded or double-stranded, DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, e.g ., operably linked.

[0043] The term “operably linked”, as used herein, denotes a physical or functional linkage between two or more elements, e.g. , polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (for example, a promoter) is functional link that allows for expression of the polynucleotide of interest. In this sense, the term “operably linked” refers to the positioning of a regulatory region and a coding sequence to be transcribed so that the regulatory region is effective for regulating transcription or translation of the coding sequence of interest. In some embodiments disclosed herein, the term “operably linked” denotes a configuration in which a regulatory sequence is placed at an appropriate position relative to a sequence that encodes a polypeptide or functional RNA such that the control sequence directs or regulates the expression or cellular localization of the mRNA encoding the polypeptide, the polypeptide, and/or the functional RNA. Thus, a promoter is in operable linkage with a nucleic acid sequence if it can mediate transcription of the nucleic acid sequence. Operably linked elements may be contiguous or non-contiguous. In addition, in the context of a polypeptide, “operably linked” refers to a physical linkage ( e.g . , directly or indirectly linked) between amino acid sequences (e.g. , different segments, modules, or domains) to provide for a described activity of the polypeptide. In the present disclosure, various segments, modules, or domains of the engineered multispecific antibodies of the disclosure may be operably linked to retain proper folding, processing, targeting, expression, binding, and other functional properties of the engineered multispecific antibodies in the cell. Unless stated otherwise, various modules, domains, and segments of the engineered multispecific antibodies of the disclosure are operably linked to each other. Operably linked modules, domains, and segments of the engineered multispecific antibodies of the disclosure may be contiguous or non-contiguous (e.g. , linked to one another through a linker).

[0044] The term “percent identity,” as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g, about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g, the NCBI web site at ncbi.nlm.nih.gov/BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the complement of a sequence. This definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., JMol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.

[0045] The term “recombinant” or “engineered” nucleic acid or polypeptide as used herein, refers to a nucleic acid molecule or polypeptide that has been altered through human intervention. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector. Other non-limiting examples of recombinant nucleic acids and recombinant polypeptides include engineered multispecific antibodies disclosed herein and the nucleic acids encoding such antibodies.

[0046] The term “vector” is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid molecule is generally linked to, e.g., inserted into, the vector nucleic acid molecule. Generally, a vector is capable of replication when associated with the proper control elements. The term “vector” includes cloning vectors and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes a regulatory region, thereby capable of expressing DNA sequences and fragments in vitro and/or in vivo. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g, DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, e.g, replication defective retroviruses and lentiviruses. In some embodiments, a vector is a gene delivery vector. In some embodiments, a vector is used as a gene delivery vehicle to transfer a gene into a cell.

[0047] As will be understood by one having ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0048] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or step.

[0049] Headings, e.g, (a), (b), (i) etc. , are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

MULTISPECIFIC ANTIBODIES

[0050] As discussed above, antibodies with multispecificity hold great promise for the next generation of therapeutic drugs against a variety of diseases, including cancers, infections, and immunological disorders. Compared to traditional monospecific antibodies that specifically recognize one ligand, multispecific antibodies can recognize two or more ligands and thus may provide an advantage in co-engaging different cell types, creating synthetic specificity, altering internalization dynamics, synergistically neutralizing virus and toxin, and simultaneously block multiple signaling pathways to maximize therapeutic benefits. In addition, multispecific antibodies can increase sensitivity and breath for recognizing target cells, tissues, or pathogens. Some bispecific antibodies were also designed to possess desirable properties other than recognition, such as enhanced production, extended half-time, or increased tissue penetration.

[0051] Production of multispecific antibodies is more complicated than monoclonal antibodies. Many different formats of bi-specific antibodies have been designed, including chemical conjugation of two different antibodies, tandem single-chain variable fragments (scFv) or Fab domains, and scFv or Fab fusion to immunoglobulins or other scaffold proteins. The design of multispecific antibodies is often derived from these bispecific formats but with substantially higher complexity. Each of these designs, and their derivatives, features distinct topology and thus may have non-identical biological functions and different pharmacokinetics, which needs to be tested experimentally. Depending on the molecular form of the building block, some formats suffer from drawbacks, ranging from poor production, low in vivo stability, and immunogenicity. Consequently, new methods and formats to produce bi- or multispecific antibodies are important topics for exploration.

[0052] Among the various designs, the IgG-like format has been an area of interest due to its resemblance to natural antibody, which often have good yields, stability, and relatively low immunogenicity. To produce these IgG-like molecules, two major problems have to be addressed: proper pairing of heavy chains from different antibodies, and correct pairing of the light chains to their corresponding heavy chains. The first issue is often addressed by the now classic knobs-into-holes (KIH) Fc mutants that enforce the formation of heavy chain heterodimers. However, the desired heterodimer in such a design is not exclusive, with homodimers still present, likely due to insufficient thermal stability of the CH3 domain and the Fc interface of the KIH heterodimer. In fact, both “knob” and “hole” Fc domains can form homodimers, causing substantial contamination in products that were generated by the KIH approach alone. As the homodimer often shares similar biophysical features as the heterodimer, such as size and isoelectric point, optimization and purification of the KIH bispecific format can be challenging and time-consuming. Additional modifications of the KIH Fc have been previously explored to enhance the formation of heterodimer.

[0053] To avoid promiscuous association of light chains to heavy chains in bispecific antibody production, different antibodies (with different ligand binding specificity) using one common light chain were previously selected from phage or yeast display libraries, providing a popular solution to the light chain pairing problem. However, this restriction on light chain reduces the sequence space that can be explored for binding affinity, specificity and downstream developability. In another example, the CrossMab format is an alternative approach for solving the light chain pairing problem, swapping only one pair of the variable or constant domains in one of the Fab fragments, and thus inhibiting chain mismatching between the switched Fab and the un-switched ones. Nonetheless, incorrect pairing of light chains are often observed, resulting in variable levels of product heterogeneity. The issue of immunogenicity of the hybrid variable- constant domain therefore remains to be investigated.

[0054] The feasibility of single-chain IgG (scIgG) has been demonstrated by previous studies and can be used to resolve the light chain pairing problem. However, an extended linker between light and heavy chains, varying between 30-60 residues in length, was retained in the final product, limiting its utility. As described herein, a new approach has been developed to produce single-chain IgG with cleavable linkers, allowing for removal of the undesired linkers in vitro, for example through the use of an enzymatic reaction ( e.g ., proteolytic reaction). As described in greater detail below, a thrombin cleavable linker has been successfully introduced between the light chain and the heavy chain, which can be removed by commercially available enzymes with high efficiency and accuracy. Antibodies produced using this approach show similar yield as the original antibodies, feature intact ligand-binding affinity, and contain only a few linker residues. Compared to an in vivo cleavage system designed in a previous study the cleavage in the disclosed system happens post-translationally and post-purification, ensuring correct pairing of the chains during cleavage. Furthermore, the linkers enable to engineer extra features in each of the two chains, such as different affinity tags, which can be used to facilitate purification of highly pure bispecific IgG molecules. Finally, the disclosed approach is entirely modular and allows facile production of multispecific antibodies, with IgG-like backbone and Fab-based binding modules.

[0055] In some embodiments, the compositions and methods disclosed herein employ single-chain IgG containing singe-chain antigen binding (scFab) fragments as a transient intermediate for proper heavy and light chain pairing and further use protease to cleave off removable linkers incorporated in the scFab fragments to generate the bispecific antibodies in an IgG-like configuration. Furthermore, the disclosed technology enables multispecific antibody generation, which has rarely been done or even attempted by other technologies due to the higher order of complexity associated with multispecific antibody generation. Indeed, by employing the compositions and methods described herein, experiments have been performed to readily use the technology disclosed herein to produce tri- and tetra-specific antibodies. Since the disclosed approach for multispecific antibody production is modular (similar to LEGO toy assembly), even higher order of specificities can be generated. Without being bound any particular theory, it is believed that the higher order of specificity enables simultaneous or synergistic recognition of multiple targets, thus maximizing therapeutic effect or creating new therapeutic opportunities.

COMPOSITIONS OF THE DISCLOSURE

[0056] As described in greater detail below, one aspect of the present disclosure relates to a new class of multispecific antibodies that specifically bind to two or more different target antigens or epitopes. In particular, provided herein, inter alia , are compositions and methods concerning the production of engineered antibodies having (i) multiple antigen-binding specificities and (ii) Fc regions that have been modified to promote heterodimer formation between heavy chains from two different specificities. In some embodiments, provided herein are engineered antibodies having (i) two singe-chain antigen binding (scFab) fragments specific for two different epitopes and (ii) Fc regions associated with one another via an interface which has been modified to promote heterodimer formation. Also provided, in other related aspects of the disclosure, are nucleic acids encoding the multispecific antibodies as disclosed herein, recombinant cells expressing the multispecific antibodies as disclosed herein, pharmaceutical compositions containing the nucleic acids and/or recombinant cells as disclosed herein.

Engineered Antibodies

[0057] In one aspect, some embodiments disclosed herein relate to a novel engineered antibodies containing multiple antigen-binding specificities and (ii) Fc regions that have been modified to promote heterodimer formation between heavy chains from two different specificities. In some embodiments, the disclosed engineered antibody includes a first and a second polypeptide chain, each of the first and second polypeptide chains including (a) one or more antigen-binding moieties and (b) an antibody Fc region, wherein the Fc regions of the first and a second polypeptide chains are associated with one another via an interface which has been modified to promote heterodimer formation. In some embodiments, the N-terminus of the first polypeptide chain and/or the second polypeptide chain is operably linked to one or more additional scFab fragments having specificity for additional antigens. In some embodiments, the engineered antibodies of the disclosure, the N-terminus of the first polypeptide chain is operably linked to at least one, e.g., 1, 2, 3, 4, 5, or 6 additional scFab fragments having specificity for additional antigens. In some embodiments, the N-terminus of the second polypeptide chain is operably linked to at least one, e.g., 1, 2, 3, 4, 5, or 6 additional scFab fragments having specificity for additional antigens.

[0058] In some embodiments, the C-terminus of the first polypeptide chain and/or the second polypeptide chain is operably linked to one or more additional scFab fragments having specificity for additional antigens. In some embodiments, the engineered antibodies of the disclosure, the C-terminus of the first polypeptide chain is operably linked to at least one, e.g., 1, 2, 3, 4, 5, or 6 additional scFab fragments having specificity for additional antigens. In some embodiments, the C-terminus of the second polypeptide chain is operably linked to at least one, e.g., 1, 2, 3, 4, 5, or 6 additional scFab fragments having specificity for additional antigens.

[0059] The scFab fragments of the engineered antibodies disclosed herein can include naturally-occurring amino acid sequences or can be engineered, designed, or modified so as to provide desired and/or improved properties, e.g. , binding affinity. Generally, binding affinity can be used as a measure of the strength of a non-covalent interaction between two molecules, e.g. , an antibody or portion thereof and an antigen (e.g, CD38, PD-L1, or HER2 antigen). In some cases, binding affinity can be used to describe monovalent interactions (intrinsic activity). Binding affinity between two molecules may be quantified by determination of the dissociation constant (K _D ). In turn, K _D can be determined by measurement of the kinetics of complex formation and dissociation using, e.g, the surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constants k _a (or k _on ) and dissociation rate constant k _d (or k _off ), respectively. K _D is related to k _a and k _d through the equation K _D = k _d / k _a. The value of the dissociation constant can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those set forth in Caceci et al. (1984, Byte 9: 340-362). For example, the KD may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428- 5432). Other standard assays to evaluate the binding ability of engineered antibodies of the present disclosure towards target antigens are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis, and other assays exemplified elsewhere herein. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as Surface Plasmon Resonance (SPR), e.g. by using a Biacore™ system, or KinExA. In some embodiments, the binding affinity of an antibody or an antigen-binding moiety for a target antigen (e.g, CD38, PD-L1, or HER2 antigen) can be calculated by the Scatchard method described by Frankel et al, Mol. Immunol , 16: 101-106,

1979. It will be understood that an antigen-binding moiety that “specifically binds” an antigen (such as HER2) is an antigen-binding moiety that does not significantly bind other antigens but binds the target antigen with high affinity, e.g, with an equilibrium constant (KD) of 100 nM or less, such as 60 nM or less, for example, 30 nM or less, such as, 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less.

[0060] In some embodiments, the antigen-binding moiety is selected from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single domain antibody (dAb), a VNAR domain, and a VHH domain, a diabody, or a functional fragment of any thereof. In some embodiments, the antigen-binding moiety includes a VH domain and a VL domain. In some embodiments, the antigen-binding moiety includes a single chain antigen-binding (scFab) fragment.

[0061] In some embodiments, the disclosed engineered antibody includes a first and a second polypeptide chain, each of the first and second polypeptide chains including: (a) a single chain antigen-binding (scFab) fragment which includes, in N-terminal to C-terminal direction, (i) a VL domain; (ii) a light chain constant domain (CL); (iii) a removable linker; (iv) a VH domain; and (v) a heavy chain constant domain (CHI), wherein the scFab fragments of the first and second polypeptide chains have specificity for different antigens; and (b) an antibody Fc region N-terminally linked to the scFab fragment in (a), wherein the Fc regions of the first and a second polypeptide chains are associated with one another via an interface which has been modified to promote heterodimer formation.

[0062] In some embodiments, the engineered antibody disclosed herein is multivalent, for example, bi-, tri-, or tetravalent (i.e., the engineered antibody includes two, three, or four antigen-binding moieties). In some embodiments, the engineered antibody of the disclosure can be a multivalent antibody ( e.g . , bivalent antibody, trivalent antibody, or tetravalent) including at least two antigen-binding moieties each having specific binding activity for a target protein. In some embodiments, the at least two antigen-binding moieties having specific binding for at least two different target proteins. Such antibody is multivalent, multispecific antibody (e.g., bispecific, tri-specific, tetra-specific, etc.) Accordingly, in some embodiments, the disclosed engineered antibody can be can be a bivalent, bispecific antibody. In some embodiments, the disclosed engineered antibody can be a trivalent, tri-specific antibody. In some embodiments, the disclosed engineered antibody can be a tetravalent, tetra-specific antibody.

[0063] In some embodiments, the at least two antigen-binding moieties have specific binding activity for the same target protein. Such antibody is multivalent, monospecific antibody. Accordingly, in some embodiments, the disclosed engineered antibody can be a bivalent, monospecific antibody. In some embodiments, the disclosed engineered antibody can be a trivalent, monospecific antibody. In some embodiments, the disclosed engineered antibody can be a tetravalent, monospecific antibody. In some embodiments, the engineered antibody disclosed herein is monospecific, e.g. all the scFab fragments bind the same antigen. In some embodiments, all the antigen-binding moieties bind the same epitope(s) of said antigen. In some embodiments, at least two antigen-binding moieties bind different epitopes on the target antigen.

Target antigens

[0064] In principle, there are no particular limitations with regard to suitable target antigens. In particular, the scFab fragments used in this disclosure can be derived from antibodies of any species (e.g. mouse, rat, rabbit, goat, human, etc.) or engineered (e.g, humanized) and having specificity to any given antigen/epitope. For simplicity, only variable domain sequences from four different antibodies are discussed in the Examples below. However, due to the modular design of the disclosed antibody constructs, the skilled artisan will appreciate that regions, domains, and segments of any known antibody may be substituted into the disclosed antibody structures.

[0065] Antigens suitable for the compositions and methods disclosed herein include, but are not limited to cell-surface antigens and extracellular antigens ( e.g ., those are not associated with cell surface). Non-limiting examples of suitable cell-surface target antigens include CD3, CD4, CD8, CD25, CD28, CD27, T-cell receptors, CD 16 A, CD38, CD47, CD56, CD14, CD16b, CDwl7, CD 18, CD71, CD79, CD68, CCR5, CCL2, SLAM, NKG2D, NKG2A, NKp46, Killer cell immunoglobulin-like receptors (KIRs), CD94, CD95, CD98, CD99, CD99R, beta 2 microglobulin, CD20, CD22, CD30, CD33, CD123, CD137, CD133, BCMA, CD19, CDla-c, CD2, CD2R, CD9, CD10, CDlla, CD11 b, CD11 c, CDwl2, CD13, CD14, CD15, CD15s, CD45, CD45A, CD45B, CD99, CD99R, CD 100, CDwlOl , CD102-CD106, CD107a-b, CDwl08, CDwl09, CD115, CDwll6, CD117, CD119, CD120a-b, CD121a-b, CD122, CDwl24, CD126, CD127, CD128, CD129, and CD130; prostate-specific membrane antigen (PSMA), B7-H3 (CD276), mesothelin, Prostate stem cell antigen (PSCA), CEA, CLEC12A, ALPPL2, ALPP, ALPI, GD2, TAG-72, EpCAM, GPC3, GPA33, GPRC5D, Her2, SSTR2 (Somatostatin Receptor 2), Mucl6, Mucl, FLT3, CD46, CD48, CD5, CD6, Mucl8, MELAN-A, DLL3, CD307, EGFRvIII, EGFR, P-cadherin, N-cadherin, ICAM-1, VLA-4, YCAM, a4/b7 integrin, an/b8 and an/b3 integrin including either a or b subunits, CD44 and CD44 splicing variants, glycoprotein llb/llla, LFA-1, CD40, 41BB, c-Met, Inducible T-cell COStimulator (ICOS), Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5), VEGF, CD80, CD86, CD55, and CD59.

[0066] Additional antigens that can be suitably used for the chimeric polypeptides disclosed herein include, but are not limited to members of receptor tyrosine kinase (RTK) family, which includes 20 classes, e.g., RTK class I (EGF receptor family) (ErbB family, e.g, EGFR, HER2, HER3 and HER4); RTK class II (Insulin receptor family, e.g, INSR, IGFR); RTK class III (PDGF receptor family, e.g. , PDGFRa, PDGFF^S, M-CSFR, KIT, FLT3L); RTK class IV (VEGF receptors family, e.g, VEGFRl, VEGFR2, VEGFR3); RTK class V (FGF receptor family, e.g., FGFR1, FGFR2, FGFR3, FGFR4); RTK class VI (CCK receptor family, e.g, CCK4); RTK class VII (NGF receptor family, e.g, TRKA, TRKB, TRKC); RTK class VIII (HGF receptor family, e.g, c-MET, RON); RTK class IX (Eph receptor family, e.g, EPHA1, EPHA2, EPH A3, EPHA4, EPHA5, EPHA6, and EPHB1 to 6); RTK class X (AXL receptor family, e.g., AXL, MER, TYR03); RTK class XI (TIE receptor family, e.g, TIE, TEK); RTK class XII (RYK receptor family, e.g., RYK); RTK class XIII (DDR receptor family, e.g, DDR1, DDR2); RTK class XIV (RET receptor family, e.g, RET); RTK class XV (ROS receptor family, e.g, ROS1); RTK class XVI (LTK receptor family, e.g, LTK, ALK); RTK class XVII (ROR receptor family, e.g, ROR1, ROR2); RTK class XVIII (MuSK receptor family, e.g, MuSK); RTK class XIX (LMR receptor, e.g, AATYKl, AATYK2, AATYK3); RTK class XX (e.g, RTK 106).

[0067] Also suitable antigens for the chimeric polypeptides disclosed herein include protein-coupled receptors (GPCRs), which include 6 classes, Class A (or 1) Rhodopsin-like; Class B (or 2) Secretin receptor family; Class C (or 3) Metabotropic glutamate/pheromone; Class D (or 4) Fungal mating pheromone receptors; Class E (or 5) Cyclic AMP receptors; and Class F (or 6) Frizzled/Smoothened. Further suitable antigens for the chimeric polypeptides and methods disclosed herein include stimulatory checkpoint molecules and inhibitory checkpoint molecules. Stimulatory checkpoint molecules include members of the tumor necrosis factor (TNF) receptor superfamily — CD27, CD40, 0X40, GITR and CD 137; members of the B7-CD28 superfamily — CD28 itself and ICOS; and the Interleukin-2 receptor beta sub-unit CD122. Inhibitory checkpoint molecules include PD- 1 (Programmed Death 1 receptor) and its ligands PD -LI and PD-L2; CTLA-4 (Cytotoxic T -Lymphocyte- Associated protein 4, aka CD152); B7-H3 (CD276); B7-H4 (VTCN1); LAG3 (Lymphocyte Activation Gene-3); TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3); VISTA (V-domain Ig suppressor of T cell activation); SIGLEC7 (Sialic acid-binding immunoglobulin-type lectin 7, aka CD328) and SIGLEC9 (Sialic acid binding immunoglobulin-type lectin 9, aka CD329); BTLA (B and T Lymphocyte Attenuator, aka CD272); A2AR (Adenosine A2A receptor ); and IDO (Indoleamine 2,3 -dioxygenase).

[0068] Additional antigens that can be suitably used for the chimeric polypeptides disclosed herein include, but are not limited to receptors for hormones or growth factors, transforming growth factor beta (TGF[:S) receptors, which include type I receptors ALK1 (ACVRLl), ALK2 (ACVR1A), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFpRl),

ALK6 (BMPRIB), and ALK7 (ACVR1C); and type II receptors TGF[:SR2, BMPR2, ACVR2A, ACVR2B, and AMHR2 (AMHR); and type III receptor TGF[:SR3, tumor-associated post- translational modifications, including polysialic acid (polySia), Gb3/CD77, GM3, GD2, GD3, the Thomsen-Friedenreich antigen (Gal-GalNAc), cancer O-glycans (Tn, sTn (sialyl Tn antigen), and T antigen), SLex and SLea (sialyl-Lewis a), altered branching and fucosylation of N-glycans, increased mucins and truncated O-glycans, altered expression of hyaluronan, changes in sulfated glycosaminoglycans, stage-specific embryonic antigen-3 (SSEA-3), SSEA-3 with fucose (Globo H), and SSEA-4.

[0069] Suitable antigens for the chimeric polypeptides disclosed herein additionally include secreted molecules such as an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g ., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g. , IL-1 to IL-10; transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-bI, TGF- [12, TGF-[:S3, TGF-[:S4, or TGF-[:i5; vascular endothelial growth factor (YEGF); RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1 -alpha); platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); insulin-like growth factor-1 and - II (IGF-I and IGF-II); des(l-3)-IGF-l (brain IGF-I), insulin-like growth factor binding proteins; biologic toxins and immunotoxins; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; a neurotrophic factor such as bone- derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-b; parathyroid hormone; thyroid stimulating hormone; rheumatoid factors; erythropoietin; osteoinductive factors; a bone morphogenetic protein (BMP); clotting factors such as factor VIIIC, factor IX, tissue factor (TF), and von Willebrands factor; anti-clotting factors such as Protein C; bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; atrial natriuretic factor; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); enkephalinase; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; DNase; inhibin; activin; superoxide dismutase; a serum albumin such as human serum albumin; a portion of the virus envelope; and addressins, lipoproteins; alpha- 1 -antitrypsin; insulin A-chain; insulin B-chain; proinsulin; lung surfactant; obesity (OB) receptor; mouse gonadotropin-associated peptide; a microbial protein, such as beta- lactamase; protein A or D; and IgE.

[0070] In some embodiments, the antigens are selected from the group consisting of CD3, CD4, CD8, CD25, CD28, CD27, T-cell receptors, CD 16 A, CD38, CD46, CD47, CD56, CD14, CD 16b, CD71, CD79, CD68, CCR5, CCL2, SLAM, NKG2D, NKG2A, NKp46, killer-cell immunoglobulin-like receptors (KIRs), CD98, beta 2 microglobulin, CD20, CD22, CD30, CD33, CD123, CD137, CD133, BCMA, CD19, CDla-c, prostate-specific membrane antigen (PSMA), B7-H3 (CD276), mesothelin, prostate stem cell antigen (PSCA), CEA, CLEC12A, ALPPL2, ALPP, ALPI, GD2, TAG-72, EpCAM, GPC3, GPA33, GPRC5D, Her2, SSTR2 (somatostatin receptor 2), Mucl 6, Mu , FLT3, Mud 8, MELAN-A, DLL3, CD307, EGFRvIII, EGFR, P- cadherin, N-cadherin, ICAM-1, VLA-4, YCAM, a4/b7 integrin, an/b8 intergrins, an/b3 integrins, CD44 and CD44 splicing variants, glycoprotein llb/llla, LFA-1, CD40, 0X40, GITR, 41BB, c-Met, inducible T-cell costimulator (ICOS), leucine rich repeat-containing G protein- coupled receptor 5 (LGR5), VEGF, CD80, CD86, CD55, CD59, members of ErbB family, members of insulin receptor family, members of PDGF receptor family, members of VEGF receptors family, members of FGF receptor family, members of CCK receptor family, members of NGF receptor family, members of HGF receptor family, members of Eph receptor family, members of AXL receptor family, members of DDR receptor family, members of RET receptor family, members of ROS receptor family, members of LTK receptor family, members of ROR receptor family, G protein-coupled receptors (GPCRs), PD-1, PD-L1, PD-L2, CTLA-4 (CD 152), B7-H3 (CD276), B7-H4 (VTCN1), LAG3, TIM-3, VISTA, SIGLEC7 (CD328), SIGLEC9 (CD329), BTLA (CD272), A2AR, IDO (indoleamine 2,3-dioxygenase), TΰRbϊa, TϋRbϋII, and TGFbR3.

[0071] As discussed above, the approach disclosed herein is highly versatile and applicable to any monoclonal antibody pair or panel, which in turns can expedite evaluation and therapeutic development of multispecific antibodies.

[0072] Generally, the scFab fragment of the first and/or second polypeptide chains includes the VL, CL, Vn, and CHI domains derived from any monoclonal antibody known in the art. Non-limiting antibodies suitable for the compositions and methods disclosed herein include abciximab, abciximab, adalimumab, aducanumab, alacizumab, alemtuzumab, alirocumab, alirocumab, ascrinvacumab, atezolizumab, atinumab, bapineuzumab, basiliximab, basiliximab, belimumab, bevacizumab, blinatumomab, blosozumab, bococizumab, brentuximab, canakinumab, caplacizumab, capromab, certolizumab, cetuximab, crenezumab, daclizumab, daratumumab, demcizumab, denosumab, denosumab, dinutuximab. Additional suitable antibodies suitable for the compositions and methods disclosed herein include, but are not limited to, ecukinumab, eculizumab, eculizumab, efalizumab, elotuzumab, enoticumab, etaracizumab, evinacumab, evolocumab, evolocumab, fasinumab, fulranumab, gantenerumab, golimumab, ibritumomab, icrucumab, idarucizumab, idarucizumab, inciacumab, infliximab, ipilimumab, mepolizumab, natalizumab, necitumumab, nesvacumab, nivolumab, obinutuzumab, ofatumumab, omalizumab, opicinumab, orticumab, ozanezumab.

[0073] Further antibodies suitable for the compositions and methods disclosed herein include, but are not limited to, palivizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, ponezumab, ralpancizumab, ramucirumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, rinucumab, rituximab, romosozumab, siltuximab, solanezumab, stamulumab, tadocizumab, tanezumab, tocilizumab, trastuzumab, ustekinumab, vedolizumab, and vesencumab. In some embodiments, the scFab fragment of the first polypeptide chain includes the VL, CL, VH, and CHI domains derived from ipilimumab and the scFab fragment of the second polypeptide chain includes the VL, CL, VH, and CHI domains derived from daratumumab. In some embodiments, the scFab fragment of the first polypeptide chain includes the VL, CL, VH, and CHI domains derived from ipilimumab and the scFab fragment of the second polypeptide chain includes the VL, CL, VH, and CHI domains derived from trastuzumab.

[0074] In some embodiments, the one or more additional scFab fragments are operably linked to the first polypeptide chain and/or the second polypeptide chain, as illustrated in Example 6. In some embodiments, only one of the first and second polypeptide chains is N- terminally linked one or more additional scFab fragments (see, e.g. , Example 6 and FIGS. 4A- 4F). In some embodiments, the one or more additional scFab fragments are N-terminally linked only to the first polypeptide chain. In some embodiments, the one or more additional scFab fragments are N-terminally linked only to the second polypeptide chain. In some embodiments, both the first polypeptide chain and the second polypeptide chain are N-terminally linked to one or more additional scFab fragments (see, e.g., Example 6 and FIGS. 5A-5G). In some embodiments, only one of the first and second polypeptide chains is N-terminally linked one or more additional scFab fragments (see, e.g, Example 6, and SEQ ID NOS: 4 and 11, and FIG. 6A). In some embodiments, the one or more additional scFab fragments are C-terminally linked only to the first polypeptide chain. In some embodiments, the one or more additional scFab fragments are C-terminally linked only to the second polypeptide chain. In some embodiments, both the first polypeptide chain and the second polypeptide chain are C-terminally linked to one or more additional scFab fragments (see, e.g ., Example 6, SEQ ID NOS: 11-12, and FIG. 6B).

[0075] Generally, the additional scFab fragments can include the VL, CL, VH, and CHI domains derived from any monoclonal antibody known in the art. Non-limiting monoclonal antibodies suitable for the compositions and methods disclosed herein include abciximab, abciximab, adalimumab, aducanumab, alacizumab, alemtuzumab, alirocumab, alirocumab, ascrinvacumab, atezolizumab, atinumab, bapineuzumab, basiliximab, basiliximab, belimumab, bevacizumab, blinatumomab, blosozumab, bococizumab, brentuximab, canakinumab, caplacizumab, capromab, certolizumab, cetuximab, crenezumab, daclizumab, daratumumab, demcizumab, denosumab, denosumab, dinutuximab, ecukinumab, eculizumab, eculizumab, efalizumab, elotuzumab, enoticumab, etaracizumab, evinacumab, evolocumab, evolocumab, fasinumab, fulranumab, gantenerumab, golimumab, ibritumomab, icrucumab, idarucizumab, idarucizumab, inciacumab, infliximab, ipilimumab, mepolizumab, natalizumab, necitumumab, nesvacumab, nivolumab, obinutuzumab, ofatumumab, omalizumab, opicinumab, orticumab, ozanezumab, palivizumab, palivizumab, panitumumab, pembrolizumab, pertuzumab, ponezumab, ralpancizumab, ramucirumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, rinucumab, rituximab, romosozumab, siltuximab, solanezumab, stamulumab, tadocizumab, tanezumab, tocilizumab, trastuzumab, ustekinumab, vedolizumab, and vesencumab. In some embodiments, at least one of the additional scFab fragments includes the VL, CL, VH, and CHI domains derived from atezolizumab.

[0076] It will be appreciated that one or more amino acid substitutions, additions and/or deletions may be made to the antibody variable domains, provided by the present disclosure, without significantly altering the ability of the antibody to bind to target antigen and to neutralize activity thereof. The effect of any amino acid substitutions, additions and/or deletions can be readily tested by one skilled in the art, for example by using known in vitro assays, for example a BIAcore assay.

[0077] The use of a single chain variable fragment (scFv) to confer specificity to a specific antigen allows for a modular construction of multispecific antibodies. The use of scFv fragment(s) fused to the terminus of IgG heavy chains and/or light chains for construction of multispecific antibodies has been previously described. This format ("IgG-scFv") allows a conventional IgG to be converted into a bispecific antibody wherein a first specificity is encoded in the variable domains of the IgG and a second specificity is encoded in the scFv domains attached through a flexible linker region. Variations of this format include fusing scFv domains at the N- or C-termini of the heavy or light chains; the scFvs may have the same or differing antigen-binding specificities (Spangler, J. B. etal, J. Mol. Biol. 422, 532-544, 2012). In addition, through the use of heavy-chain heterodimers (for example, using knob-hole or similar constructs), scFvs of differing specificities may be attached to the N- or C-terminus of each heavy chain.

[0078] Any modifications that could be made to an IgG could also be made to the disclosed structures, including: the addition or deletion of constant region domains; point mutations; and fusion of additional proteins such as cytokines, enzymes or toxins. Specific modifications given as examples herein are the addition of an anchor domain (AD) or dimerization and docking domain (DDD) to convert the fusion protein to a DNL module. The DNL module could then be further enhanced by the conjugation of additional functional groups using the Dock-and-Lock (DNL) method.

[0079] The engineered antibodies disclosed herein may be incorporated as naked antibodies, alone or in combination with one or more therapeutic agents. Alternatively, the disclosed engineered antibodies may be utilized as immunoconjugates, attached to one or more therapeutic agents. (For methods of making immunoconjugates, see, e.g ., U.S. Pat. Nos. 4,699,784; 4,824,659; 5,525,338; 5,677,427; 5,697,902; 5,716,595; 6,071,490; 6,187,284; 6,306,393; 6,548,275; 6,653,104; 6,962,702; 7,033,572; 7,147,856; and 7,259,240, the Examples section of each incorporated herein by reference.) Exemplary therapeutic agents may be selected from the group consisting of a radionuclide, a cytotoxin, a chemotherapeutic agent, a drug, a pro- drug, a toxin, an enzyme, an immunomodulator, an anti-angiogenic agent, a pro-apoptotic agent, a cytokine, a hormone, an oligonucleotide molecule (e.g, an antisense molecule or a gene) or a second antibody or fragment thereof. Removable Linkers and Connectors

[0080] In some embodiments, the CL domain and VH domain of each scFab fragment are operably linked to one another via a linker. In some embodiments, the linker is a synthetic compound linker such as, for example, a chemical cross-linking agent. In some embodiments, the CL domain and VH domain of each scFab fragment are operably linked to one another via a linker polypeptide sequence ( e.g ., peptidal linkage). In principle, there are no particular limitations to the length and/or amino acid composition of the linker polypeptide sequence. In some embodiments, any arbitrary single-chain peptide comprising about one to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a polypeptide linker. In some embodiments, the linker polypeptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker polypeptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker polypeptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker polypeptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residue.

[0081] In some embodiments, the CL domain and VH domain of each scFab fragment are operably linked to one another via a removable linker. In some embodiments, the amino acid sequence of the removable linker includes one or more proteolytic cleavage sites. Generally, any proteolytic cleavage site known in the art can be incorporated into the engineered antibodies of the disclosure and can be, for example, proteolytic cleavage sequences that are cleaved post production by a protease. Further suitable proteolytic cleavage sites also include proteolytic cleavage sequences that can be cleaved following addition of an external protease.

[0082] In some embodiments, at least one of the one or more proteolytic cleavage sites can be cleaved by a protease selected from the group consisting of thrombin, PreScission™ protease, and tobacco etch virus (TEV) protease. In some embodiments, the protease is thrombin. In some embodiments, the removable linker includes a sc36TMB linker. In some embodiments, the removable linker includes the polypeptide sequence of SEQ ID NO: 80. [0083] In some embodiments, at least one of the one or more proteolytic cleavage sites can be cleaved by an endopeptidase, which is sometimes referred to as endoproteinase or proteolytic peptidase that breaks peptide bonds of nonterminal amino acids {i.e., within the molecule), in contrast to exopeptidase, which breaks peptide bonds from end-pieces of terminal amino acids. Endopeptidases suitable for the disclosed antibodies include, but are not limited to, trypsin, chymotrypsin, elastase, thermo lysin, pepsin, glutamyl endopeptidase, or neprilysin.

[0084] In some embodiments, the one or more additional scFab fragments are operably linked to the C- or N-terminus of the first and/or second polypeptide chain via a connector. In some embodiments, the connector is a synthetic compound linker such as, for example, a chemical cross-linking agent. In some embodiments, one or more additional scFab fragments are operably linked to the C- or N-terminus of the first and/or second polypeptide chain via a peptide connector. In principle, there are no particular limitations to the length and/or amino acid composition of the connector polypeptide sequence. In some embodiments, any arbitrary single chain peptide comprising about one to 100 amino acid residues ( e.g ., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a polypeptide linker.

In some embodiments, the linker polypeptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues.

[0085] In certain embodiments, the peptide connector contains only glycine and/or serine residues (e.g., glycine-serine linker). Examples of such peptide connectors include: Gly, Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser; Ser Gly Gly Gly; Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly; Gly Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly Gly; Gly Gly Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly Gly Gly; (Gly Gly Gly Gly Ser)n, wherein n is an integer of one or more; and (Ser Gly Gly Gly Gly)n, wherein n is an integer of one or more. In some embodiments, the peptide connectors are modified such that the amino acid sequence GSG (that occurs at the junction of traditional Gly/Ser linker peptide repeats) is not present. For example, in some embodiments, the peptide connector includes an amino acid sequence selected from the group consisting of: (GGGXX)nGGGGS and GGGGS(XGGGS)n, where X is any amino acid that can be inserted into the sequence and not result in a polypeptide comprising the sequence GSG, and n is 0 to 4. In some embodiments, the sequence of a peptide connector is (GGGXlX2)nGGGGS and XI is P and X2 is S and n is 0 to 4. In some other embodiments, the sequence of a peptide connector is (GGGXlX2)nGGGGS and Xi is G and X2 is Q and n is 0 to 4. In some other embodiments, the sequence of a peptide connector is (GGGXlX2)nGGGGS and XI is G and X2 is A and n is 0 to 4. In yet some other embodiments, the sequence of a peptide connector is GGGGS(XGGGS)n, and X is P and n is 0 to 4. In some embodiments, a peptide connector of the disclosure comprises or consists of the amino acid sequence (GGGGA)2GGGGS. In some embodiments, the peptide connector comprises or consists of the amino acid sequence (GGGGQ)2GGGGS. In another embodiment, a peptide connector comprises or consists of the amino acid sequence (GGGPS)2GGGGS. In another embodiment, the peptide connector comprises or consists of the amino acid sequence GGGGS(PGGGS)2. In some embodiments, the peptide connector is (GxS)n or (GxS)nGm with G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m=0, 1, 2 or 3), preferably x=4 and n=2 or 3, more preferably with x=4, n=2. In some embodiments, the peptide connector is (G ₄ S) _2. In some embodiments, the connector includes the sequence -GGGSGGGSGGGSG- (SEQ ID NO: 82). In some embodiments, the connector includes the sequence -ASTKGPSGSG- (SEQ ID NO: 81). Affinity Tags

[0086] In some embodiments, the removable linker incorporated in each scFab fragment further includes one or more heterologous affinity tags. In some embodiments, at least one of the one or more heterologous affinity tags is incorporated at a position selected from the group consisting of the N-terminus of removable linker sequence, the C- terminus of removable linker sequence, and internal to the removable linker sequence. In some embodiments, the one or more heterologous affinity tags is selected from the group consisting of polyhistidine (poly -His) tags, Hemagglutinin (HA) tags, AviTag™, Protein C tags, FLAG tags, Strep-tag® II, and Twin-Strep- tag®, glutathione -S -transferase (GST), C-myc tag, Chitin-binding domain, Streptavidin binding proteins (SBP), maltose binding protein (MBP), Cellulose-binding domains, Calmodulin-binding peptides, and S-tags. In some embodiments, at least one of the one or more affinity tags includes a poly-His tag. In some embodiments, at least one of the one or more affinity tags includes a Twin- Strep-tag®. In some embodiments, the removable linkers of the scFab fragments of the first and second polypeptide chains includes the same affinity tags. In some embodiments, the removable linkers of the scFab fragments of the first and second polypeptide chains includes different affinity tags.

[0087] In some embodiments, the removable linker further comprises one or more additional polypeptide motifs that promote dimer formation. In some embodiments, the one or more additional polypeptide motifs are selected from the group consisting of homodimerization motifs, heterodimerization motifs, leucine zipper motifs, and combinations of any thereof. In some embodiments, at least one of the additional polypeptide motifs comprises a heterodimerization motif of the constant region of T-cell receptor alpha and/or beta chains. Heavv-Chain Heterodimerization

[0088] As described above, the compositions and methods disclosed herein provide advantageous approaches to improving the efficiency of multispecific antibody production. An approach to circumvent the problem of mispaired byproducts, which is known as “knobs-into- holes” or “protuberance-into-cavity,” aims at promoting the pairing of two different antibody heavy chains by introducing mutations into the constant domains to modify their contact interface. For example, in some embodiments, the disclosed compositions and methods involve induction of heavy chain heterodimer formation and inhibition of heavy chain homodimer formation by substituting an amino acid side chain present in a constant region (e.g. , CH2 or CH3 region) of one of the heavy chains to a larger side chain to create a “knob,” and substituting the amino acid side chain present in the CH3 region of the other heavy chain to a smaller side chain to create a “hole,” such that the knob can be positioned into the hole. By combining these two antibody heavy chains (e.g, co-expressing in recombinant cells), high yields of heterodimer formation (“knob-hole”) versus homodimer formation (“hole-hole” or “knob-knob”) can be achieved. Additional information in this regard can be found in, for example, U.S. Patent No. 8,216,805. Furthermore, the percentage of heterodimer may be further increased by the introduction of a disulfide bridge to stabilize the heterodimers or by remodeling the interaction surfaces of the two CH3 domains using a phage display approach. Additional approaches for the KIH technology can be found in, e.g. European Patent Publication No. EP 1870459 Al.

[0089] In some embodiments, the Fc regions of the first and the second polypeptide chains are associated with one another via a modified interface within a constant domain of the Fc regions. In some embodiments, the first and second polypeptide chains each include an antibody constant domain such as the CH2 domain or CH3 domain of a human IgGl . In some embodiments, the first and second polypeptide chains each include a CH2 domain. In some embodiments, the first and second polypeptide chains each include a CH3 domain. In some embodiments, the first and second polypeptide chains each include the interface residues of the CH3 domain of IgG. In some embodiments, the modified interface of the first polypeptide chain includes a protuberance which is positionable in a cavity in the modified interface of the second polypeptide chain. In some embodiments, the amino acid sequence of an original interface has been modified so as to introduce the protuberance and/or cavity into the modified interface such that a greater ratio of heterodimenhomodimer forms than that for a dimer having a non-modified interface.

[0090] One skilled in the art will appreciate that the “interface” of the engineered antibodies disclosed herein includes those “contact” amino acid residues in the first polypeptide which interact with one or more “contact” amino acid residues in the interface of the second polypeptide. The interface can be a domain of an immunoglobulin such as a variable domain or constant domain (or regions thereof). In some embodiments, the interface includes the CH2 domain and/or CH3 domain of an immunoglobulin. In some embodiments, the interface includes the CH2 domain and/or CH3 domain of an IgG antibody. In some embodiments, the interface includes the CH2 domain and/or CH3 domain of a human IgGl antibody.

[0091] A “cavity” refers to at least one amino acid side chain which is recessed from the interface of the second polypeptide and therefore accommodates a corresponding protuberance on the adjacent interface of the first polypeptide. A “protuberance” refers to at least one amino acid side chain which projects from the interface of the first polypeptide and is therefore positionable in a compensatory cavity in the adjacent interface (i.e., the interface of the second polypeptide) so as to stabilize the heterodimer, and thereby favor heteromultimer formation over homodimer formation, for example. “Protuberances” are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. , tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the protuberances are optionally created on the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g, alanine or threonine). In instances where a suitably positioned and dimensioned protuberance or cavity already exists at the unmodified interface of either the first or second polypeptide, it is only necessary to engineer a corresponding cavity or protuberance, respectively, at the adjacent interface. Additional information regarding KIH technology can be found in, e.g ., U.S. Patent No. 8,679,785, which is hereby incorporated by referenced.

[0092] In some embodiments, the cavity includes an amino acid residue substituted into the interface of the second polypeptide, and wherein the substituted amino acid residue is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). In some embodiments, the protuberance includes an amino acid residue substituted into the interface of the first polypeptide, and wherein the substituted amino acid residue is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some embodiments, the amino acid residue is substituted into the interface of the first polypeptide at a position corresponding to a contact residue of the CEB domain of human EgGl, as indicated in Figure 7 of U.S. Patent No. 8,679,786, which is incorporated herein by reference. In some embodiments, the amino acid residue is substituted into the interface of the first polypeptide at position 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409 of the CEB domain of human EgGl. In some embodiments, the protuberance includes a T366W amino acid substitution within the constant domain CH3 of the Fc region of the first polypeptide. In some embodiments, the cavity includes an amino acid substitution selected from the group consisting of S354C, T366S, L368A, and Y407V present within the constant domain CH3 of the Fc region of the second polypeptide.

[0093] In some embodiments, the engineered antibody or functional fragment thereof as described herein includes a single-chain polypeptide with an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences disclosed herein. In some embodiments, at least one of the first and second polypeptides includes a single-chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to any one of the amino acid sequences disclosed herein. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to any one of the amino acid sequences identified in the Sequence Listing. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to any one of SEQ ID NOS: 1-12. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 1. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 2. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 3. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 4. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 5. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 6.

[0094] In some embodiments, the engineered antibody or functional fragment thereof as described herein includes at least one of the first and second polypeptides with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO:

7. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO:

8. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO:

9. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO:

10. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 11. In some embodiments, at least one of the first and second polypeptides includes an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 12.

[0095] It is also contemplated that alternative methodologies can be used to promote heavy-chain heterodimerization of the first and second polypeptide chains of the engineered antibodies of the disclosure. For example, in some embodiments, the heavy-chain heterodimerization of the first and second polypeptide chains of the engineered antibodies as disclosed herein can be achieved by a controlled Fab arm exchange method as described in Aran FL et al., Proc. Natl. Acad. Sci. U.S.A, March 26, 2013, vol. 110, no. 13, 5145-5150, which is hereby incorporated by referenced in its entirety.

Making antibodies

[0096] One skilled in the art will appreciate that the complete amino acid sequence of an antibody can be used to construct a back-translated gene. For example, a DNA oligomer containing a nucleotide sequence coding for a given polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides generally contain 5' or 3' overhangs for complementary assembly. In some cases, the individual oligonucleotides can contain 5' and 3' blunt-ends, which can also be assembled by using, e.g., blunt-end ligases.

[0097] In addition to generating polypeptides via expression of nucleic acid molecules that have been assembled by recombinant molecular biological techniques, a subject engineered antibody in accordance with the present disclosure can be chemically synthesized. Chemically synthesized polypeptides are routinely generated by those of skill in the art.

[0098] Once assembled (by synthesis, site-directed mutagenesis or another method), the DNA sequences encoding an engineered antibody as disclosed herein can be inserted into an expression vector and operably linked to an expression control sequence appropriate for expression of the engineered antibody in the desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of the engineered antibody in a suitable host. As is known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

[0099] The binding activity of the engineered antibodies of the disclosure can be assayed by any suitable method known in the art. For example, the binding activity of the engineered antibodies of the disclosure can be determined by, e.g. , Scatchard analysis (Munsen, etal. 1980 Analyt. Biochem. 107:220-239). Specific binding may be assessed using techniques known in the art including but not limited to competition ELISA, BIACORE® assays and/or KINEXA® assays. An antibody that "preferentially binds" or "specifically binds" (used interchangeably herein) to a target antigen or target epitope is a term well understood in the art, and methods to determine such specific or preferential binding are also known in the art. An antibody is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen or epitope than it does with alternative antigens or epitopes. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. Also, an antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration to that target in a sample than it binds to other substances present in the sample. For example, an antibody that specifically or preferentially binds to a HER2 epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other HER2 epitopes or non-HER2 epitopes. It is also understood by reading this definition, for example, that an antibody which specifically or preferentially binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding.

[0100] A variety of assay formats may be used to select an antibody that specifically binds an antigen of interest. For example, solid-phase ELISA immunoassay, immunoprecipitation, Biacore™ (GE Healthcare, Piscataway, NJ), KinExA, fluorescence -activated cell sorting (FACS), Octet™ (ForteBio, Inc., Menlo Park, CA) and Western blot analysis are among many assays that may be used to identify an antibody that specifically reacts with an antigen.

Generally, a specific or selective reaction will be at least twice the background signal or noise, more typically more than 10 times background, even more typically, more than 50 times background, more typically, more than 100 times background, yet more typically, more than 500 times background, even more typically, more than 1000 times background, and even more typically, more than 10,000 times background.

Nucleic Acids and Recombinant Cells

[0101] In one aspect, some embodiments disclosed herein relate to nucleic acid molecules encoding the multispecific antibodies of the disclosure, expression cassettes, and expression vectors containing these nucleic acid molecules operably linked to regulator sequences which allow expression of the multispecific antibodies in a host cell or ex-vivo cell-free expression system. [0102] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules including cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid molecule can be double-stranded or single- stranded ( e.g ., a sense strand or an antisense strand). A nucleic acid molecule may contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a polynucleotide molecule. The nomenclature for nucleotide bases as set forth in 37 CFR §1.822 is used herein.

[0103] Nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, including nucleic acid molecules that are generally between about 5 Kb and about 50 Kb, for example between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0104] In some embodiments, the nucleic acid molecules of the disclosure are recombinant, e.g. nucleic acid molecules that have been altered through human intervention. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector. As non-limiting examples, a recombinant nucleic acid molecule: 1) has been synthesized or modified in vitro , for example, using chemical or enzymatic techniques (for example, by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g. , methylation), or recombination (including homologous and site-specific recombination)) of nucleic acid molecules; 2) includes conjoined nucleotide sequences that are not conjoined in nature, 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleic acid molecule sequence, and/or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleic acid sequence. [0105] In some embodiments, provided herein are nucleic acid molecules including a nucleotide sequence that encodes the first and/or the second polypeptide chain of an engineered antibody as disclosed herein. In some embodiments, a nucleic acid molecule as disclosed herein encodes at least one of the first and the second polypeptide chain of an engineered antibody as disclosed herein. In some embodiments, a nucleic acid molecule as disclosed herein encodes both the first and the second polypeptide chain of an engineered antibody as disclosed herein.

[0106] In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to the first or the second polypeptide chain of an engineered antibody as disclosed herein. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to any one of SEQ ID NOS: 1-12 in the Sequence Listing. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 2. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 4. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 5. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 6.

[0107] In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 7. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 8. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%,

95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 9. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 10. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%,

95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 11. In some embodiments, the nucleic acid molecules include a nucleotide sequence encoding a single chain polypeptide with an amino acid sequence having at least 80%, 90%, 95%, 96%, 97, 98%, 99% sequence identity to SEQ ID NO: 12.

[0108] Some embodiments disclosed herein relate to vectors or expression cassettes including a recombinant nucleic acid molecule encoding the engineered antibodies as disclosed herein. The term expression cassette generally refers to a construct of genetic material that contains coding sequences and sufficient regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo. The expression cassette may be inserted into a vector for targeting to a desired host cell and/or into a subject. As such, the term expression cassette may be used interchangeably with the term “expression construct.” As used herein, the term “construct” is intended to mean any recombinant nucleic acid molecule such as an expression cassette, plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular, single-stranded or double-stranded, DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, e.g. operably linked.

[0109] Also provided herein are vectors, plasmids, or viruses containing one or more of the nucleic acid molecules encoding any of the engineered antibodies disclosed herein. The nucleic acid molecules described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed/transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in Ausubel, F. M., etal. , Current Protocols in Molecular Biology, New York, NY: Wiley (including supplements through 2014), and Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, L, & Russel, D. W. (2001).

[0110] It should be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. For example, vectors that can be used include those that allow the DNA encoding the engineered antibodies of the present disclosure to be amplified in copy number. Such amplifiable vectors are known in the art.

[0111] Accordingly, in some embodiments, the engineered antibodies as described herein, can be expressed from vectors, preferably expression vectors. The vectors are useful for autonomous replication in a host cell or may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g. , non- episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g, replication defective retroviruses, adenoviruses, and adeno-associated viruses) are also included. Exemplary recombinant expression vectors can include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, operably linked to the nucleic acid sequence to be expressed.

[0112] DNA vector can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (2001, supra) and other standard molecular biology laboratory manuals.

[0113] The nucleic acid sequences encoding the engineered antibodies of the disclosure, can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon optimization are known in the art. Codon usages within the coding sequence of the engineered antibodies disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.

[0114] Vectors suitable for use include T7 -based vectors for use in bacteria, the pMSXND expression vector for use in mammalian cells, and baculovirus-derived vectors for use in insect cells. In some embodiments, nucleic acid inserts, which encode the engineered antibody in such vectors, can be operably linked to a promoter, which is selected based on, for example, the cell type in which expression is sought.

[0115] In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject polypeptide, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this disclosure, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences.

[0116] Within these parameters one of skill in the art may select various vector/expression control sequence/host combinations that will express the desired DNA sequences on fermentation or in large scale animal culture, for example, using CHO cells or COS 7 cells.

[0117] The choice of expression control sequence and expression vector, in some embodiments, will depend upon the choice of host. A wide variety of expression host/vector combinations can be employed. Non-limiting examples of useful expression vectors for eukaryotic hosts, include, for example, vectors with expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Non-limiting examples of useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli , including col El, pCRI, pER32z, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g. , the numerous derivatives of phage lambda, e.g. , NM989, and other DNA phages, such as Ml 3 and filamentous single stranded DNA phages. Non-limiting examples of useful expression vectors for yeast cells include the 2m plasmid and derivatives thereof. Non-limiting examples of useful vectors for insect cells include pVL 941 and pFastBac™ 1.

[0118] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain origins of replication, and other genes that encode a selectable marker. For example, the neomycin-resistance ( neoR ) gene imparts G418 resistance to cells in which it is expressed, and thus permits phenotypic selection of the transfected cells. Those of skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context.

[0119] Viral vectors that can be used in the disclosure include, for example, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors , CSH Laboratory Press, Cold Spring Harbor, N. Y.).

[0120] In one aspect of the disclosure, recombinant prokaryotic or eukaryotic cells that contain an engineered antibody as disclosed herein, and/or contain and express a nucleic acid molecule that encodes any one of the engineered antibody disclosed herein are also features of the disclosure. In some embodiments, provided herein are recombinant cells including the first polypeptide chain of an engineered antibody as disclosed herein, or a scFab fragment thereof. In some embodiments, provided herein are recombinant cells including the second polypeptide chain of an engineered antibody as disclosed herein, or a scFab fragment thereof. In some embodiments, provided herein are recombinant cells including both the first and second polypeptide chains of an engineered antibody as disclosed herein. In some embodiments, the recombinant cells include a recombinant nucleic acid as disclosed herein.

[0121] In some embodiments, a recombinant cell of the disclosure is a transfected cell, e. g., a cell into which a nucleic acid molecule, for example a nucleic acid molecule encoding an engineered antibody disclosed herein, has been introduced by means of recombinant methodologies and techniques. The progeny of such a cell are also considered within the scope of the disclosure. In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the eukaryotic cell is a HEK293 A cell, a Jurkat cell, or an MCF7 cell.

[0122] Cell cultures containing at least one recombinant cell as disclosed herein are also within the scope of the present disclosure. The terms, "cell", "cell culture", "cell line", "recombinant host cell", "recipient cell" and "host cell" as used herein, include the primary subject cells and any progeny thereof, without regard to the number of transfers. It should be understood that not all progeny are exactly identical to the parental cell (due to deliberate or inadvertent mutations or differences in environment); however, such altered progeny are included in these terms, so long as the progeny retain the same functionality as that of the originally transformed cell.

[0123] The precise components of the expression system are not critical. For example, an engineered antibody as disclosed herein can be produced in a prokaryotic host, such as the bacterium A. coli , or in a eukaryotic host, such as an insect cell (e.g, an S£21 cell), or mammalian cells (e.g, COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, care should be taken to ensure that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans may consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1993) and Pouwels el a/. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0124] The expressed antibody can be purified from the expression system using routine biochemical procedures, and can be used, e.g. , as therapeutic agents, as described herein.

[0125] In some embodiments, engineered antibodies obtained will be glycosylated or unglycosylated depending on the host organism used to produce the engineered antibodies. If bacteria are chosen as the host then the engineered antibodies produced will be unglycosylated. Eukaryotic cells, on the other hand, will glycosylate the engineered antibodies, although perhaps not in the same way as native polypeptides is glycosylated. The recombinant antibodies produced by the transformed host can be purified according to any suitable methods known in the art. Produced recombinant antibodies can be isolated from inclusion bodies generated in bacteria such as E. coli , or from conditioned medium from either mammalian or yeast cultures producing an engineered antibody of the disclosure using cation exchange, gel filtration, and or reverse phase liquid chromatography.

[0126] In addition or alternatively, another exemplary method of constructing a DNA sequence encoding the engineered antibodies of the disclosure is by chemical synthesis. This includes direct synthesis of a peptide by chemical means of the amino acid sequence encoding for an engineered antibody exhibiting the properties described. This method can incorporate both natural and unnatural amino acids at positions that affect the binding affinity of the engineered antibodies with a target protein. Alternatively, a gene which encodes the desired engineered antibodies can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired engineered antibodies, and preferably selecting those codons that are favored in the host cell in which the engineered antibody of the disclosure will be produced. In this regard, it is well recognized in the art that the genetic code is degenerate such that an amino acid may be coded for by more than one codon. For example, Phe (F) is coded for by two codons, TIC or TTT, Tyr (Y) is coded for by TAC or TAT and his (H) is coded for by CAC or CAT. Trp (W) is coded for by a single codon, TGG. Accordingly, it will be appreciated by those skilled in the art that for a given DNA sequence encoding a particular engineered antibody, there will be many DNA degenerate sequences that will code for that engineered antibody. For example, it will be appreciated that in addition to the DNA sequences for engineered antibodies provided herein, there will be many degenerate DNA sequences that code for the engineered antibodies disclosed herein. These degenerate DNA sequences are considered within the scope of this disclosure. Therefore, "degenerate variants thereof' in the context of this disclosure means all DNA sequences that code for and thereby enable expression of a particular engineered antibody.

[0127] The DNA sequence encoding the subject engineered antibody, whether prepared by site directed mutagenesis, chemical synthesis or other methods, can also include DNA sequences that encode a signal sequence. Such signal sequence, if present, should be one recognized by the cell chosen for expression of the engineered antibody. It can be prokaryotic, eukaryotic or a combination of the two. In general, the inclusion of a signal sequence depends on whether it is desired to secrete the engineered antibody as disclosed herein from the recombinant cells in which it is made. If the chosen cells are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence. If the chosen cells are eukaryotic, it generally is preferred that a signal sequence be included.

[0128] The nucleic acid molecules provided can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide, e.g. , antibody. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single- stranded (e.g, either a sense or an antisense strand).

[0129] The nucleic acid molecules are not limited to sequences that encode polypeptides (e.g, antibodies); some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g. , the coding sequence of an engineered antibody) can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by performance of the polymerase chain reaction (PCR). In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription.

[0130] Exemplary isolated nucleic acid molecules of the present disclosure can include fragments not found as such in the natural state. Thus, this disclosure encompasses recombinant molecules, such as those in which a nucleic acid sequence (for example, a sequence encoding an engineered antibody disclosed herein) is incorporated into a vector (e.g, a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a position other than the natural chromosomal location).

Pharmaceutical Compositions

[0131] In some embodiments, the engineered antibodies of the disclosure, and/or nucleic acids as described herein, can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include the engineered antibodies and/or nucleic acids of the disclosure and, optionally, a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds ( e.g ., antibiotics) can also be incorporated into the compositions.

[0132] 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 dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g. , sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, 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.

[0133] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0134] Oral compositions, if used, generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound ( e.g ., engineered antibodies, and/or nucleic acid molecules of the disclosure) can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like, can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel™, or corn starch; a lubricant such as magnesium stearate or Sterotes™; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0135] In the event of administration by inhalation, the subject engineered antibodies of the disclosure are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g, a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[0136] Systemic administration of the subject engineered antibodies of the disclosure can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0137] In some embodiments, the engineered antibodies of the disclosure can also be prepared in the form of suppositories (e.g, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0138] In some embodiments, the engineered antibodies of the disclosure can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol. 20: 1006-1010, 2002), or Putnam (Am. J. Health Syst. Pharm. 53: 151-160, 1996, erratum at Am. J. Health Syst. Pharm. 53:325, 1996).

[0139] In one embodiment, the subject engineered antibodies of the disclosure are prepared with carriers that will protect the engineered antibodies against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0140] In some embodiments, the engineered antibodies of the disclosure can be further modified to prolong their half-life in vivo and/or ex vivo. Non-limiting examples of known strategies and methodologies suitable for modifying the engineered antibodies of the disclosure include (1) chemical modification of an engineered antibody described herein with highly soluble macromolecules such as polyethylene glycol ("PEG") which prevents the engineered antibody from contacting with proteases; and (2) covalently linking or conjugating an engineered antibody described herein with a stable protein such as, for example, albumin. Accordingly, in some embodiments, the engineered antibodies of the disclosure can be fused to a stable protein, such as, albumin. For example, human albumin is known as one of the most effective proteins for enhancing the stability of polypeptides fused thereto and there are many such fusion proteins reported.

[0141] In some embodiments, the engineered antibodies of the disclosure are chemically modified with one or more polyethylene glycol moieties, e.g ., PEGylated; or with similar modifications, e.g. PASylated. In some embodiments, the PEG molecule or PAS molecule is conjugated to one or more amino acid side chains of the interferon. In some embodiments, the PEGylated or PASylated antibody contains a PEG or PAS moiety on only one amino acid. In other embodiments, the PEGylated or PASylated antibody contains a PEG or PAS moiety on two or more amino acids, e.g ., attached to two or more, five or more, ten or more, fifteen or more, or twenty or more different amino acid residues. In some embodiments, the PEG or PAS chain is 2000, greater than 2000, 5000, greater than 5,000, 10,000, greater than 10,000, greater than 10,000, 20,000, greater than 20,000, and 30,000 Da. The engineered antibodies may be coupled directly to PEG or PAS (e.g. , without a linking group) through an amino group, a sulfhydryl group, a hydroxyl group, or a carboxyl group.

[0142] In some embodiments, the pharmaceutical compositions of the disclosure includes one or more pegylation reagent. As used herein, the term "PEGylation" means and refers to modifying a protein by covalently attaching polyethylene glycol (PEG) to the protein, with "PEGylated" referring to a protein having a PEG attached. A range of PEG, or PEG derivative sizes with optional ranges of from about 10,000 Daltons to about 40,000 Daltons may be attached to the engineered antibodies of the disclosure using a variety of chemistries. In some embodiments, the pegylation reagent is selected from methoxy polyethylene glycol -succinimidyl propionate (mPEG-SPA), mPEG-succinimidyl butyrate (mPEG-SBA), mPEG-succinimidyl succinate (mPEG-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG-Succinimidyl Glutarate (mPEG-SG), mPEG-N-hydroxyl-succinimide (mPEG-NHS), mPEG-tresylate and mPEG-aldehyde. In some embodiments, the pegylation reagent is methoxy polyethylene glycol- succinimidyl propionate; preferably said pegylation reagent is methoxy polyethylene glycol- succinimidyl propionate 5000 with an average molecular weight of 5,000 Daltons.

METHODS OF THE DISCLOSURE

Methods for Preparing Engineered Antibodies

[0143] As described above, multispecific antibodies are capable of recognizing multiple ligands simultaneously or synergistically, creating complex biological interactions not achievable by monoclonal antibodies, thus expanding opportunities for novel therapy development. With the large number of monoclonal antibodies either approved or under clinical development, there are numerous opportunities to combine their specificities to further improve therapeutic potential. Although seemingly simple in concept, clinical development of multi specific antibodies face several challenges, chief of which is how to efficiently and reliably produce bispecific and multispecific antibodies with expected specificity and desired biophysical properties. Some embodiments of the disclosure provide a modular approach that uses temporary linkers to enforce proper chain pairing and proteases such as thrombin to remove those linkers from the final product. When combined with the “knob-into-hole” design, IgG-like, multispecific antibodies can be generated from any pre-existing monoclonal antibodies. The approach disclosed herein is highly versatile and applicable to any monoclonal antibody pair or panel, expediting evaluation and therapeutic development of multispecific antibodies.

[0144] In some embodiments, the present disclosure describes a technology that enables modular construction of multispecific antibodies. An exemplary production scheme involves the use of a protease sensitive linker to connect the cognate heavy and light chains to re-enforce the correct pairing, which is then removed by protease such as thrombin. As discussed above, affinity tags can also be built into the linker for further purification of the desired heterodimer before protease cutting. The technology allows facile production of IgG-like bispecific as well as multispecific antibodies with Fab as the binding unit. The technology is modular in nature, analogous to building LEGO toys. Hence, the multispecific antibodies generated by this technology are termed "LegoBodies".

[0145] As described in greater detail herein, an advantageous and modular platform has been developed to generate bispecific and multispecific antibodies from any pre-existing monoclonal antibody. The use of thrombin-removable linkers enforces correct pairing of light and heavy chains and enables efficient in vitro removal of these linkers following purification. Comparing other bispecific formats, such as the CrossMAb or common light chain IgG molecules, the disclosed format requires minimal efforts in design and optimization, imposes no restrictions on the underlying monoclonal antibody (the building block), and can be used as tools to readily generate and evaluate bispecific antibodies from any two monoclonal antibodies of interest. The linker employed herein to enforce proper chain paring is very malleable and can be used to introduce versatile features to aid purification. As an exemplification, different affinity tags have been designed into the linker, and demonstrated that sequential purification by affinity chromatography can effectively eliminate homodimer contaminations. Other features such as asymmetric length or charge (isoelectric point) can be incorporated into the linker to customize the purification scheme. [0146] The disclosed approach, which is modular in nature and permits Lego-like assembly, has been expanded to multispecific antibody generation. As described in Examples 6, two classes of tri-specific and tetra-specific antibodies have been generated by appending additional Fab domains to either the N-terminus of the light chain or the C-terminus of the heavy chain in the starting bispecific molecule. The added Fab domains also use thrombin -removable linkers to enforce correct heavy and light chain pairing. Several tri- and tetra-specific molecules have been successfully generated and their binding verified to corresponding ligands. Given the modular nature of the process, the approach disclosed herein should be applicable to the generation of even higher order of specificities. Given the explosion of information on the molecular mechanism of human diseases and the rapid expansion of the list of potential targets, multispecific antibodies are likely to become an expanding class of novel therapeutics due to their unique ability to generate synergistic or synthetic interactions among targets, thus uncovering new biology and targeting opportunities.

[0147] In one aspect, provided herein are various methods for preparing an engineered antibody, including: (a) providing an engineered antibody as disclosed herein; and (b) removing the removable linker to produce an antibody product that does not contain the removable linker.

[0148] Non-limiting exemplary embodiments of the disclosed methods for preparing an engineered antibody include one or more of the following features. In some embodiments, the engineered antibody is provided by culturing a host cell that co-expresses the first and the second polypeptide chains of the engineered antibody. In some embodiments, the engineered antibody is provided by co-expressing the first and the second polypeptide chains in the same recombinant cell. Alternatively, in some embodiments, the first and the second polypeptide chains are individually expressed from different recombinant cells, and subsequently combined, ex vivo or in vitro , to generate a fully assembled antibody having the first and the second polypeptide chains associated with one another.

[0149] In some embodiments, the methods for preparing an engineered antibody further include a purification process. The purification process can be carried out by essentially separating the engineered antibody from undesirable impurities present in the expression/processing system, such as host cell debris, aggregated unfolded polypeptides, homodimers, and/or incorrectly folded polypeptides which should not be present in the intermediate or final product. The term "impurity" as used herein, in the broadest sense, to refer to any substance which differs from the engineered antibody such that the engineered antibody is not pure. The impurity can include host cell substances such as nucleic acids, lipids, polysaccharides, proteins, etc. ; culture medium, and additives which are used in the preparation and processing of the engineered antibody. In some embodiments, the impurity can include one or more of the following substances: monomeric scIgG, incorrectly folded molecules of scIgG, mispaired byproducts ( e.g ., “hole-hole” or “knob-knob” homodimers), antibodies with incomplete removal of the linker, and host cell proteins.

[0150] The purity of the multispecific antibody products can be assessed by using any suitable analytical techniques known in the art, such as SDS-PAGE, hydrophobic interaction chromatography (HIC), or HIC-HPLC as described in Examples 2 and 4-5 and illustrated in FIGS. 2-3

[0151] In some embodiments, the antibody produced by the disclosed methods includes the engineered antibody with a purity of greater than about 70%. In some embodiments, the antibody produced by the disclosed methods includes the engineered antibody with a purity of greater than about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the antibody produced by the disclosed methods includes the engineered antibody with a purity ranging from about 70% to about 100%, from about 80% to about 95%, from about 90% to about 99%, from about 75% to about 95%, from about 85% to about 100%, or from about 95% to about 100%. In some embodiments, the antibody produced by the disclosed methods includes the engineered antibody lacking the removable linker with a purity of greater than about 70%. In some embodiments, the antibody produced by the disclosed methods includes the engineered antibody lacking the removable linker with a purity of greater than about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the antibody produced by the disclosed methods includes the engineered antibody lacking the removable linker with a purity ranging from about 70% to about 100%, from about 80% to about 95%, from about 90% to about 99%, from about 75% to about 95%, from about 85% to about 100%, or from about 95% to about 100%.

[0152] In some embodiments, the first and the second polypeptide chains are individually expressed from different recombinant cells and purified before they are subsequently combined, ex vivo or in vitro , to generate a fully assembled antibody having the first and the second polypeptide chains are associated with one another. In some embodiments, the engineered antibody is further purified before the removal of the removable linker originally embedded within the scFab fragments. In some embodiments, the engineered antibody is purified after the removal of the removable linker originally embedded within the scFab fragments. In some embodiments, various purification processed can be performed both prior to and after removal of the removable linker(s).

[0153] In some embodiments, the purifying process includes one or more techniques selected from the group consisting of affinity chromatography, ion-exchange chromatography (IEC), anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydroxyapatite chromatography, hydrophobic interaction chromatography (HIC), metal affinity chromatography, and mixed mode chromatography (MMC).

[0154] In some embodiments, the purifying process includes affinity chromatography, e.g ., subjecting a sample containing the engineered antibody to a suitable affinity chromatographic support. Non-limiting examples of such chromatographic supports include, but are not limited to Protein A resin, Protein G resin, affinity supports comprising an antigen against which the antibody of interest was raised, and affinity supports comprising an Fc binding protein. In some embodiments, the affinity chromatography includes protein A affinity chromatography.

[0155] In some embodiments, the purifying process can be carried out using ion-exchange chromatography (IEC) in order to remove other contaminants. In principle, cation exchange chromatography (CEX) and/or anion exchange chromatography (AEX) can be suitably used. Generally, the AEX chromatography can be performed by using any one of functional groups known for AEX chromatography of proteins. These groups include diethylaminoethyl (DEAE), trimethylaminoethyl (TMAE), quaterny aminomethyl (Q), and quatemy aminoethyl (QAE). These are commonly used functional anion exchange groups for biochromatographic processes. Suitable functional groups used for CEX chromatography include, but are not limited to, carboxymethyl (CM), sulfonate (S), sulfopropyl (SP) and sulfoethyl (SE). These are commonly used cation exchange functional groups for biochromatographic processes.

[0156] Throughout this specification, various patents, patent applications and other types of publications (e.g, journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0157] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0158] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

[0159] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

EXAMPLES EXAMPLE 1

General experimental procedures

[0160] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology . New York, NY: Wiley (including supplements through 2014); Bollag, D. M. etal. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. el al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. etal. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, C A: Academic Press; Doyle, A. etal. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. etal. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

EXAMPLE 2

Additional experimental procedures Monoclonal antibody expression and purification

[0161] Genes encoding antibody variable domains were synthesized by gBlock® (Integrated DNA Technologies). Plasmids for the heavy and light chains for each antibody were separately cloned in the Abvec vector as previously described (Lee NK et al. Scientific Reports 2018; 8:766, and Smith K. et al. Nat Protoc 2009; 4:372-84). Generally, antibodies were produced by co-transfecting plasmids expressing the heavy and light chains at a 1:1 ratio in HEK293 A cells for 6-8 days, followed by purification of culture supernatant on protein A agarose (Pierce/Thermo Scientific).

Monoclonal single-chain IsG expression and purification

[0162] The sc36TMB linker (SEQ ID NO: 80) and the heavy chain variable domains were fused by PCR reaction. The resultant DNA fragment was subcloned into Ig-g Abvec vector (NovoPro) and subsequently fused with the heavy chain constant domain and Fc fragment to generate the complete single-chain IgG (scIgG) (see, e.g. , SEQ ID NOs: 1-2 and FIGS. 1A-1D). The resulting plasmid was transfected into HEK293 A cells and scIgG molecules were purified from culture supernatant by protein A agarose (Pierce - Thermo Scientific).

Thrombin cleavase and removal of the enzyme [0163] To remove the sc36TMB linker, the scIgG prepared as described above was mixed with thrombin at the ratio of 50-100 pg antibody per unit of thrombin (Millipore, 605160), and the mixture was incubated at 37°C for 2 hours. The processed IgG molecule was re-purified by protein A agarose to remove the enzyme. The conversion of the scIgG to IgG was verified by SDS-PAGE with reducing agent added to the sample.

Bisyecific antibody expression and purification

[0164] The “knob” Fc (T366W) or “hole” Fc (T366S/L368A/Y407V) fragments ^{41, 42} were introduced to the Ig-g chain by site-directed mutagenesis. The fused gene encoding the complete light chain, sc36TMB linker, and heavy chain variable domain was subcloned into either the “knob” or “hole” vector as described in greater detail below, for example in Examples 3-6, SEQ ID NOS: 3-5, and FIGS: 2A-2L. To produce the scIgG bispecific antibody, a pair of the “knob” and “hole” vectors, each encoding one scIgG antibody chain, were co-transfected into HEK293 A cells at a 1:1 ratio. After culturing for 6-8 days, the bispecific scIgG was purified from culture supernatant by protein A agarose. The sc36TMB linkers in the bispecific scIgG were then removed by thrombin cleavage as described above, yielding the bispecific IgG. Detailed information for various exemplary single-chain polypeptides of the disclosure can be found in Table 1 below.

TABLE 1: This table provides a brief description for each of the single-chain polypeptides, their corresponding components, as well as corresponding sequence identifiers as set forth in the Sequence Listing. Atez: Atezolizumab; Dara: Daratumumab; Her: Herceptin; Ipil: Ipilimumab.

Tri-specific and Tetra-specific antibody expression and purification

[0165] The Agel restriction site between the sequences encoding the signal peptide and N- terminus of the antibody gene in the Abvec vector was retrained in the construct for scIgG with either the “knob” or “hole” Fc fragment. This Agel site was used to introduce an additional Fab gene (with a thrombin removable linker in-between the light and heavy chain) and the N-linker (- ASTKGPSGSG-; SEQ ID NO: 81) by a ligase independent cloning technique. To produce the tri- and tetra-specific antibodies, the pair of “knob” and “hole” vector were co-transfected into HEK293 A cells. Supernatant collected after 6-8 days was purified on protein A agarose, and the sc36TMB linkers were removed by thrombin treatment.

[0166] For producing the tri-specific antibody, the tandem Fab (Ipilimumab and Herceptin) with the “hole” Fc vector was paired the single-chain Daratumumab vector with “knob” Fc fragment (see, e.g. , FIGS. 4A-4F). The resulting tri-specific antibody was termed Tri-N-Fab antibody. For the tetra-specific, the tandem Fab (Ipilimumab and Herceptin) with the “hole” Fc vector was paired the tandem Fab (Daratumumab and Atezolizumab) with “knob” Fc vector (see, e.g. , FIGS. 5A-5G). The resulting tetra-specific antibody was termed Tetra-N-Fab antibody.

Both Tri-N-Fabs and Tetra-N-Fabs molecules were submitted for ion-exchange chromatography for additional purification.

[0167] Alternatively, a Hin dill restriction site located at the C-terminus of the antibody gene was used to introduce the C-linker (-GGGSGGGSGGGSG-; SEQ ID NO: 82) and an extra Fab domain to the “knob” or “hole” vector (see, e.g, FIGS. 6A-6J). To produce the Tri-C-Fabs molecule, the “hole” vector with two Fab modules (Ipilimumab at N-terminus and Herceptin at C-terminus) was co-expressed with the “knob” Fc vector for the single-chain Daratumumab Fab in HEK293A cells (see, e.g, FIG. 6A). To produce the Tetra-C-Fabs molecule, the above “hole” vector was co-expressed with the “knob” Fc vector with two Fab modules (Daratumumab at N- terminus and Atezolizumab at C-terminus) in HEK293A cells (see, e.g, FIG. 6B). Following purification by protein A agarose, the linkers were removed by thrombin treatment. Ion- exchange chromatography was applied to improve the purity.

Purification by ion-exchange chromatography

[0168] The tri-specific and tetra-specific molecules were further purified by ion-exchange chromatography after the removal of intra-Fab linkers. In brief, the molecules were applied to a Mono S™ 5/50 GL column (GE Healthcare) on AKTA (GE Healthcare). The following buffers were used: mobile phase buffer A: 20 mM MES (2-morpholin-4-ylethanesulfonic acid), pH 6.0; and mobile phase buffer B: 20 mM MES, 1 M NaCl, pH 6.0. After loading and washing the samples with buffer A, gradient elution with 10% to 50% B were used for fractionation.

Fractions were collected and analyzed by SDS-PAGE.

Ligand preparation and biotinylation

[0169] Extracellular domain Fc fusions for human CTLA-4 (CTLA4-Fc) and ErbB2 (ErbB2-Fc) were purchased from Abeam (ab 180054 and ab 168896). These ligands were then biotinylated by the EZ-link® Sulfo-NHS-LC-Biotin according to the manufacturer's protocol (Thermo Scientific). Excessive biotin was removed by buffer-exchange into PBS, and biotinylated ligands were concentrated and stored at -80°C. The recombinant extracellular domain of CD38 and PD-L1 was produced as a 6-His and AviTag™ fusion in HEK293 cells and purified by Ni-NTA followed by in vitro biotinylation by BirA biotin ligase (Avidity).

ELISA and EC so estimation

[0170] To accommodate the differences in valency between monoclonal and bi-, tri-, or tetra-specific antibodies, an ELISA assay was performed by immobilizing the antibody on a microtiter plate and testing binding to ligands in solution. The antibodies were diluted to 1 pg/ml in PBS and 100 mΐ of this diluted solution per well were applied to the Nunc MaxiSorp ELISA plate (Thermo Fisher) for coating overnight. The plate was washed three times with PBS, blocked with 4% BSA at RT for 1 hour, and incubated with corresponding biotinylated ligands at various concentrations at RT for 1 hour, each condition in triplicates. The plate was then washed five times with the washing buffer (0.05% Tween-20 in PBS), incubated with 0.1 pg/ml HRP- conjugated streptavidin (Pierce-Thermo Fisher) at RT for 30 minutes, washed 3 times with the washing buffer, and incubated with the peroxidase substrate solution (SureBlue®, Seracare) at RT for 3-5 min before the reaction was terminated by adding equal volume of 1 M HC1. The absorbance at 450 nm of each well was detected by a microtiter plate reader (Synergy BioTek® instrument). The absorbance value as a function of the ligand concentrations was analyzed to obtain the EC50 value by curve fit (Prism, GraphPad).

Purity assessment by analytical hydrophobic interaction chromatography

[0171] Purified antibodies were analyzed by HIC-HPLC with the infinity 1220 LC System (Agilent). Mobile phase A consisted of 25 mM phosphate and 1.5 M ammonium sulfate, pH 7.0. Mobile phase B consisted of 25% (v/v) isopropanol in 25 mM phosphate, pH 7.0. Antibody samples were loaded onto a TSK gel HIC column (Tosoh Bioscience) and operated at 0.5 mL/min with a gradient from 10% to 100% B. Absorbance was detected at 280 nm. Purity was estimated by area integration using OpenLab CDS software (Agilent).

Cell binding analysis by flow cytometry

[0172] Jurkat and MCF7 cell lines obtained from American Type Culture Collection (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal calf serum and 100 pg/ml penicillin- streptomycin in humidified atmosphere of 95% air and 5% CO2 at 37°C. For flow cytometry analysis, approximately 50,000 cells were incubated with different monoclonal, bispecific or multispecific antibodies (the highest concentration of 200 nM with serial 4-fold dilutions) at RT for 1 hour, washed 3 times with PBS and further incubated with the secondary antibody solution (Alexa Fluor 647®-conjugated goat anti-human IgG, final concentration of 1 pg/ml) at RT for 1 hour, washed 3 times with PBS and analyzed by flow cytometry (Accuri™C6, BD Biosciences). The median fluorescence intensity (MFI) values were analyzed by Prism (GraphPad) to obtain EC 50 values by curve fitting.

Kd determination by bio-layer interferometry

[0173] A Gator instrument (Probe Life) was used to determine K _d of antibody-ligand interactions by bio-layer interferometry. Biotinylated ligands were diluted to 5-10 pg/ml in the provided kinetics buffer (Probe Life) and immobilized onto streptavidin-coated biosensors. The sensors were sequentially incubated with antibodies (200 nM) and kinetics buffers for 180 seconds to assess rates of association and disassociation. K _d values were analyzed by the Gator software (Probe Life).

EXAMPLE 3

Monoclonal antibodies produced from thrombin-removable single-chain IgGs show similar biochemical features as native IgGs

[0174] This Example describes experiments performed to develop an efficient system for removing peptide linker to obtain a true IgG-like molecule. This is because single-chain IgGs using peptide linkers to join the light chain and heavy chain have been investigated previously for production and ligand binding.

[0175] In these experiments, several commercially available proteases were tested and determined that thrombin is a suitable enzyme because of its efficiency, accuracy, and compatibility with non-reducing environments. Two clinically established antibodies,

Ipilimumab (Ipili) and Daratumumab (Dara), were selected as the study antibodies to generate scIgGs of Ipili and Dara with a linker positioned between the light chain and heavy chain. The linker, sc36TMB, is a flexible 36-residue peptide adapted from a previous study, with additional thrombin cleavage sites on both the N- and C-terminus (FIG. 7). As shown in Table 2 below, the engineered scIgG displayed similar yield as the original antibodies. In these experiments, antibodies were purified by protein A affinity capture from culture supernatant collected from cell culture dish (100 mm in diameter) 4 days following transient transfection of HEK293 A cells.

TABLE 2:

[0176] Furthermore, the linker was successfully removed by thrombin cleavage, yielding a -150 kDa antibody product formed by disulfide-bonded light (-25 kDa) and heavy chains (-50 kDa), same as the natural antibody (Table 2). Following thrombin cleavage, a 5-mer peptide (- GLVPR, SEQ ID NO: 83) remained at the C-terminus of the light chain, as well as two residues (-GS) at the N-terminus of the heavy chain (see, e.g ., FIG. 7). To evaluate if the processed IgG molecule possesses an intact paratope, the ligand binding was tested by ELISA (FIGS. 1C-1D). Ipilimumab was found to bind to the CTLA4-Fc with an EC50 of approximately 0.99 ± 0.13 nM, while the EC50 of the cleaved Ipili-sc36TMB was estimated to be about 0.83 ± 0.11 nM (FIG. 1C). Likewise, Daratumumab was found to bind its ligand, CD38, with EC50 of approximately 1.56 ± 0.11 nM, and the cleaved Dara-sc36TMB had an EC50 of 1.24 ± 0.09 nM (FIG. ID). The similar EC50 values between the processed scIgGs and the original antibodies indicate that the engineered linker and the cleavage process did not interfere with the formation of the antibody or the integrity of its paratope.

EXAMPLE 4

Bispecific antibodies produced from thrombin-cleavable single-chain IgGs with the “knobs-into- holes” Fc heterodimer

[0177] This Example describe experiments performed to expand the study from monoclonal to bispecific antibody generation. The “knobs-into-holes” (KIH) design features critically paired mutations in the “knob” Fc (T366W) and “hole” Fc (T366S/L368A/Y407V), which enforces pairing of the KIH Fc heterodimer. However, this KIH design does not enforce proper light chain pairing.

[0178] The successful production and thrombin-cleavage of the scIgGs to IgG molecules provides a plausible solution for enforcing correct pairing between light and heavy chains. The experiments described herein were performed to generate bispecific antibodies using the KIH Fc system in conjunction with a thrombin-removable linker. Two sets of antibodies, Ipilimumab pairing with Daratumumab, and Ipilimumab pairing with Herceptin, were selected as the test samples. The scheme to produce bispecific antibodies is shown in FIG. 2A. In brief, the light chain of each antibody is fused with the corresponding heavy chain via a thrombin-removable linker. The Fc region used for Ipilimumab was the “hole” Fc region, while the Fc for either Daratumumab or Herceptin was the “knob” Fc region. Bispecific antibodies were produced by co-transfecting HEK293 A cells with the “knob” and “hole” scIgG constructs at a 1:1 ratio, and purified from culture supernatant by protein A agarose, and processed by thrombin to remove the linker.

[0179] Using this system, experiments described herein have illustrated that it was obtain relatively pure IgG-like molecules for both bispecific antibodies with estimated molecular weights of -150 kDa under non-reducing condition. As shown in FIG. 2B, the KIH product could be separated into light chain (-25 kDa) and heavy chain (-50 kDa) by reduction with b- mercapto ethanol (b-ME). The purity of the KIH product was assessed using analytical hydrophobic interaction chromatography (HIC). Both Ipili-Dara-KIH and Ipili-Her-KIH antibodies displayed a dominant bispecific component, comprising 71% and 73% of the total product, respectively. FIGS: 2G-2L summarize the results of a purity assessment of the bi specific antibodies Ipili-Dara-KIH and Ipili-Her-KIH by analytical hydrophobic interaction chromatography. The main peak of Ipili Dara KIH (FIG. 2G) shows an elution time in-between of Ipilimumab (FIG. 2H) and Daratumumab (FIG. 21), representing the desired bispecific product that is estimated by area integration (OpenLab CDS, Agilent) to be 71% of total protein obtained from one-step protein A purification. For the Ipili-Her-KIH antibody, the main peak of Ipili Her KIH (FIG. 2J) shows an elution time in-between of Ipilimumab (FIG. 2K) and Herceptin (FIG. 2L), representing the desired bispecific product that is estimated to be 73% of total protein obtained from one-step protein A purification. The integrity of the paratopes of each Fab in the bispecific antibody was also investigated by ELISA. Unlike an IgG molecule that binds bivalently to one ligand, the binding of the IgG-like bispecific antibody to each of its two ligands is monovalent. To minimize the influence of valency in the ELISA assay, the antibodies were mobilized on plates and the binding to ligands was assessed in solution. As shown in FIGS. 2C-2D, and Table 3 below, the Ipili-Dara-KIH was capable of binding to both CTLA4 and CD38, in manners similar to the parental monoclonal antibodies (Ipilimumab and Daratumumab), indicating that the bispecific antibodies possessed intact paratopes. Similar results were observed for the bispecific Ipili-Her-KIH (FIGS. 2E-2F, and Table 4).

TABLE 3 :

TABLE 4:

EXAMPLE 5

Use of linker-embedded affinity tags for further purification of bispecific antibodies

[0180] This Example describes the use of linker-embedded affinity tags for further purification of bispecific antibodies described above. Contamination in bispecific antibodies produced by the KIH approach has been observed previously, which primarily arises from “hole- hole” or “knob-knob” homodimers. Common procedures for antibody purification, such as protein A or G affinity chromatography, reply on recognition of the Fc domain and thus cannot effectively distinguish the bispecific KIH heterodimer from contaminating homodimers. Prior approaches to enhance the efficiency of correct pairing of the KIH heterodimer or eliminate the non-bispecific contamination often involve introducing additional mutations and additional steps in purification.

[0181] Some experiments described herein took advantage of the removable nature of the linker and embedded a pair of affinity tags, the 10-His tag and Twin-Strep-Tag®, into the linkers to enable the purification of the bispecific product (see, e.g ., FIG. 3A and SEQ ID NOS: 6-7). The KIH heterodimer would possess both affinity tags and thus be readily distinguished from homodimers. Two following plasmids have been constructed: the first plasmid expressing Ipilimumab with a Twin-Strep-Tag® linker and the “hole” Fc mutant region, and the second plasmid expressing Daratumumab or Herceptin with a 10-His tag linker and the “knob” Fc mutant region. The resulting plasmids were co-transfected HEK293 A cells to generate bispecific antibodies, i.e., Ipili-Dara-KIH and Ipili-Her-KIH. The supernatant was sequentially purified using Ni-NTA and Strep-Tactin®XT resin to obtain antibody products with both tags and thus both specificities. The linkers were removed by thrombin cleavage to generate the final product. As shown in FIG. 3B, SDS-PAGE results showed that bispecific antibodies produced in this manner contained substantially lower amounts of containments (labeled with *). In addition, HIC-HPLC analysis showed that the purities of the Ipili-Dara-KIH and Ipili-Her-KIH were nearly 100% and about 92%, respectively (FIGS. 3C-3D).

[0182] Subsequent experiments were performed to investigate how bispecific antibodies recognized cells expressing target antigens. The Jurkat cell line has been previously used to study CD38 binding. In the experiments described herein, it was found that indeed Jurkat cells express CD38 were bound by Daratumumab (FIG. 3E). However, little to no expression of CTLA4 was detected by Ipilimumab (FIG. 3E). Thus Jurkat cells are useful mainly for assessing the CD38 binding arm. As shown in Table 5 below, the bispecific antibody, Ipili-Dara-KIH, which is monovalent in each specificity, was found to bind to Jurkat cells with EC ₅₀ of 2.26 ± 0.26 nM. Daratumumab, a bivalent IgG, bound to Jurkat cells with EC ₅₀ of 0.31 ± 0.05 nM. The bispecific Ipili-Dara-KIH bound to Jurkat cells with a higher median fluorescence intensity (MFI) value compared with Daratumumab, consistent with its monovalent binding mode.

TABLE 5: Binding to CD38 and ErbB2 was tested on Jurkat cells and MCF7 cells, respectively. The apparent Kd values were obtained by curve fitting MFI values (Prism, GraphPad). [0183] Similarly, additional experiments were performed to investigate how the bispecific Ipili-Her-KIH antibody binds to the breast cancer cell line MCF7 that has been reported to express both ErbB2 (Her2) and CTLA4. It was observed that a high staining signal by flow cytometry on MCF7 by Herceptin, but a rather low signal by Ipilimumab. Thus, MCF7 cells were used to evaluate the Her2 -binding arm only. The bispecific Ipili-Her-KIH binds to MCF7 (FIG. 3F) with EC50 of 1.39 ± 0.21 nM (Table 5). Herceptin, a bivalent IgG, binds to MCF7 (FIG. 3F) with EC50 of 0.60 ± 0.18 nM (Table 5). The total MFI for the bispecific Ipili-Her-KIH is higher than Herceptin, consistent with its monovalent binding mode.

[0184] To evaluate binding of bispecific antibodies to CTLA4 on cell surface, HEK293A cells were transiently transfected with a construct expressing the human CTLA4 gene. After culturing for 18 hours, the cells were harvest and analyzed by flow cytometry for binding by Ipilimumab IgG, Ipilimumab Fab, Ipili-Dara-KIH, and Ipili-Her-KIH. Like Ipilimumab, it was observed that both bispecific antibodies were able to bind to the CTLA4-transfected cells (data not shown), confirming that the anti-CTLA-4 arm is functional. As shown in Table 6 below, the apparent binding affinities of the bispecific antibodies were lower than that of the bivalent Ipilimumab IgG, an expected result given the valency difference, but higher than the Ipilimumab Fab that is also monovalent. MFI values at saturation binding were higher for the bispecific (and the Ipilimumab Fab) than the Ipilimumab IgG, again consistent with the monovalent vs. bivalent binding mode.

TABLE 6: The apparent Kd values were obtained by curve fitting MFI values (Prism, GraphPad). EXAMPLE 6

Facile generation of multispecific antibodies using thrombin-removable linkers

[0185] This Example describes experiments performed taking advantage of the modular nature of the linker-enforced chain assembly to generate multispecific antibodies using the enzymatically cleavable linker. The generation and application of antibodies with even higher order of specificity and complexity are challenging tasks that have only been attempted a limited number of times. In general, as the complexity of the molecule grows, the difficulty grows disproportionally or exponentially in proper assembly of six, eight, or more chains into one antibody. As described in greater detail below, multispecific antibodies were designed based on the IgG-like bispecific with additional specificities introduced by appending Fab domains to either the N-terminus (FIGS. 4-5, tandem Fabs) or C-terminus (FIG. 6) of the bispecific molecule.

[0186] In the case of tandem Fab constructs shown in FIG. 4 (tri-specific) and FIG. 5 (tetra-specific), the four different chains (two heavy and two light chains) of two antibodies (I, III) were genetically fused into one polypeptide in the following configuration:

[0187] Light chain-sc36TMB linker-heavy chain-N-linker-light chain (I)-sc36TMB linker-heavy chain (I) and “hole” Fc mutant region (as illustrated in FIG. 4A, SEQ ID NO: 9).

[0188] This tandem Fab construct was paired with an antibody construct (position II) with the “knob” Fc mutant to produce the tri-specific antibodies (FIG. 4A, SEQ ID NO: 4 and 9). To produce a tetra-specific molecule, two other antibodies (II, IV) were constructed in a second tandem Fab construct with the “knob” mutant in a configuration similar to that of the “hole” Fc mutant (FIG. 5A, and SEQ ID NO: 9-10).

[0189] Co-expression of resulting constructs produced the precursors for either tri-specific (FIG. 4A, SEQ ID NO: 4 and 9) or tetra-specific (FIG. 5A, and SEQ ID NO: 9-10) molecules, which were then processed by thrombin cleavage to remove the linker scaffold within each Fab unit. In this way, the following antibodies were generated: the asymmetric tri-specific antibody (Tri-N-Fabs) and the symmetric tetra-specific antibody (Tetra-N-Fabs), with Ipilimumab in position I, Daratumumab in position II, Herceptin in position III, and Atezolizumab in position IV (in the case of Tetra-N-Fabs). [0190] By SDS-PAGE analysis, it was found that purified Tri-N-Fabs and Tetra-N-Fabs displayed correct molecular weights, estimated to be 200 kDa and 250 kDa respectively, under non-reducing conditions and were separated into polypeptides with molecular weights of approximately 50 kDa and 25 kDa under reducing conditions (FIGS. 4B, 5B). Purity of the final products was assessed by analytical HIC-HPLC and found to be approximately 87% for Tri-N- Fabs (FIG. 4C) and 95% for Tetra-N-Fabs (FIG. 5C). Antigen binding was evaluated by ELISA, where the antibody was immobilized on plate, and tested for binding to biotinylated ligands in solution (CTLA4-Fc, CD38, ErbB2-Fc, and PD-L1). Both the Tri-N-Fabs and the Tetra-N-Fabs showed binding to all corresponding ligands with apparent affinity similar to that of the parental antibodies (see, FIGS. 4D-4F and Table 7 for Tri-N-Fabs; FIGS. 5D-5G and Table 8 for Tetra-N-Fabs). In these experiments, estimated EC50 values of ligand binding by Tri-N-Fabs along with the parental antibodies were analyzed by ELISA. OD450 values were curve-fit (Prism, GraphPad) to obtain EC50 values. These results indicate that the Tri-N-Fabs and the Tetra-N-Fabs possess the designed paratopes that are functional.

TABLE 7: Assessment of ligand binding of tri-specific Tri-N-Fabs antibodies.

TABLE 8: Assessment of ligand binding of tetra-specific Tetra-N-Fabs antibodies.

[0191] Experimental data presented in FIGS. 6A-6J illustrates an alternative way to arrange the different Fab modules in a tri- or tetra-specific antibody is to append the extra Fabs to the C-terminus of the Fc domain (FIG. 6A for the tri-specific, Tri-C-Fabs; and FIG. 6B for the tetra-specific, Tetra-C-Fabs). For the asymmetric Tri-C-Fabs, the two chains were in the following configuration:

[0192] Light chain (I)-sc36TMB linker-Fab heavy chain (I)-”hole” Fc mutant-[C- linker]-light chain (III)-sc36TMB linker-Fab heavy chain (III); and light chain (II)-sc36TMB linker-Fab heavy chain (II)-”knob” Fc mutant (FIG. 6A and SEQ ID NOS: 4 and 11).

[0193] The C-linker was designed as a 13-residue long Gly-Ser peptide as shown in SEQ ID NO: 82 of the Sequence Listing.

[0194] For the symmetric Tetra-C-Fabs, the two chains were in the following configuration:

[0195] Light chain (I/II)-sc36TMB linker-Fab heavy chain (I/II)-”hole”/”knob” Fc mutant-C-linker-light chain (III/IV)-sc36TMB linker-Fab heavy chain (III/IV) (FIG. 6B; SEQ ID NOS: 11 and 12).

[0196] These multispecific antibodies were produced by co-transfecting cells with plasmids expressing the corresponding polypeptide chains followed by purification on protein A agarose. The linkers positioned between light and heavy chains were removed by thrombin cleavage prior to analysis.

[0197] As shown in FIGS. 6A-6J, the following antibodies were successfully produced: a tri-specific antibody (Tri-C-Fabs) with Ipilimumab Fab at position I, Daratumumab Fab at II, and Herceptin Fab at III; and a tetra-specific antibody (Tetra-C-Fabs) with the addition of Atezolizumab Fab at position IV. The main products for both Tri-C-Fabs and Tetra-C-Fabs displayed the correct molecular weights by SDS-PAGE analysis (FIG. 6C). FIGS. 6K-6L summarize the results of a purity assessment of tri-specific and tetra-specific antibodies by analytical hydrophobic interaction chromatography. Purities of Tri-C-Fabs (FIG. 6K) and Tetra- CFabs (FIG. 6L) are estimated to be 93% and 79%, respectively. Both Tri-C-Fabs and Tetra-C- Fabs were able to bind their intended ligands by ELISA, with EC ₅₀ similar to that of the parental antibodies, i.e., Ipilimumab, Daratumumab, and Atezolizumab (see, FIGS. 6D-6J, and Tables 9- 10). In these experiments, estimated EC50 values of ligand binding by Tri-N-Fabs along with the parental antibodies were analyzed by ELISA. OD ₄₅₀ values were curve-fit (Prism, GraphPad) to obtain EC ₅₀ values. It was observed that there was a reduction in target binding by the Herceptin Fab in these multispecific products. With regard to ErbB2 binding, the Tri-C-Fabs and Tetra-C- Fabs display approximately 9- and 3- fold higher EC ₅₀ values compared to the parental antibody Herceptin. However, the effect seems to be unique to the Herceptin Fab as the Atezolizumab Fab in a similar position in Tetra-C-Fabs was not affected (FIG. 6J).

TABLE 9: Assessment of ligand binding of tri-specific Tri-C-Fabs antibodies.

TABLE 10: Assessment of ligand binding of tetra-specific Tetra-C-Fabs antibodies.

[0198] The binding kinetics of the bispecific or multispecific antibodies to corresponding ligands was also evaluated by biolayer-interferometry (BLI). In these experiments, the biotinylated ligands were immobilized on the streptavidin-coated sensor and measured antibody binding. It was found that the bispecific and multispecific antibodies bind to their ligands in manners expected from their monovalent binding mode Tables 11-12). The parent monoclonal IgGs showed lower Kd values due to avidity (bivalent vs. monovalent), but the parent monovalent Fab (Ipilimumab Fab) showed similar Kd as the bispecific and multispecific antibodies.

Table 11 : Evaluation of antibody binding kinetics (K _d ) by biolayer-interferometry.

Table 12: Evaluation of antibody binding kinetics (K _0ff) by biolayer-interferometry.

[0199] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented. REFERENCES

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