FOR IMMUNOREGULATION

FIELD

[001] The present disclosure relates to antibody-cytokine engrafted compositions that bind to interleukin-ten receptor (IL10R), and stimulate signaling through this receptor.

BACKGROUND

[002] Interleukin 10 (IL10) was identified as a cytokine synthesis inhibition factor, exerting effects on Thl helper cells and antigen presenting cells (APC's). IL10, a 161 amino acid protein that exists as a domain-swapped non-covalent homodimer, binds to the high affinity IL-10 receptor I (IL-10RI) with a stoichiometry of one homodimer to four IL-10RI subunits that recruits IL-10R2 and activates signal transduction through JAKI/TYK2 pathway. IL10 inhibits monocyte and macrophage synthesis of pro-inflammatory cytokines, e.g., IL-1, IL-6, IL-8, IL- 12, TNF-alpha, GM-CSF, and reactive oxygen and nitrogen intermediates, and has shown strong efficacy in numerous preclinical models of autoimmunity. IL10 efficacy is thought to result from its ability to inhibit activation and effector function of monocytes, macrophages, and T cells. However, IL10 is a pleiotropic immunoregulatory cytokine with a very broad spectrum of biological activities. IL10 has also been shown to promote growth and differentiation of B cells, NK cells, cytotoxic and helper T cells, mast cells, granulocytes, dendritic cells, keratinocytes, and even endothelial cells, indicating it also possesses pro-inflammatory functions (Moore et al, Annu Rev Immunol. 2001;19:683-765).

[003] IL10 as a therapeutic for the treatment of autoimmune and inflammatory conditions, specifically inflammatory bowel disease (IBD), is supported in animal model systems as well as genetics. For example, knockout of IL10 in mice and mice with a defect in the IL-1 OR develop a spontaneous onset of colitis. In humans, 9-16% of Crohn's Disease patients have a NOD2 mutation that is associated with defective or reduced IL10 production, and 20% of ulcerative colitis (UC) patients carry a disease-associated IL10 SNP in the 3'UTR. Lastly, certain mutations in IL-10R1 in humans cause a rare, severe form of Crohn's Disease (Glocker at al, N Engl J Med. 2009; 361(21):2033-45). [004] Despite the strong genetic linkage data, recombinant human IL10 demonstrated tolerability and safety in healthy volunteers and specific patient populations up to 25 μg/kg, and mild effects in a subset of recipients up to 100 μg/kg; however only mild clinical efficacy was found for the specific patient populations and clinical development was discontinued for lack of efficacy. Reviews of the studies have been undertaken and several possibilities for the results have been identified, for example, limited efficacy is due to poor exposure of the target organ (colon) due to short half-life of IL10; the pro-inflammatory effects of IL10 that manifested at high doses and counterbalanced the anti-inflammatory efficacy of low dose IL10. (Lindsay and Hodgson, Aliment Pharmacol Ther. 2001; 15(11) 1709-1716) Consistent with this possibility, high doses of IL10 were associated with elevated systemic IFNy, granzyme and neopterin levels, which are associated with increased inflammation (Tilg et al, Gut 2002; 50(2)191-5).

[005] Antibody cytokine engrafted proteins have been developed to generate a more effective IL10 therapeutic that confers an improved profile that address the clinical failings of recombinant human IL10. This improved therapeutic profile includes strong anti-inflammatory IL10 potency with a marked reduction in pro-inflammatory activity, extended half-life, ease of administration and stability. Thus, the disclosure provides improved IL10 antibody cytokine engrafted proteins and methods of treating immune related disorders.

SUMMARY

[006] The disclosure provides for antibody-cytokine engrafted proteins having preferred therapeutic profiles over recombinant IL10 and IL10 constructs known and used in the clinic. In particular, provided are antibody cytokine engrafted proteins that maintain the desired anti-inflammatory activity of native or recombinant human IL10; however, a marked reduction in pro-inflammatory activity is retained. Additionally, the antibody cytokine engrafted proteins convey improved half-life, stability and ease of administration over recombinant human IL10 protein (rhlLlO). The preferred properties of these compositions result in preferable therapeutic compositions over those previously used in the clinic or described in the literature. The present disclosure thus provides antibody cytokine engrafted proteins that bind to and promote signalling through IL10 receptors and stimulate certain cell types. Provided are antibody cytokine engrafted proteins comprising: (i) an immunoglobulin heavy chain sequence comprising a heavy chain variable region (VH) and a heavy chain constant region comprising CHI, CH2 and CH3 regions, and (ii) an immunoglobulin light chain sequence comprising a light chain variable region (VL), and a light chain constant region (CL); wherein a monomeric IL10 molecule is engrafted into a complementarity determining region (CDR) of the VH or the VL.

[007] In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 13 and a variable light chain comprising SEQ ID NO: 14. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 29 and a variable light chain comprising SEQ ID NO: 30. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 45 and a variable light chain comprising SEQ ID NO: 46. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 61 and a variable light chain comprising SEQ ID NO: 62. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 77 and a variable light chain comprising SEQ ID NO: 78. In some embodiments, the antibody cytokine engrafted protein a variable heavy chain comprising SEQ ID NO: 93 and a variable light chain comprising SEQ ID NO: 94. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 109 and a variable light chain comprising SEQ ID NO: 110. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 125 and a variable light chain comprising SEQ ID NO: 126. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 141 and a variable light chain comprising SEQ ID NO: 142. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 157 and a variable light chain comprising SEQ ID NO: 158. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 173 and a variable light chain comprising SEQ ID NO: 174. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 189 and a variable light chain comprising SEQ ID NO: 190. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 205 and a variable light chain comprising SEQ ID NO: 206. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 222 and a variable light chain comprising SEQ ID NO: 223. In some embodiments, the antibody cytokine engrafted protein includes a variable heavy chain comprising SEQ ID NO: 238 and a variable light chain comprising SEQ ID NO: 239. [008] In certain embodiments, the antibody cytokine engrafted protein comprises an

IgG class antibody Fc region. In particular embodiments, the immunoglobulin is selected from IgGl , IgG2, or IgG4 subclass Fc region. In some embodiments, the antibody cytokine engrafted protein optionally contains at least one modification that modulates (i.e., increases or decreases) binding of the antibody or antibody fragment to an Fc receptor. The immunoglobulin heavy chain of the antibody cytokine engrafted protein optionally comprises a modification conferring modified effector function. In particular embodiments the immunoglobulin heavy chain comprises a mutation conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[009] In a related aspect, the disclosure further provides polynucleotides encoding at least a heavy chain and/or a light chain protein of an antibody cytokine engrafted protein as described herein. In another related aspect, the disclosure further provides host cells suitable for the production of an antibody cytokine engrafted protein as described herein. In particular embodiments, host cells comprise nucleic acids encoding a heavy chain and/or light chain polypeptide of the antibody cytokine engrafted protein. In still another aspect, methods for producing antibody cytokine engrafted proteins of the disclosure are provided, comprising culturing provided host cells as described herein under conditions suitable for expression, formation, and secretion of the antibody cytokine engrafted protein and recovering the antibody cytokine engrafted protein from the culture. In a further aspect, the disclosure further provides kits comprising an antibody cytokine engrafted protein, as described herein.

[0010] In another related aspect, the disclosure further provides compositions comprising an antibody cytokine engrafted protein, as described herein, and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides pharmaceutical compositions comprising an antibody cytokine engrafted protein of the disclosure for administering to an individual.

[0011] In another aspect, the disclosure provides methods of treating an immune related disorder in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody cytokine engrafted protein of the disclosure, as described herein. In a further aspect, the disclosure provides an antibody cytokine engrafted protein for use in treatment or prophylaxis of an immune related disorder in an individual. [0012] In some embodiments, the patient has an immune related disorder, for example, inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, type I diabetes, acute pancreatitis, uveitis, Sjogren's disease, Behcet's disease, sarcoidosis, and graft versus host disease (GVHD). In particular embodiments the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0013] In some embodiments the disclosure provides an antibody cytokine engrafted protein comprising:

(a) a heavy chain variable region (VH), comprising Complementarity Determining Regions (CDR) HCDR1, HCDR2, HCDR3; and

(b) a light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3; and

(c) an Interleukin 10 (IL10) monomeric molecule engrafted into a CDR of the VH or the VL.

[0014] The antibody cytokine engrafted protein wherein the IL10 monomeric molecule is engrafted into a heavy chain CDR.

[0015] The antibody cytokine engrafted protein wherein the heavy chain CDR is selected from HCDR1, HCDR2 or HCDR3.

[0016] The antibody cytokine engrafted protein wherein the IL10 monomeric molecule is engrafted into HCDR1.

[0017] The antibody cytokine engrafted protein wherein the IL10 monomeric molecule is engrafted into a light chain CDR.

[0018] The antibody cytokine engrafted protein wherein the light chain CDR is selected from LCDR1, LCDR2 or LCDR3.

[0019] The antibody cytokine engrafted protein wherein the IL10 monomeric molecule is engrafted into a LCDR1.

[0020] The antibody cytokine engrafted protein wherein the antibody cytokine engrafted protein has less activation of T cells or NK cells when compared to IL10.

[0021] The antibody cytokine engrafted protein wherein the antibody cytokine engrafted protein has a longer half-life than rhlLlO.

[0022] The antibody cytokine engrafted protein wherein the IL10 monomeric molecule consists of SEQ ID NO: 209.

[0023] The antibody cytokine engrafted protein further comprising an IgG class antibody heavy chain. [0024] The antibody cytokine engrafted protein wherein the IgG class heavy chain is selected from IgGl, IgG2, or IgG4.

[0025] The antibody cytokine engrafted protein wherein the binding specificity of the

CDRs to a target protein is reduced by the presence of the engrafted IL10 monomeric molecule.

[0026] The antibody cytokine engrafted protein wherein the binding specificity of the

CDRs to a target protein is retained in the presence of the engrafted IL10 monomeric molecule.

[0027] The antibody cytokine engrafted protein wherein the binding specificity of the

CDRs is to a target protein distinct from the cytokine-receptor binding specificity of the IL10 monomeric molecule.

[0028] The antibody cytokine engrafted protein wherein the binding specificity of the

CDRs is to a non-human target.

[0029] The antibody cytokine engrafted protein wherein the antibody cytokine engrafted protein is humanized or human.

[0030] An antibody cytokine engrafted protein comprising:

(i) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 193, (b) a HCDR2 of SEQ ID NO: 194, (c) a HCDR3 of SEQ ID NO: 195 and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 196, (e) a LCDR2 of SEQ ID NO: 197, and (f) a LCDR3 of SEQ ID NO: 198;

(ii) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 97, (b) a HCDR2 of SEQ ID NO:98, (c) a HCDR3 of SEQ ID NO:99; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 100, (e) a LCDR2 of SEQ ID NO: 101, and (f) a LCDR3 of SEQ ID NO: 102;

(iii) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 1, (b) a HCDR2 of SEQ ID NO:2, (c) a HCDR3 of SEQ ID NO:3; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 4, (e) a LCDR2 of SEQ ID NO: 5, and (f) a LCDR3 of SEQ ID NO: 6; a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 17, t HCDR2 of SEQ ID NO: 18, (c) a HCDR3 of SEQ ID NO: 19; and a light chain variable region that comprises: (d) a LCDRl of SEQ ID NO: 20, (e) a LCDR2 of SEQ ID NO:21, and (f) a LCDR3 of SEQ ID NO:22;

(v) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 33, (b) a HCDR2 of SEQ ID NO:34, (c) a HCDR3 of SEQ ID NO:35; and a light chain variable region that comprises: (d) a LCDRl of SEQ ID NO: 36, (e) a LCDR2 of SEQ ID NO: 37, and (f) a LCDR3 of SEQ ID NO: 38;

(vi) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 49, (b) a HCDR2 of SEQ ID NO: 50, (c) a HCDR3 of SEQ ID NO: 51 ; and a light chain variable region that comprises: (d) a LCDRl of SEQ ID NO: 52, (e) a LCDR2 of SEQ ID NO: 53, and (f) a LCDR3 of SEQ ID NO: 54;

(vii) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 65, (b) a HCDR2 of SEQ ID NO:66, (c) a HCDR3 of SEQ ID NO:67; and a light chain variable region that comprises: (d) a LCDRl of SEQ ID NO:68, (e) a LCDR2 of SEQ ID NO:69, and (f) a LCDR3 of SEQ ID NO:70;

(viii) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO:81, (b) a HCDR2 of SEQ ID NO:82, (c) a HCDR3 of SEQ ID NO:83; and a light chain variable region that comprises: (d) a LCDRl of SEQ ID NO: 84, (e) a LCDR2 of SEQ ID NO: 85, and (f) a LCDR3 of SEQ ID NO: 86;

(ix) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID

NO: 113, (b) a HCDR2 of SEQ ID NO: 114, (c) a HCDR3 of SEQ ID NO: 115; and a light chain variable region that comprises: (d) a LCDRl of SEQ ID NO: 116, (e) a LCDR2 of SEQ ID NO: 117, and (f) a LCDR3 of SEQ ID NO: 118;

(x) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 129, (b) a HCDR2 of SEQ ID NO: 130, (c) a HCDR3 of SEQ ID NO: 131; and a light chain variable region that comprises: (d) a LCDRl of SEQ ID NO: 132, (e) a LCDR2 of SEQ ID NO: 133, and (f) a LCDR3 of SEQ ID NO: 134; (xi) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID

NO: 145, (b) a HCDR2 of SEQ ID NO: 146, (c) a HCDR3 of SEQ ID NO: 147; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 148, (e) a LCDR2 of SEQ ID NO : 149, and (f) a LCDR3 of SEQ ID NO : 150;

(xii) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID

NO: 161, (b) a HCDR2 of SEQ ID NO: 162, (c) a HCDR3 of SEQ ID NO: 163; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 164, (e) a LCDR2 of SEQ ID NO: 165, and (f) a LCDR3 of SEQ ID NO: 166;

(xiii) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID

NO: 177, (b) a HCDR2 of SEQ ID NO: 178, (c) a HCDR3 of SEQ ID NO: 179; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 180, (e) a LCDR2 of SEQ ID NO : 181 , and (f) a LCDR3 of SEQ ID NO : 182;

(xiv) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO:210, (b) a HCDR2 of SEQ ID NO:211, (c) a HCDR3 of SEQ ID NO:212; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO:213, (e) a LCDR2 of SEQ ID NO:214, and (f) a LCDR3 of SEQ ID NO:215; and

(xv) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO:226, (b) a HCDR2 of SEQ ID NO:227, (c) a HCDR3 of SEQ ID NO:228; and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 229, (e) a LCDR2 of SEQ ID NO : 230, and (f) a LCDR3 of SEQ ID NO : 231.

An antibody cytokine engrafted protein comprising:

(i) a heavy chain variable region (VH) that comprises SEQ ID NO:205, and a light chain variable region (VL) that comprises SEQ ID NO:206;

(ii) a heavy chain variable region (VH) that comprises SEQ ID NO: 109, and a light chain variable region (VL) that comprises SEQ ID NO: 110; (iii) a heavy chain variable region (VH) that comprises SEQ ID NO: 13, and a light chain variable region (VL) that comprises SEQ ID NO: 14;

(iv) a heavy chain variable region (VH) that comprises SEQ ID NO: 29, and a light chain variable region (VL) that comprises SEQ ID NO: 30;

(v) a heavy chain variable region (VH) that comprises SEQ ID NO:45, and a light chain variable region (VL) that comprises SEQ ID NO: 46;

(vi) a heavy chain variable region (VH) that comprises SEQ ID NO: 61, and a light chain variable region (VL) that comprises SEQ ID NO: 62;

(vii) a heavy chain variable region (VH) that comprises SEQ ID NO: 77, and a light chain variable region (VL) that comprises SEQ ID NO: 78;

(viii) a heavy chain variable region (VH) that comprises SEQ ID NO: 93, and a light chain variable region (VL) that comprises SEQ ID NO: 94;

(ix) a heavy chain variable region (VH) that comprises SEQ ID NO: 125, and a light chain variable region (VL) that comprises SEQ ID NO: 126;

(x) a heavy chain variable region (VH) that comprises SEQ ID NO: 141, and a light chain variable region (VL) that comprises SEQ ID NO: 142;

(xi) a heavy chain variable region (VH) that comprises SEQ ID NO: 157, and a light chain variable region (VL) that comprises SEQ ID NO: 158;

(xii) a heavy chain variable region (VH) that comprises SEQ ID NO: 173, and a light chain variable region (VL) that comprises SEQ ID NO: 174;

(xiii) a heavy chain variable region (VH) that comprises SEQ ID NO: 189, and a light chain variable region (VL) that comprises SEQ ID NO: 190;

(xiv) a heavy chain variable region (VH) that comprises SEQ ID NO: 222, and a light chain variable region (VL) that comprises SEQ ID NO: 223; and (xv) a heavy chain variable region (VH) that comprises SEQ ID NO: 238, and a light chain variable region (VL) that comprises SEQ ID NO:239.

[0032] The antibody cytokine engrafted protein further comprising a modified Fc region corresponding with reduced effector function.

[0033] The antibody cytokine engrafted protein wherein the modified Fc region comprises a mutation selected from one or more of D265A, P329A, P329G, N297A, L234A, and L235A.

[0034] The antibody cytokine engrafted protein wherein the modified Fc region is selected from the group consisting of D265A/P329A, D265A N297A, L234A/L235A,

P329A/L234A/L235A, and P329G/L234A/L235A.

[0035] An antibody cytokine engrafted protein consisting of a HCDR1 of SEQ ID NO:

193, a HCDR2 of SEQ ID NO: 194, a HCDR3 of SEQ ID NO: 195, a LCDR1 of SEQ ID

NO: 196, a LCDR2 of SEQ ID NO: 197, a LCDR3 of SEQ ID NO: 198, a modified Fc region containing the mutation D265A/P329A, and wherein the antibody cytokine engrafted protein has less activation of T cells or NK cells when compared to IL10.

[0036] The antibody cytokine engrafted protein wherein the binding specificity of the

CDRs is to a non-human target.

[0037] The antibody cytokine engrafted protein wherein the antibody cytokine engrafted protein has a longer half-life than rhlLlO.

[0038] An isolated nucleic acid comprising:

(i) a heavy chain variable region encoding polynucleotide sequence of SEQ ID NO: 246 and a light chain variable region encoding polynucleotide sequence of SEQ ID NO: 247;

(ii) a heavy chain encoding polynucleotide sequence of SEQ ID NO: 248 and a light chain encoding polynucleotide sequence of SEQ ID NO: 249;

(iii) a heavy chain variable region encoding polynucleotide sequence of SEQ ID NO: 242 and a light chain variable region encoding polynucleotide sequence of SEQ ID NO: 243; or

(iv) a heavy chain encoding polynucleotide sequence of SEQ ID NO: 244 and a light chain encoding polynucleotide sequence of SEQ ID NO:245.

[0039] A recombinant host cell suitable for the production of an antibody cytokine engrafted protein, comprising the nucleic acids encoding the heavy and light chain polypeptides of the antibody cytokine engrafted protein, and optionally, secretion signals.

[0040] The host cell wherein the cell is mammalian. [0041] A pharmaceutical composition comprising the antibody cytokine engrafted protein and a pharmaceutically acceptable carrier.

[0042] A method of treatment or prophylaxis of an immune related disorder in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of the antibody cytokine engrafted protein.

[0043] The method wherein the immune related disorder is selected from the group consisting of: inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, type I diabetes, acute pancreatitis, uveitis, Sjogren's disease, Behcet's disease, sarcoidosis, and graft versus host disease (GVHD).

[0044] The method wherein the antibody cytokine engrafted protein is administered in combination with another therapeutic agent.

[0045] The method wherein the therapeutic agent is an anti-TNF agent selected from the group consisting of: infliximab, adalimumab, certolizumab, golimumab, natalizumab, and vedolizumab.

[0046] The method wherein the therapeutic agent is an aminosalicylate agent selected from the group consisting of: sulfasalazine, mesalamine, balsalazide, olsalazine and other derivatives of 5-aminosalicylic acid.

[0047] The method wherein the therapeutic agent is a corticosteroid selected from the group consisting of: methylprednisolone, hydrocortisone, prednisone, budenisonide, mesalamine, and dexamethasone.

[0048] The method wherein the therapeutic agent is an antibacterial agent.

[0049] A method of activating monocytes in a patient in need thereof, comprising administering an antibody cytokine engrafted protein.

[0050] The method wherein monocytes are activated and T cells or NK cells are not activated.

[0051] The method wherein administration of the antibody cytokine engrafted protein reduces TNFa production.

[0052] The use of an antibody cytokine engrafted protein comprising:

(i) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 193, (b) a HCDR2 of SEQ ID NO: 194, (c) a HCDR3 of SEQ ID NO: 195 and a light chain variable region that comprises: (d) a LCDR1 of SEQ ID NO: 196, (e) a LCDR2 of SEQ ID NO: 197, and (f) a LCDR3 of SEQ ID NO: 198; and (ii) a heavy chain variable region that comprises (a) a HCDR1 of SEQ ID NO: 97, (b) a HCDR2 of SEQ ID NO:98, (c) a HCDR3 of SEQ ID NO:99; and a light chain variable region that comprises: (d) a LCDRl of SEQ ID NO: 100, (e) a LCDR2 of SEQ ID NO: 101, and (f) a LCDR3 of SEQ ID NO: 102 in the treatment of immune related disorders.

[0053] The antibody cytokine engrafted protein for use as a therapeutic in treatment of an immune related disorder selected from the group consisting of: inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, type I diabetes, acute pancreatitis, uveitis, Sjogren's disease, Behcet's disease, sarcoidosis, and graft versus host disease (GVHD).

[0054] The use wherein the antibody cytokine engrafted protein is administered in combination with another therapeutic agent.

[0055] The use wherein the therapeutic agent is an anti-TNF agent selected from the group consisting of: infliximab, adalimumab, certolizumab, golimumab, natalizumab, and vedolizumab.

[0056] The use wherein the therapeutic agent is an aminosalicylate agent selected from the group consisting of: sulfasalazine, mesalamine, balsalazide, olsalazine and other derivatives of 5 -aminosalicylic acid.

[0057] The use wherein the therapeutic agent is a corticosteroid selected from the group consisting of: methylprednisolone, hydrocortisone, prednisone, budenisonide, mesalamine, and dexamethasone.

[0058] The use wherein the therapeutic agent is an antibacterial agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 illustrates the biological effects of IL10, the cell types it acts on, and whether those biological effects are anti-inflammatory or pro-inflammatory. Antibody cytokine engrafted proteins reduce the pro- inflammatory biological effects depicted on the right side of the diagram.

[0060] Figure 2 illustrates the structure of an IL10 antibody cytokine engrafted protein.

The insert panel depicts monomeric IL10 inserted into the LCDRl of an antibody. Dark residues depict exemplary Fc modifications optionally incorporated in the antibody cytokine engrafted protein. [0061] Figure 3A-3B depicts results of in vitro biological assays of recombinant human

IL10 (rhILlO, gray square) and the IgGIL10M13 antibody cytokine engrafted protein (black triangle). Figure 3 A illustrates that IgGIL10M13 demonstrated decreased pro- inflammatory activity as compared to rhILlO as measured by IFN gamma induction in CD8 T cell assays. Similar differential activity was found on human primary NK cells, B cells, and mast cells, as well as using granzyme-B as a readout measurement. Figure 3B illustrates that rhILlO and IgGIL10M13 demonstrate similar anti-inflammatory activity as measured by inhibition of TNFa in whole blood assays.

[0062] Figure 4 depicts results of CyTOF analysis of IL10 dependent pSTAT3 signaling in human whole blood stimulated with equal molar amounts recombinant human IL10 rhILlO (left panel) or IgGIL10M13 (right panel). IL10 induces anti-inflammatory activities in monocytes; and activation of T, B or NK cells induces pro-inflammatory cytokines. Results of fold change in activity of cells over unstimulated are depicted by heat map (changes in shading). Left panel indicates rhILlO confers stimulation across all IL10 sensitive cell types (with outline); however, as seen in the right panel IgGIL10M13 confers less potent stimulation on T, B, and NK cells, with levels similar or slightly above unstimulated cells; while a similar potency of stimulation of monocytes (outlined) and mDC cells was demonstrated with IgG-ILlOM and rhILlO. These relevant cell types (monocytes, mDC) are key cells for maintenance of gut homeostatis in inflammatory bowel disease.

[0063] Figure 5A-5D illustrates improved characteristics of antibody cytokine engrafted protein IgGIL10M13 in in vivo assays. Figure 5A-B depicts results of

pharmacokinetic studies of rhILlO and IgGIL10M13. Following intravenous administration, IgGIL10M13 demonstrates prolonged pharmacokinetics (half-life) as antibody cytokine engrafted protein is still detectable after 4.4 days (Figure 5B), while rhILlO had a half-life of approximately 1 hour (Figure 5 A). Figure 5C and 5D depict results of pharmacodynamic assays of in vivo activity of antibody cytokine engrafted proteins. Figure 5C depicts in vivo activity in colon tissue as measured by pSTAT3 signaling seventy-two (72) hours post dosing. Figure 5D depicts improved duration of in vivo response of IgGIL10M13 as compared to rhILlO as measured by inhibition of TNFa in response to LPS challenge following administration of IgGIL10M13.

[0064] Figure 6 is the results of an LPS challenge model, demonstrating IgGIL10M13 reduces TNFa induction 48 hours after LPS challenge. [0065] Figure 7 is a graph representing the improved %CMAX of ILIO antibody cytokine engrafted proteins.

[0066] Figure 8 depicts CyTOF data of pSTAT3 activity in various immune cells from healthy subjects and patients when stimulated with rhILlO or with IgGIL10M13.

[0067] Figures 9A-9C are graphical representations demonstrating IgGIL10M13 has reduced pro- inflammatory activity in PHA stimulated human whole blood compared to rhILlO. Figure 9D shows the graphs of a titration experiment with rhILlO and IgGIL10M13.

[0068] Figures 10A-10B depict the aggregation properties of IL10 wild type or monomeric when conjugated via a linker to an Fc, compared to the aggregation properties of an antibody cytokine engrafted protein.

[0069] Figure 11 is ELISA data showing that the IL10 antibody cytokine engrafted protein still binds to RSV.

[0070] Figure 12 is a representation of the mechanism of action of an IL10 antibody cytokine engrafted protein. The left panel shows how a normal rhILlO dimer binds IL-10R1, and initiates strong pSTAT3 signaling. The right panel depicts how an IL10 monomer engrafted into a CDR of an antibody is constrained to have less efficient binding to IL-10R1 and thus produces a weaker pSTAT3 signal.

[0071] Figure 13A-C is the crystal structure resolution of IL10 monomer engrafted into

LCDR1 of palivizumab.

DEFINITIONS

[0072] An "antibody" refers to a molecule of the immunoglobulin family comprising a tetrameric structural unit. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD), connected through a disulfide bond. Recognized immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

Antibodies can be of any isotype/class (e.g., IgG, IgM, IgA, IgD, and IgE), or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2).

[0073] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used structurally and functionally. The N-terminus of each chain defines a variable (V) region or domain of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V _L ) and variable heavy chain (V _H ) refer to these regions of light and heavy chains respectively. The pairing of a V _H and V _L together forms a single antigen-binding site. In addition to V regions, both heavy chains and light chains contain a constant (C) region or domain. A secreted form of a immunoglobulin C region is made up of three C domains, CHI, CH2, CH3, optionally CH4 ( μ), and a hinge region. A membrane-bound form of an immunoglobulin C region also has membrane and intracellular domains. Each light chain has a V _L at the N-terminus followed by a constant domain (C) at its other end. The constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N- terminus is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy-terminal domains of the heavy and light chain, respectively. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain. As used herein, an "antibody" encompasses conventional antibody structures and variations of antibodies. Thus, within the scope of this concept are full length antibodies, chimeric antibodies, humanized antibodies, human antibodies, and antibody fragments thereof.

[0074] Antibodies exist as intact immunoglobulin chains or as a number of well- characterized antibody fragments produced by digestion with various peptidases. The term "antibody fragment," as used herein, refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' ₂ , a dimer of Fab' which itself is a light chain joined to V _H -C _H 1 by a disulfide bond. The F(ab)' ₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' ₂ dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region. Paul, Fundamental Immunology 3d ed. (1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. As used herein, an "antibody fragment" refers to one or more portions of an antibody, either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies, that retain binding specificity and functional activity. Examples of antibody fragments include Fv fragments, single chain antibodies (ScFv), Fab, Fab', Fd (Vh and CHI domains), dAb (Vh and an isolated CDR); and multimeric versions of these fragments {e.g., F(ab') _2, ) with the same binding specificity. Antibody fragments can also be incorporated into cytokine engrafted proteins to achieve the binding specificity and activity provided in the present disclosure.

[0075] A "Fab" domain as used herein comprises a heavy chain variable domain, a constant region CHI domain, a light chain variable domain, and a light chain constant region CL domain. The interaction of the domains is stabilized by a disulfide bond between the CHI and CL domains. In some embodiments, the heavy chain domains of the Fab are in the order, from N-terminus to C-terminus, VH-CH and the light chain domains of a Fab are in the order, from N- terminus to C-terminus, VL-CL. In some embodiments, the heavy chain domains of the Fab are in the order, from N-terminus to C-terminus, CH-VH and the light chain domains of the Fab are in the order CL-VL. Although Fabs were historically identified by papain digestion of an intact immunoglobulin, in the context of this disclosure, a "Fab" is typically produced recombinantly by any method. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen- binding site.

[0076] "Complementarity-determining domains" or "complementarity-determining regions" ("CDRs") interchangeably refer to the hypervariable regions of V _L and V _H . CDRS are the target protein-binding site of antibody chains that harbors specificity for such target protein. There are three CDRs (CDRl-3, numbered sequentially from the N-terminus) in each human V _L or V _H , constituting about 15-20% of the variable domains. CDRs are structurally complementary to the epitope of the target protein and are thus directly responsible for the binding specificity. The remaining stretches of the V _L or V _H , the so-called framework regions (FR), exhibit less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. W.H. Freeman & Co., New York, 2000).

[0077] Positions of CDRs and framework regions can be determined using various well known definitions in the art, e.g., Kabat, Chothia, international ImMunoGeneTics database (IMGT) and AbM (see, e.g., Johnson et al, Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol, 196:901-917 (1987); Chothia et al, Nature, 342:877-883 (1989); Chothia et al, J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al, J.Mol.Biol., 273:927-748 (1997)). Definitions of antigen combining sites are also described in the following: Ruiz et ah, Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M.P., Nucleic Acids Res., 29:207-209 (2001); MacCallum ei a/., J. Mol. Biol, 262:732-745 (1996); and Martin et ah, Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et ah, Methods Enzymol, 203: 121-153 (1991); and Rees et ah, In Sternberg M. J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).

[0078] Under Kabat, CDR amino acid residues in the V _H are numbered 31-35

(HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the V _L are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, CDR amino acids in the V _H are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in V _L are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL.

[0079] An "antibody variable light chain" or an "antibody variable heavy chain" as used herein refers to a polypeptide comprising the V _L or V _H , respectively. The endogenous V _L is encoded by the gene segments V (variable) and J (junctional), and the endogenous V _H by V, D (diversity), and J. Each of V _L or V _H includes the CDRs as well as the framework regions (FR). The term "variable region" or "V-region" interchangeably refer to a heavy or light chain comprising FR1 -CDR1 -FR2-CDR2-FR3 -CDR3 -FR4. A V-region can be naturally occurring, recombinant or synthetic. In this application, antibody light chains and/or antibody heavy chains may, from time to time, be collectively referred to as "antibody chains." As provided and further described herein, an "antibody variable light chain" or an "antibody variable heavy chain" and/or a "variable region" and/or an "antibody chain" optionally comprises a cytokine polypeptide sequence engrafted into a CDR.

[0080] The C-terminal portion of an immunoglobulin heavy chain as disclosed herein, comprising, e.g., CH2 and CH3 domains, is the "Fc" domain. An "Fc region" as used herein refers to the constant region of an antibody excluding the first constant region (CHI)

immunoglobulin domain. Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and Cy. It is understood in the art that boundaries of the Fc region may vary, however, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, using the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). "Fc region" may refer to this region in isolation or this region in the context of an antibody or antibody fragment. "Fc region" includes naturally occurring allelic variants of the Fc region, e.g., in the CH2 and CH3 region, including, e.g., modifications that modulate effector function. Fc regions also include variants that don't result in alterations to biological function. For example, one or more amino acids are deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. For example, in certain embodiments a C-terminal lysine is modified replaced or removed. In particular embodiments one or more C-terminal residues in the Fc region is altered or removed. In certain embodiments one or more C-terminal residues in the Fc (e.g., a terminal lysine) is deleted. In certain other embodiments one or more C-terminal residues in the Fc is substituted with an alternate amino acid (e.g., a terminal lysine is replaced). Such variants are selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, et al, Science 247:306-1310, 1990). The Fc domain is the portion of the immunoglobulin (Ig) recognized by cell receptors, such as the FcR, and to which the complement-activating protein, CI q, binds. The lower hinge region, which is encoded in the 5' portion of the CH2 exon, provides flexibility within the antibody for binding to FcR receptors.

[0081] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, and drug; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

[0082] A "humanized" antibody is an antibody that retains the reactivity (e.g., binding specificity, activity) of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining non-human CDR regions and replacing remaining parts of an antibody with human counterparts. See, e.g., Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988); Padlan, Molec. Immua, 28:489-498 (1991); Padlan, Molec. Immua, 31(3): 169-217 (1994).

[0083] A "human antibody" includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if an antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000). Human antibodies may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing).

[0084] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. A corresponding human germline sequence can also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison to all other evaluated variable region amino acid sequences. A corresponding human germline sequence can be framework regions only, complementary determining regions only, framework and complementary determining regions, a variable segment (as defined above), or other combinations of sequences or subsequences that comprise a variable region. Sequence identity can be determined using the methods described herein, for example, aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference variable region nucleic acid or amino acid sequence. Corresponding human germline sequences can be determined, for example, through the publicly available international ImMunoGeneTics database (IMGT) and V-base.

[0085] The term "valency" as used herein refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds one target molecule or a specific site on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site may specifically bind the same or different molecules (e.g., may bind to different molecules, e.g., different antigens, or different epitopes on the same molecule). A conventional antibody, for example, has two binding sites and is bivalent; "trivalent" and "tetravalent" refer to the presence of three binding sites and four binding sites, respectively, in an antibody molecule. The antibody cytokine engrafted protein can be monovalent (i.e., bind one target molecule), bivalent, or multivalent (i.e., bind more than one target molecule).

[0086] The phrase "specifically binds" or "binding specificity," when used in the context of describing the interaction between the original antibody target (e.g., an antigen) and an antibody cytokine engrafted protein before and after the cytokine engrafting. Under certain designated conditions, an antibody cytokine engrafted protein with a particular binding specificity binds to its original target at least two times the background and does not substantially bind in a significant amount to other targets present in a sample. In one embodiment, under designated conditions, an antibody cytokine engrafted protein with a particular binding specificity binds to its original target at least ten (10) times the background and does not substantially bind in a significant amount to other targets present in the sample. Specific binding of an antibody cytokine engrafted protein under such conditions can require an antibody cytokine engrafted protein to have been selected for its specificity to a particular target. Methods for determining binding specific are, for example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0087] As used herein, "cytokine-receptor binding specificity" or "cytokine-receptor specificity" includes antibody cytokine engrafted proteins that selectively bind to IL10 receptor and does not include antibody cytokine engrafted proteins that cross-react with other cytokine receptor superfamily members. In some embodiments, antibody cytokine engrafted proteins are selected that selectively bind to human IL10R and cross-react with non- human primate IL10R (e.g., cynomolgus IL10R). In some embodiments, antibody engrafted proteins are selected that selectively bind to human IL10R1.

[0088] The term "equilibrium dissociation constant (K _D , M)" refers to the dissociation rate constant (k _d , time ^"1 ) divided by the association rate constant (k _a , time ^"1 , M ^"1 ). Equilibrium dissociation constants can be measured using any known method in the art. The antibody cytokine engrafted proteins of the present disclosure generally will have an equilibrium 7 8 9

dissociation constant of less than about 10 ^" or 10 ^" M, for example, less than about 10 ^" M or l O ^{" 10} M, in some embodiments, less than about 10 ^"11 M, 10 ^"12 M or 10 ^"13 M.

[0089] The term "IL10" or "interleukin 10" or "interleukin-10 (IL-10)",

interchangeably, refer to a potent anti-inflammatory cytokine, capable of down regulating activation of macrophages as well as reducing antigen presentation and maturation of dendritic cells. IL10 comprising residues 19-178 of the full length native human is utilized in construction of the agonist antibody cytokine engrafted proteins. The human IL10 as disclosed herein has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence published as GenBank Accession No: NP 000563. The human IL10 nucleic acid encoding for the IL10 protein has over its full length at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence published under GenBank Accession No: NM_000572.

[0090] "Monomeric IL10" or "ILl 0M" refers to a molecule has at least 85%, 86%,

87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 209. In some embodiments, the monomeric IL10 molecule comprises the sequence of SEQ ID NO: 209. In some embodiments, the monomeric IL10 molecule consists of the sequence of SEQ ID NO: 209.

[0091] The term "agonist" refers to an antibody cytokine engrafted protein capable of activating a receptor to induce a full or partial receptor-mediated response. For example, an agonist of IL10R binds to IL10R and induces ILl OR-mediated intracellular signaling and/or cell activation. The antibody cytokine engrafted protein stimulates signaling through IL10R similar in some respects to the native ligand, human IL10. Binding of hILlO to IL10R induces NFKB activation due to degradation of ΙκΒ. In some embodiments, an antibody cytokine engrafted protein agonist can be identified by its ability to bind IL10R and induce STAT3 phosphorylation, suppress production of pro- inflammatory cytokines (e.g. TNFa, ILl, IL6, IL12, IFNy) and/or differentiation and proliferation in macrophages; induce T cell (e.g., CD8 ⁺ CTLs or CD4 ⁺ Th cells) proliferation, survival, cytolytic activity and/or cytokine production (e.g., IFNy, IL10, IL- 13, TNFa), or as otherwise described herein.

[0092] The term "isolated," when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. It can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[0093] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids

(DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolim et al, Mol. Cell. Probes 8:91-98 (1994)).

[0094] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

[0095] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0096] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0097] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. The following eight groups each contain amino acids that are

conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). [0098] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (e.g. , a polypeptide ), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0099] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The disclosure provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein (e.g., the variable regions exemplified in any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:61 , SEQ ID NO:62, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO:205, SEQ ID NO:206; SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:238 SEQ ID

NO: 239; the variable regions exemplified in any one of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:79, SEQ ID NO: 80, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO: l l l, SEQ ID NO: 1 12, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 240, SEQ ID NO: 241 ; the CDRs exemplified in any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:46, SEQ ID N0:61, SEQ ID NO:62, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:238 and SEQ ID NO:239. Optionally, the identity exists over a region that is at least about 15, 25 or 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or over the full length of the reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence. With respect to shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.

[00100] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[00101] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA

85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al, Current Protocols in Molecular Biology (1995 supplement)).

[00102] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Hemkoff and Hemkoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[00103] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[00104] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[00105] The term "antibody cytokine engrafted protein" or "antibody cytokine graft" or

"engrafted" means that at least one cytokine is incorporated directly within a CDR of the antibody, interrupting the sequence of the CDR. The cytokine can be incorporated within HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3. The cytokine can be incorporated within HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 and incorporated toward the N- terminal sequence of the CDR or toward the C-terminal sequence of the CDR. The cytokine incorporated within a CDR can reduce the specific binding of the antibody portion to its target protein or the antibody cytokine engrafted protein can retain its specific binding to its target protein.

[00106] An "immune related disorder" or "immune disease" refers to a dysfunction of the immune system, in which the body's own immune system attacks healthy tissue. The dysfunction can be in components of the immune cells, and includes both overactive and underactive immune systems.

[00107] The terms "subject," "patient," and "individual" interchangeably refer to a mammal, for example, a human or a non-human primate mammal. The mammal can also be a laboratory mammal, e.g., mouse, rat, rabbit, hamster. In some embodiments, the mammal can be an agricultural mammal (e.g., equine, ovine, bovine, porcine, camelid) or domestic mammal (e.g., canine, feline).

[00108] As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder refer in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, "treat," "treating," or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treat," "treating," or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom),

physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, "treat," "treating," or "treatment" or "prophylaxis" refers to preventing or delaying the onset or development or progression of a disease or disorder.

[00109] The term "therapeutically acceptable amount" or "therapeutically effective dose" interchangeably refer to an amount sufficient to effect the desired result (i.e. , a reduction in inflammation, inhibition of pain, prevention of inflammation, inhibition or prevention of inflammatory response). In some embodiments, a therapeutically acceptable amount does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved. A "prophylactically effective dosage," and a "therapeutically effective dosage," of an IL10 antibody cytokine engrafted protein can prevent the onset of, or result in a decrease in severity of, respectively, disease symptoms, including symptoms associated with immune related disorder.

[00110] The term "co-administer" refers to the simultaneous presence of two (or more) active agents in an individual. Active agents that are co-administered can be concurrently or sequentially delivered.

[00111] As used herein, the phrase "consisting essentially of refers to the genera or species of active pharmaceutical agents included in a method or composition, as well as any inactive carrier or excipients for the intended purpose of the methods or compositions. In some embodiments, the phrase "consisting essentially of expressly excludes the inclusion of one or more additional active agents other than an IL10 antibody cytokine engrafted protein. In some embodiments, the phrase "consisting essentially of expressly excludes the inclusion of more additional active agents other than an IL10 antibody cytokine engrafted protein and a second coadministered agent.

[00112] The terms "a," "an," and "the" include plural referents, unless the context clearly indicates otherwise.

DETAILED DESCRIPTION ILIO Antibody Cytokine engrafted proteins

[00113] Interleukin- 10 (ILIO) is a multifunctional, homodimeric immunomodulatory cytokine that has both immunostimulatory and immunosuppressive activities. ILIO inhibits the induction of a number of proinflammatory cytokines in activated monocytes and macrophages but is a costimulator for immature and mature lymphocytes, mast cells and B cells (reviewed in Mosmann, Adv. Immunol. 1994; 56: 1-26; Moore et al, Annu Rev Immunol. 2001 ;19:683-765). Provided herein are antibody cytokine engrafted proteins comprising monomeric IL10 engrafted into the complementarity determining region (CDR) of an antibody. The antibody cytokine engrafted proteins show suitable properties to be used in human patients, for example, they retain immunosuppressive activity similar to that of native or recombinant human IL10, however, the pro-inflammatory effects are reduced.

[00114] Accordingly, the present disclosure provides antibody cytokine engrafted proteins that are agonists of the IL10 receptor, with selective activity profiles. Provided antibody cytokine engrafted proteins comprise an immunoglobulin heavy chain sequence and an immunoglobulin light chain sequence. Each immunoglobulin heavy chain sequence comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), wherein the heavy chain constant region consists of CHI, CH2, and CH3 constant regions. Each immunoglobulin light chain sequence comprises a light chain variable region (VL) and a light chain constant region (CL). In each antibody cytokine engrafted protein a monomeric IL10 molecule is engrafted into a complementarity determining region (CDR) of the VH or VL region.

[00115] In some embodiments, the antibody cytokine engrafted protein comprises a monomeric IL10 engrafted into a heavy chain CDR. In certain embodiments monomeric IL10 is engrafted into heavy chain complementarity determining region 1 (HCDR1). In certain embodiments monomeric IL10 is engrafted into heavy chain complementarity determining region 2 (HCDR2). In certain embodiments monomeric IL10 is engrafted into heavy chain complementarity determining region 3 (HCDR3).

[00116] In some embodiments, the antibody cytokine engrafted protein comprises a monomeric IL10 engrafted into a light chain CDR. In certain embodiments monomeric IL10 is engrafted into light chain complementarity determining region 1 (LCDR1). In certain embodiments monomeric IL10 is engrafted into light chain complementarity determining region 2 (LCDR2). In certain embodiments monomeric IL10 is engrafted into light chain complementarity determining region 3 (LCDR3). In a preferred embodiment, monomeric IL10 is engrafted into light chain complementarity determining region 1 (LCDR1).

[00117] In some embodiments, the antibody cytokine engrafted protein comprises a monomeric IL10 engrafted into a CDR, whereby the IL10 sequence is inserted into the CDR sequence. The engrafted IL10 can be at or near the N-terminal portion of the CDR, in the center region of the CDR or at or near the C-terminal portion of the CDR. In other embodiments, the antibody cytokine engrafted protein comprises a monomeric IL10 incorporated into a CDR, wherein the IL10 sequence replaces all or part of a CDR sequence. A replacement can be at or near the beginning of the CDR, in the middle region of the CDR or at or near the end of the CDR. A replacement can be as few as one or two amino acids of a CDR sequence, or as many as an entire CDR sequence.

[00118] In some embodiments monomeric IL10 is engrafted directly into a CDR without a peptide linker.

[00119] In some embodiments the antibody cytokine engrafted protein comprises immunoglobulin heavy chains of an IgG class antibody heavy chain. In certain embodiments an IgG heavy chain is any one of an IgGl, an IgG2 or an IgG4 subclass.

[00120] In some embodiments antibody cytokine engrafted proteins comprise heavy and light chain immunoglobulin sequences selected from a known, clinically utilized

immunoglobulin sequence. In certain embodiments antibody cytokine engrafted proteins comprise heavy and light chain immunoglobulin sequences which are humanized sequences. In other certain embodiments antibody cytokine engrafted proteins comprise heavy and light chain immunoglobulin sequences which are human sequences.

[00121] In some embodiments antibody cytokine engrafted proteins comprise heavy and light chain immunoglobulin sequences selected from germline immunoglobulin sequences.

[00122] In some embodiments antibody cytokine engrafted proteins comprise heavy and light chain immunoglobulin sequences having binding specificity of the immunoglobulin variable domains to a target distinct from the cytokine receptor binding specificity of the IL10 monomer. In some embodiments the binding specificity of the immunoglobulin variable domain is retained in the presence of the engrafted cytokine. In certain embodiments the antibody binding specificity is to a non-human antigen. In other embodiments the binding specificity is to a target having therapeutic utility in conjunction with IL10 therapy. In certain embodiments modulating the binding specificity of the immunoglobulin conveys additional therapeutic benefit to the IL10 component. In certain embodiments the binding specificity of the immunoglobulin conveys synergistic activity with IL10 monomer.

[00123] In still other embodiments, the binding specificity of the immunoglobulin is reduced in the presence of the IL10 engrafted cytokine.

[00124] In some embodiments, the antibody cytokine engrafted protein confers antiinflammatory properties similar to human IL10 or recombinant human IL10, and the engrafted protein confers reduced proportional pro- inflammatory activity as compared to human IL10 or recombinant human IL10. In some embodiments the proportional pro-inflammatory activity is reduced by at least twenty five percent (25%) as compared with IL10. In some embodiments the proportional pro-inflammatory activity is reduced by at least thirty five percent (35%) as compared with IL10. In some embodiments the proportional pro- inflammatory activity is reduced by at least about fifty percent (50%) as compared with IL10. In some embodiments the proportional pro-inflammatory activity is reduced by at least about sixty five percent (65%) as compared with IL10. In some embodiments the proportional pro- inflammatory activity is reduced by at least about seventy five percent (75%) as compared with IL10. In some embodiments the proportional pro-inflammatory activity is reduced by at least about eighty percent (80%) as compared with IL10. In some embodiments the proportional pro-inflammatory activity is reduced by more than eighty percent (80%) as compared with IL10.

[00125] In some embodiments, the antibody cytokine engrafted proteins comprise a modified immunoglobulin IgG having a modified Fc conferring modified effector function. In certain embodiments the modified Fc region comprises a mutation selected from one or more of D265A, P329A, P329G, N297A, L234A, and L235A. In particular embodiments the

immunoglobulin heavy chain may comprise a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00126] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 13 and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 14. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,

P329A/L234A/L235A, and P329G/L234A/L235A.

[00127] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:29 and (n) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 30. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,

P329A/L234A/L235A, and P329G/L234A/L235A.

[00128] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:45 and (n) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO:46. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,

P329A/L234A/L235A, and P329G/L234A/L235A.

[00129] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 61 and (n) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 62. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00130] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 77 and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 78. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,

P329A/L234A/L235A, and P329G/L234A/L235A.

[00131] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 93 and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 94. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234A/L235A,

P329A/L234A/L235A, and P329G/L234A/L235A.

[00132] In some embodiments, the antibody cytokine engrafted protein comprises each comprising (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 109 and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 110. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A,

D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A. [00133] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 125 and (h) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 126. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00134] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 141 and (h) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 142. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00135] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 157 and (h) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 158. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00136] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 173 and (h) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 174. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00137] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO: 189 and (h) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 190. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00138] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:205 and (h) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 206. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00139] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:222 and (h) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 223. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00140] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a heavy chain variable region of SEQ ID NO:238 and (h) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to a light chain variable region of SEQ ID NO: 239. The immunoglobulin chain is an IgG class selected from IgGl, IgG2, or IgG4. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00141] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 15 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 16. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00142] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 31 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 32. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A. [00143] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 47 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO:48. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00144] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 63 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 64. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00145] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 79 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 80. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00146] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 95 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 96. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A. [00147] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 111 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 112. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00148] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 127 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 128. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00149] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 143 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 144. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00150] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 159 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 160. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A. [00151] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 175 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 176. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00152] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 191 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 192. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00153] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 207 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 208. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00154] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 224 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 225. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A. [00155] In some embodiments, the antibody cytokine engrafted protein comprises (i) a heavy chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 240 and (ii) a light chain having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 241. In certain embodiments the immunoglobulin heavy chain optionally comprises a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A N297A, L234A/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

Engineered and Modified Antibody Cytokine Engrafted Proteins

[00156] Antibody cytokine engrafted proteins are generated by engrafting a monomeric

IL10 sequence into a CDR region of an immunoglobulin scaffold. Both heavy and light chain immunoglobulin chains are produced to generate final antibody engrafted proteins. Antibody cytokine engrafted proteins confer preferred therapeutic anti-inflammatory properties of IL10; however, antibody cytokine engrafted proteins have reduced proportional pro-inflammatory activity as compared with native or recombinant human IL10 (rhlLlO).

[00157] To engineer engrafted constructs, monomeric IL10 (IL10M, SEQ ID NO: 209), comprising residues 19-178 of full length IL10 and a six amino acid sequence between residues 134 and 135 was engrafted into a CDR loop of an immunoglobulin scaffold. Engrafted constructs can be prepared using any of a variety of known immunoglobulin sequences which have been utilized in clinical settings, known immunoglobulin sequences which are in current discovery and/or clinical development, human germline antibody sequences, as well as sequences of novel antibody immunoglobulin chains. Antibody cytokine engrafted proteins are produced using standard molecular biology methodology utilizing recombinant DNA encoding relevant sequences. Sequences of IL10M in two exemplary scaffolds, referred to as GFTXl and GFTX3, and are depicted in TABLE 1. Insertion points for IL10 monomer were selected to be the mid-point of the loop based on available structural or homology model data, however, insertion points may be adjusted toward one or another end of a CDR loop.

[00158] Thus the present disclosure provides antibody cytokine engrafted proteins or fragments thereof that cytokine- receptor specifically bind to an IL10R protein, comprising an IL10M protein recombinantly inserted into to a heterologous antibody protein or polypeptide to generate antibody cytokine engrafted proteins. In particular, the disclosure provides engrafted proteins comprising an antibody or antigen-binding fragment of an antibody described herein or any other relevant scaffold antibody polypeptide (e.g., a full antibody immunoglobulin protein, a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain, a VL CDR, etc.) and a heterologous cytokine protein, polypeptide, or peptide, e.g., IL10M. Methods for conjugating proteins, polypeptides, or peptides to an antibody or an antibody fragment are known in the art. See, e.g., U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al, 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al, 1995, J. Immunol. 154:5590- 5600; and Vil et al, 1992, Proc. Natl. Acad. Sci. USA 89: 11337- 11341. Additionally, antibody cytokine engrafted proteins may be generated through the techniques of gene-shuffling, motif- shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to prepare antibody cytokine engrafted proteins and/or to alter the activities of antibodies or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al, 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson, et al, 1999, J. Mol. Biol. 287:265- 76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308- 313 (each of these patents and publications are hereby incorporated by reference in its entirety). Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. A polynucleotide encoding an antibody or fragment thereof that specifically binds to an antigen protein of interest may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous cytokine molecules, e.g., IL10M, for preparation of antibody cytokine engrafted proteins as provided herein.

[00159] An antibody Fab contains six CDR loops, 3 in the light chain (CDRLl, CDRL2,

CDRL3) and 3 in the heavy chain (CDRHl, CDRH2, CDRH3) which can serve as potential insertion sites for a cytokine protein. Structural and functional considerations are taken into account in order to determine which CDR loop(s) to insert the cytokine. As a CDR loop size and conformation vary greatly across different antibodies, the optimal CDR for insertion may be determined empirically for each particular antibody /protein combination. Additionally, since a cytokine protein will be inserted into a CDR loop, this can put additional constraints on the structure of the cytokine protein. For example, the amino and carboxy terminal of the cytokine protein should allow for possibility to be constrained relatively close in space (-25 A).

Additionally, an antibody cytokine engrafted protein should not rely on oligomerization for biological activity.

[00160] IL10 is a homodimeric cytokine that employs a domain swapped architecture. In native form, IL10 would not be suitable for antibody grafting since the domain swapped structure would not be able to form (i.e. both N and C termini are constrained in the loop).

However, an engineered monomeric IL10 (IL10M), created by inserting a six residue sequence ((GGGSGG) (SEQ ID N0.251)) between helices D and E allows helices E and F (which swap into the other IL10 monomer in the native dimeric structure) to fold back into the same monomer (Josephson et al J. Biol.Chem. 2000; 275(18) 13552-7) and create the structure present in the antibody cytokine engrafted protein as shown in Figure 2 and Figure 13. Josephson et al., demonstrated that the IL10 monomer with the six amino acid insertion is more thermostable than the wild-type dimer and is also able to form a 1 : 1 interaction with the IL10 receptor chain (IL10R1). IL10M also has in vitro activity, though it is nine to eighteen-fold less active than wild-type IL10. Another feature of IL10M is that the N and C-termini are close to each other in space, which makes the modified cytokine protein suitable for antibody grafting.

[00161] An antibody cytokine engrafted protein of the disclosure further can be prepared using an antibody having one or more of the CDRs and/or VH and/or VL sequences shown herein {e.g., TABLE 1) as starting material to engineer a modified antibody cytokine engrafted protein, which may have altered properties from the starting antibody. Alternatively, any known antibody sequences may be utilized as a scaffold to engineer modified antibody cytokine engrafted proteins. For example, any known, clinically utilized antibody may be utilized as a starting materials scaffold for preparation of antibody cytokine engrafted protein. Known antibodies and corresponding immunoglobulin sequences include, e.g., palivizumab, alirocumab, mepolizumab, necitumumab, nivolumab, dinutuximab, secukinumab, evolocumab,

blinatumomab, pembrolizumab, ramucirumab vedolizumab, siltuximab, obinutuzumab, trastuzumab, raxibacumab, pertuzumab, belimumab, ipilimumab. denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certolizumab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab tiuxetan, adalimumab, alemtuzumab, gemtuzumab, infliximab, basiliximab, daclizumab, rituximab, abciximab, muromonab, or modifications thereof. Known antibodies and immunoglobulin sequences also include germline antibody sequences. Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, as well as in Kabat, E. A., et ah, 1991 Sequences of Proteins of

Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et ah, 1992 J. fol. Biol. 227:776-798; and Cox, J. P. L. et ah, 1994 Eur. J Immunol. 24:827-836. In still other examples, antibody and corresponding immunoglobulin sequences from other known entities which may be in early discovery and/or drug development may be similarly adapted as starting material to engineer a modified antibody cytokine engrafted protein .

[00162] A wide variety of antibody/ immunoglobulin frameworks or scaffolds can be employed so long as the resulting polypeptide includes at least one binding region which accommodates incorporation of a cytokine (e.g., IL10M). Such frameworks or scaffolds include the 5 main idiotypes of human immunoglobulins, or fragments thereof, and include

immunoglobulins of other animal species, preferably having humanized and/or human aspects. Novel antibodies, frameworks, scaffolds and fragments continue to be discovered and developed by those skilled in the art.

[00163] Antibodies can be generated using methods that are known in the art. For preparation of monoclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et ah, Immunology Today 4:72 (1983); Cole et ah, Monoclonal Antibodies and Cancer Therapy, pp. 77-96. Alan R. Liss, Inc. 1985). Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce antibodies for use in antibody cytokine engrafted proteins of this disclosure. Also, transgenic mice, or other organisms such as other mammals, may be used to express and identify primatized or humanized or human antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens for use in antibody cytokine engrafted proteins, (see, e.g., McCafferty et ah, supra; Marks et ah, Biotechnology, 10:779-783, (1992)).

[00164] Methods for primatizing or humanizing non-human antibodies are well known in the art. Generally, a primatized or humanized antibody has one or more amino acid residues introduced into it from a source which is non-primate or non-human. Such non-primate or non- human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Jones et ah, Nature 321 :522-525 (1986); Riechmann et ah, Nature 332:323-327 (1988); Verhoeyen et ah, Science 239: 1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, primatized or humanized antibodies are typically primate or human antibodies in which some complementary determining region ("CDR") residues and possibly some framework ("FR") residues are substituted by residues from analogous sites in an originating species (e.g., rodent antibodies) to confer binding specificity.

[00165] Alternatively or additionally, an in vivo method for replacing a nonhuman antibody variable region with a human variable region in an antibody while maintaining the same or providing better binding characteristics relative to that of the nonhuman antibody can be utilized to convert non-human antibodies into engineered human antibodies. See, e.g., U.S. Patent Publication No. 20050008625, U.S. Patent Publication No. 2005/0255552. Alternatively, human V segment libraries can be generated by sequential cassette replacement in which only part of the reference antibody V segment is initially replaced by a library of human sequences; and identified human "cassettes" supporting binding in the context of residual reference antibody amino acid sequences are then recombined in a second library screen to generate completely human V segments (see, U.S. Patent Publication No. 2006/0134098).

[00166] Various antibodies or antigen-binding fragments for use in preparation of antibody cytokine engrafted proteins can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv), or identified using phage display libraries (see, e.g., McCafferty et ah, Nature 348:552-554, 1990). For example, minibodies can be generated using methods described in the art, e.g., Vaughan and Sollazzo, Comb Chem High Throughput Screen. 4:417-30 2001.

Bispecific antibodies can be produced by a variety of methods including engrafted of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp.

Immunol. 79:315-321 (1990); Kostelny et ah, J. Immunol. 148, 1547-1553 (1992). Single chain antibodies can be identified using phage display libraries or ribosome display libraries, gene shuffled libraries. Such libraries can be constructed from synthetic, semi-synthetic or native and immunocompetent sources. Selected immunoglobulin sequences may thus be utilized in preparation of antibody cytokine engrafted proteins as provided herein.

[00167] Antibodies or antigen-binding molecules of use in the present disclosure further include bispecific antibodies. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Other antigen-binding fragments or antibody portions include bivalent scFv (diabody), bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (dAbs), and minibodies. Selected immunoglobulin sequences can be utilized in preparation of antibody cytokine engrafted proteins as provided herein.

[00168] Antigen-binding fragments of antibodies e.g., a Fab fragment, scFv, can be used as building blocks to construct antibody cytokine engrafted proteins, and optionally include multivalent formats. In some embodiments, such multivalent molecules comprise a constant region of an antibody (e.g., Fc).

[00169] Antibody cytokine engrafted proteins can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and/or VL) of an antibody, for example within one or more CDR regions, and such adapted VH and/or VL region sequences utilized for incorporation of a cytokine for preparation of antibody cytokine engrafted protein. One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific antibody by constructing expression vectors that include CDR sequences from a specific antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al, 1998 Nature 332:323-327; Jones, P. et al, 1986 Nature 321 :522- 525; Queen, C. et al, 1989 Proc. Natl. Acad., U.S.A. 86: 10029-10033; U.S. Patent No.

5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.). In certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al). [00170] In some aspects mutation of amino acid residues within the VH and/or VL

CDR1, CDR2, and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest, known as "affinity maturation," can be beneficial, e.g., to optimize antigen binding of an antibody in conjunction with the context of the cytokine engrafted protein. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein and provided in the Examples and/or alternative or additional assays known in the art. Conservative modifications can be introduced. The mutations may be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.

[00171] Engineered antibodies or antibody fragments include those in which

modifications have been made to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. In some embodiments such framework modifications are made to decrease immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation contains framework residues that differ from germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site- directed mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the disclosure. Additional framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No.

2003/0153043 by Carr et al.

[00172] Constant regions of the antibodies or antibody fragments utilized for preparation of the antibody cytokine engrafted proteins can be any type or subtype, as appropriate, and can be selected to be from the species of the subject to be treated by the present methods (e.g. , human, non- human primate or other mammal, for example, agricultural mammal (e.g., equine, ovine, bovine, porcine, camelid), domestic mammal (e.g., canine, feline) or rodent (e.g., rat, mouse, hamster, rabbit). In some embodiments antibodies utilized in antibody cytokine engrafted proteins are engineered to generate humanized or Humaneered® antibodies. In some embodiments antibodies utilized in antibody cytokine engrafted proteins are human antibodies. In some embodiments, antibody constant region isotype is IgG, for example, IgGl, IgG2, IgG3, IgG4. In certain embodiments the constant region isotype is IgGi. In some embodiments, antibody cytokine engrafted proteins comprise an IgG. In some embodiments, antibody cytokine engrafted proteins comprise an IgGl Fc. In some embodiments, antibody cytokine engrafted proteins comprise an IgG2 Fc.

[00173] In addition or alternative to modifications made within framework or CDR regions, antibodies or antibody fragments utilized in preparation of antibody cytokine engrafted proteins can be engineered to include modifications within an Fc region, typically to alter one or more functional properties of the antibody, such as, e.g., serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody or antibody fragment can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody cytokine engrafted protein.

[00174] In one embodiment, a hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. For example, by the approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. wherein the number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody cytokine engrafted protein. In another embodiment, an Fc hinge region of an antibody is mutated to alter the biological half-life of the antibody cytokine engrafted protein. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc- hinge fragment such that the antibody cytokine engrafted protein has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[00175] The present disclosure provides for antibody cytokine engrafted proteins that specifically bind to IL10R protein which have an extended half-life in vivo. In another embodiment, an antibody cytokine engrafted protein is modified to increase its biological half- life. Various approaches are possible. Antibody cytokine engrafted proteins having an increased half-life in vivo can also be generated introducing one or more amino acid modifications (i.e., substitutions, insertions or deletions) into an IgG constant domain, or FcRn binding fragment thereof (preferably a Fc or hinge Fc domain fragment). For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. See, e.g., International Publication No. WO 98/23289; International Publication No. WO 97/34631; and U.S. Patent No. 6,277,375. Alternatively, to increase the biological half-life, the antibody cytokine engrafted protein is altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al. In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody cytokine engrafted protein. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody cytokine engrafted protein has an altered affinity for an effector ligand but retains antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor (FcR) or the CI component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[00176] In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody cytokine engrafted protein has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.

[00177] Antibody cytokine engrafted proteins containing such mutations mediate reduced or no antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some embodiments, amino acid residues L234 and L235 of the IgGl constant region are substituted to Ala234 and Ala235. In some embodiments, amino acid residue N267 of the IgGl constant region is substituted to Ala267.

[00178] In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody cytokine engrafted protein to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.

[00179] In yet another embodiment, an Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody cytokine engrafted protein for an Fey receptor by modifying one or more amino acids. This approach is described further in PCT Publication WO00/42072 by Presta. Moreover, binding sites on human IgGl for FcyRl, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et ah, 2001 J. Biol. Chem. 276:6591-6604).

[00180] In still another embodiment, glycosylation of an antibody cytokine engrafted protein is modified. For example, an aglycoslated antibody cytokine engrafted protein can be made (i.e., the antibody cytokine engrafted protein lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for "antigen." Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[00181] Additionally or alternatively, an antibody cytokine engrafted protein can be made that has an altered type of glycosylation, such as a hypofucosylated antibody cytokine engrafted protein having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody cytokine engrafted protein in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibody cytokine engrafted proteins to thereby produce an antibody cytokine engrafted protein with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibody cytokine engrafted proteins expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibody cytokine engrafted proteins expressed in that host cell (see also Shields, RL. et ah, 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N

acetylglucosaminyltransferase III (GnTIII)) such that antibody cytokine engrafted proteins expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al, 1999 Nat. Biotech. 17: 176- 180).

[00182] In some embodiments, one or more domains, or regions, of an antibody cytokine engrafted protein are connected via a linker, for example, a peptide linker, such as those that are well known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ., et al. (1994) Structure 2: 1121-1123). A peptide linker may vary in length, e.g., a linker can be 1-100 amino acids in length, typically a linker is from five to 50 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.

[00183] In some embodiments, monomeric IL10 is incorporated into the CDR sequence optionally with one or more peptide linker sequences. In certain embodiments one or more peptide linkers is independently selected from a (Gly _n -Ser) _m sequence, a (Gly _n -Ala) _m sequence, or any combination of a (Gly _n -Ser) _m /(Gly _n -Ala) _m sequence, wherein each n is independently an integer from 1 to 5 and each m is independently an integer from 0 to 10. In some embodiments a peptide linker is (Gly ₄ -Ser) _m wherein m is an integer from 0 to 10. In some embodiments a peptide linker is (Gly ₄ -Ala) _m wherein m is an integer from 0 to 10. Examples of linkers include, but are not limited to, certain embodiments one or more linkers include G ₄ S repeats, e.g., the Gly-Ser linker GGGGS (SEQ ID NO:252), or (GGGGS) _m wherein m is a positive integer equal to or greater than 1. For example, m=l, m=2, m=3. m=4, m=5 and m=6, m=7, m=8, m=9 and m=10. In some embodiments, the linker includes multiple repeats of GGGGS (SEQ ID

NO:252), including, but is not limited to (GGGGS)3 or (GGGGS) ₄ . In some embodiments, Ser can be replaced with Ala e.g., linkers G/A such as (GGGGA) (SEQ ID NO: 253), or (GGGGA) _m wherein m is a positive integer equal to or greater than 1. In some embodiments, the linker includes multiple repeats of GGGGA (SEQ ID NO:253). In other embodiments, a linker includes combinations and multiples of GGGGS (SEQ ID NO:252) and GGGGA (SEQ ID NO:253).

[00184] Moreover, the antibody cytokine engrafted proteins can be linked to marker sequences, such as a peptide to facilitate purification of antibody cytokine engrafted proteins. In preferred embodiments, a marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al, 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient purification of the engrafted protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et ah, 1984, Cell 37:767), and the "flag" tag.

[00185] Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or

polypropylene.

Assays for Antibody Cytokine Engrafted Protein activity

[00186] Assays for identifying antibody cytokine engrafted proteins are known in the art and are described herein. The antibody cytokine engrafted proteins bind to IL10 receptor (IL10R), and promote, induce and stimulate intracellular signaling resulting in anti-inflammatory effects as well as immunostimulatory effects.

[00187] Binding of the antibody cytokine engrafted proteins to IL-10R can be determined using any method known in the art. For example, binding to IL-10R can be determined using known techniques, including without limitation ELISA, Western blots, surface plasmon resonance (e.g., BIAcore), and flow cytometry.

[00188] Intracellular signaling through IL-1 OR can be measured using any method known in the art. For example, activation through IL-1 OR promotes STAT3 phosphorylation and signaling. Methods for measuring STAT3 activation are standard in the art (e.g.,

phosphorylation status of STAT3 protein, reporter gene assays, downstream signaling assays, etc.). Activation through IL-10R has anti-inflammatory effects, including suppression of proinflammatory cytokines as well as differentiation and proliferation of macrophages.

Additionally, activation through IL-1 OR has immunostimulatory effects including stimulation of B cell proliferation, mast cell proliferation (e.g., MC/9), and natural killer (NK) cell proliferation and proliferation of activated CD8 ⁺ T cells, as well as induction of certain pro-inflammatory cytokines. Methods for measuring proliferation of cells are standard in the art (e.g., ³ H- thymidine incorporation assays, CFSE labeling). Methods for measuring cytokine production are well known in the art (e.g., ELISA assays, ELISpot assays). In performing in vitro assays, test cells or culture supernatant from test cells contacted with an agonist antibody cytokine engrafted proteins can be compared to control cells or culture supernatants from control cells that have not been contacted with an agonist antibody cytokine engrafted proteins and/or those that have been contacted with recombinant human IL10 (rhlLlO).

[00189] IL10 receptor agonist activity of the antibody cytokine engrafted proteins can also be measured ex vivo and/or in vivo. In some aspects, methods for measuring STAT3 activation across various cell types ex vivo from animals treated with antibody cytokine engrafted proteins as compared to untreated control animals and/or animals similarly treated with rhlLlO may be used to show differential activity of the antibody engrafted proteins across cell types. Preferred antibody cytokine engrafted proteins have the ability to activate and expand monocytes. For example, in vivo activation and expansion of monocytes can be measured using any method known in the art, e.g., by flow cytometry. Antibody cytokine engrafted proteins can be therapeutically useful in suppressing levels of pro-inflammatory response following stimulation. Levels of pro-inflammatory cytokines can be measured using any method known in the art in samples isolated from animals treated with an antibody cytokine engrafted protein before, during and/or after treatment with a stimulatory agent (eg., LPS), and results may be compared to non-treated control animals and/or animals similarly treated with recombinant human ILlO(rhlLlO) therapy. Preferred agonist antibody cytokine engrafted proteins can be therapeutically useful in preventing, reducing, inhibiting or eliminating inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, type I diabetes, acute pancreatitis, uveitis, Sjogren's disease, Behcet's disease, sarcoidosis, and graft versus host disease (GVHD). The efficacy of the agonist antibody cytokine engrafted proteins can be determined by administering a therapeutically effective amount of the antibody cytokine engrafted protein to a subject and comparing the subject before and after administration of the antibody cytokine engrafted protein. Efficacy of the agonist antibody cytokine engrafted protein in therapy for inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, type I diabetes, acute pancreatitis, uveitis, Sjogren's disease, Behcet's disease, sarcoidosis, and graft versus host disease (GVHD) also can be determined by administering a therapeutically effective amount of an antibody cytokine engrafted protein to a test subject and comparing the test subject to a control subject who has not been administered the antibody cytokine engrafted protein and/or comparison to a subject similarly treated with rhlLlO.

Polynucleotides Encoding Antibody Cytokine Engrafted Proteins

[00190] In another aspect, isolated nucleic acids encoding heavy and light chain proteins of the antibody cytokine engrafted proteins are provided. Antibody cytokine engrafted proteins can be produced by any means known in the art, including but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers. Recombinant expression can be from any appropriate host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[00191] Provided herein are polynucleotides that encode the variable regions exemplified in any one of SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:205, and SEQ ID NO: 206; the variable regions exemplified in any one of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO:207, SEQ ID NO:208, the CDRs exemplified in any one of SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:205, and SEQ ID NO:206; the heavy and light chains exemplified in any one of SEQ ID NO: l l l, SEQ ID NO: 112, SEQ ID NO:207 and SEQ ID NO:208.

[00192] The disclosure thus provides polynucleotides encoding the heavy and/or light chain polypeptides of the antibody cytokine engrafted proteins described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising the complementary determining regions as described herein. In some embodiments, the

polynucleotide encoding the heavy chain variable regions comprises a sequence having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO: 242. In some embodiments, the polynucleotide encoding the light chain variable regions comprises a sequence having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO: 243.

[00193] In some embodiments, the polynucleotide encoding the heavy chain variable regions comprises a sequence having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:246. In some embodiments, the polynucleotide encoding the light chain variable regions comprises a sequence having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:247.

[00194] In some embodiments, the polynucleotide encoding the heavy chain has at least

85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO: 244. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:245. [00195] In some embodiments, the polynucleotide encoding the heavy chain has at least

85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:248. In some embodiments, the polynucleotide encoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected of SEQ ID NO:249.

[00196] Polynucleotides can encode the variable region sequence of an antibody cytokine engrafted protein. They can also encode both a variable region and a constant region of the antibody cytokine engrafted protein. Some of the polynucleotide sequences encode a polypeptide that comprises variable regions of both the heavy chain and the light chain of one of the antibody cytokine engrafted proteins. Some other polynucleotides encode two polypeptide segments that respectively are substantially identical to the variable regions of the heavy chain and the light chain of one of the antibody protein engrafted proteins.

[00197] In certain embodiments polynucleotides or nucleic acids comprise DNA. In other embodiments polynucleotides or nucleic acids comprise RNA, which may be single stranded or double stranded.

[00198] In some embodiments a recombinant host cell comprising the nucleic acids encoding one or more cytokine engrafted protein or fragment thereof, and optionally, secretion signals are provided. In certain embodiments a recombinant host cell comprises a vector encoding an antibody cytokine engrafted protein or fragment thereof and secretion signals. In other certain embodiments a recombinant host cell comprises one or more vectors encoding two immunoglobulin protein chains of the antibody cytokine engrafted protein and secretion signals. In some embodiments a recombinant host cell comprises a single vector encoding two immunoglobulin protein chains of the antibody cytokine engrafted protein and secretion signals. In some embodiments a recombinant host cell comprises two vectors, one encoding a heavy chain immunoglobulin protein chain, and another encoding a light chain immunoglobulin protein chain of the heterodimer of the antibody cytokine engrafted protein, with each including secretion signals. A recombinant host cell may be a prokaryotic or eukaryotic cell. In some embodiments a host cell is a eukaryotic cell line. In some embodiments a host cell is a mammalian cell line.

[00199] Additionally provided are methods for producing the antibody cytokine engrafted proteins. In some embodiments the method comprises the steps of (i) culturing a host cell comprising one or more vectors encoding immunoglobulin protein chains of an antibody cytokine engrafted protein under conditions suitable for expression, formation, and secretion of the antibody cytokine engrafted protein and (ii) recovering the antibody cytokine engrafted protein.

[00200] The polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e.g., sequences as described herein) encoding a polypeptide chain of an antibody cytokine engrafted protein. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al, Meth. Enzymol. 68:90, 1979; the phosphodiester method of Brown et al, Meth. Enzymol. 68: 109, 1979; the diethylphosphoramidite method of Beaucage et al, Tetra. Lett., 22: 1859, 1981; and the solid support method of U.S. Patent No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H.A. Er ch (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and

Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al, Nucleic Acids Res. 19:967, 1991; and Eckert ei a/., PCR Methods and Applications 1 : 17, 1991.

[00201] Also provided in the disclosure are expression vectors and host cells for producing the antibody cytokine engrafted proteins described above. Various expression vectors can be employed to express polynucleotides encoding the immunoglobulin polypeptide chains, or fragments, of the antibody cytokine engrafted proteins. Both viral-based and nonviral expression vectors can be used to produce the immunoglobulin proteins in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al, Nat Genet 15:345, 1997). For example, nonviral vectors useful for expression of the antibody cytokine engrafted protein polynucleotides and polypeptides in mammalian {e.g., human) cells include pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al, supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al, Cell 68: 143, 1992. [00202] The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, the expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides encoding the antibody cytokine engrafted protein. In some embodiments, an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions.

Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under noninducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of an immunoglobulin chain or fragment of the antibody cytokine engrafted proteins. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al, Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al, Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.

[00203] Expression vectors can also provide a secretion signal sequence to form a heterologous protein in addition to sequences encoded by the antibody cytokine engrafted protein sequences. More often, the inserted immunoglobulin sequences of the antibody cytokine engrafted proteins are operably linked to a signal sequence before inclusion in the vector.

Vectors to be used to receive sequences encoding immunoglobulin light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as antibody cytokine engrafted proteins with the constant regions, thereby leading to production of intact antibody cytokine engrafted proteins or fragments thereof. Typically, such constant regions are human.

[00204] Host cells for harboring and expressing the antibody cytokine engrafted protein chains can be either prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which typically contain expression control sequences compatible with the host cell {e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation. Other microbes, such as yeast, can also be employed to express antibody cytokine engrafted proteins. Insect cells in combination with baculovirus vectors can also be used.

[00205] In some preferred embodiments, mammalian host cells are used to express and produce the antibody cytokine engrafted proteins of the present disclosure. For example, they can be a mammalian cell line harboring an exogenous expression vector. These include any normal mortal or normal or abnormal immortal animal or human cell. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including the CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen et al, Immunol. Rev. 89:49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone- inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[00206] Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (see generally Sambrook et al, supra). Other methods include, e.g., electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, poly cation: nucleic acid conjugates, naked DNA, artificial virions, engrafted to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression will often be desired. For example, cell lines which stably express antibody cytokine engrafted protein immunoglobulin chains can be prepared using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media. Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate to the cell type.

Compositions Comprising Antibody Cytokine Engrafted Proteins

[00207] Provided are pharmaceutical compositions comprising an antibody cytokine engrafted protein formulated together with a pharmaceutically acceptable carrier. Optionally, pharmaceutical compositions additionally contain other therapeutic agents that are suitable for treating or preventing a given disorder. Pharmaceutically acceptable carriers enhance or stabilize the composition, or facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[00208] A pharmaceutical composition of the present disclosure can be administered by a variety of methods known in the art. Route and/or mode of administration vary depending upon the desired results. It is preferred that administration be by parenteral administration (e.g., selected from any of intravenous, intramuscular, intraperitoneal, intrathecal, intraarterial, or subcutaneous), or administered proximal to the site of the target. A pharmaceutically acceptable carrier is suitable for administration by any one or more of intravenous, intramuscular, intraperitoneal, intrathecal, intraarterial, subcutaneous, intranasal, inhalational, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of

administration, active compound, e.g., antibody cytokine engrafted protein, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. In some embodiments the pharmaceutical composition is formulated for intravenous administration. In some embodiments the pharmaceutical composition is formulation for subcutaneous administration. [00209] An antibody cytokine engrafted protein, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[00210] In some embodiments, a pharmaceutical composition is sterile and fluid. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. In certain embodiments

compositions can be prepared for storage in a lyophilized form using appropriate excipients (e.g., sucrose).

[00211] Pharmaceutical compositions can be prepared in accordance with methods well known and routinely practiced in the art. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present disclosure. Applicable methods for formulating an antibody cytokine engrafted protein and determining appropriate dosing and scheduling can be found, for example, in Remington: The Science and Practice of Pharmacy, 21 ^st Ed., University of the Sciences in Philadelphia, Eds., Lippincott Williams & Wilkins (2005); and mMartindale: The Complete Drug Reference , Sweetman, 2005, London: Pharmaceutical Press., and in

Martindal e, Martindale: The Extra Pharmacopoeia, 31st Edition., 1996, Amer Pharmaceutical Assn, and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, each of which are hereby incorporated herein by reference.

Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of an antibody cytokine engrafted protein is employed in the pharmaceutical compositions. An antibody cytokine engrafted protein is formulated into pharmaceutically acceptable dosage form by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the desired response (e.g., a therapeutic response). In determining a therapeutically or prophylactically effective dose, a low dose can be administered and then incrementally increased until a desired response is achieved with minimal or no undesired side effects. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[00212] Actual dosage levels of active ingredients in the pharmaceutical compositions of the present disclosure can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.

Co-Formulation with Second Agent

[00213] In some embodiments, the pharmacological compositions comprise a mixture of an antibody cytokine engrafted protein and one or more additional pharmacological agent(s). Exemplary second agents for inclusion in mixtures with the present antibody cytokine engrafted protein include without limitation anti-inflammatory agents, immunomodulatory agents, aminosalicylates, and antibiotics. Appropriate selection may depend on preferred formulation, dosage and/or delivery method.

[00214] In some embodiments an antibody cytokine engrafted protein is co-formulated

{i.e., provided as a mixture or prepared in a mixture) with an anti-inflammatory agent. In particular embodiments, corticosteroid anti-inflammatory agents can be used in conjunction with the antibody cytokine engrafted protein. Corticosteroids for use can be selected from any of methylprednisolone, hydrocortisone, prednisone, budenisonide, mesalamine, and

dexamethasone. Appropriate selection will depend on formulation and delivery preferences. [00215] In some embodiments, an antibody cytokine engrafted protein is co-formulated with an immunomodulatory agent. In particular embodiments, the immunomodulatory agent is selected from any of 6-mercaptopurine, azathioprine, cyclosporine A, tacrolimus, and methotrexate. In a particular embodiment, the immunomodulatory agent is selected from an anti-TNF agent (e.g., infliximab, adalimumab, certolizumab, golimumab), natalizumab, and vedolizumab.

[00216] In some embodiments an antibody cytokine engrafted protein is co-formulated with an aminosalicylate agent. In particular embodiments, an aminosalicylate is selected from sulfasalazine, mesalamine, balsalazide, olsalazine or other derivatives of 5-aminosalicylic acid.

[00217] In some embodiments an antibody cytokine engrafted protein is co-formulated with an antibacterial agent. Exemplary antibacterial agents include without limitation sulfonamides (e.g., sulfanilamide, sulfadiazine, sulfamethoxazole, sulfisoxazole, sulfacetamide), trimethoprim, quinolones (e.g., nalidixic acid, cinoxacin, norfloxacin, ciprofloxacin, ofloxacin, sparfloxacin, fleroxacin, perloxacin, levofloxacin, garenoxacin and gemifloxacin), methenamine, nitrofurantoin, penicillins (e.g., penicillin G, penicillin V, methicilin oxacillin, cloxacillin, dicloxacillin, nafcilin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, and piperacillin), cephalosporins (e.g., cefazolin, cephalexin, cefadroxil, cefoxitin, cefaclor, cefprozil, cefuroxime, cefuroxime acetil, loracarbef, cefotetan, ceforanide, cefotaxime, cefpodoxime proxetil, cefibuten, cefdinir, cefditoren pivorxil, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, and cefepine), carbapenems (e.g., imipenem, aztreonam), and aminoglycosides (e.g., neomycin, kanamycin, streptomycin, gentamicin, toramycin, netilmicin, and amikacin).

Articles of Manufacture / Kits

[00218] In some aspects, an antibody cytokine engrafted protein is provided in an article of manufacture (i.e., a kit). The antibody cytokine engrafted protein is generally in a vial or a container. Thus, an article of manufacture comprises a container and a label or package insert, on or associated with the container. Suitable containers include, for example, a bottle, vial, syringe, solution bag, etc. As appropriate, the antibody cytokine engrafted protein can be in liquid or dried (e.g., lyophilized) form. The container holds a composition which, by itself or combined with another composition, is effective for preparing a composition for treating, preventing and/or ameliorating an immune related disorder. The label or package insert indicates the composition is used for treating, preventing and/or ameliorating an immune related disorder. Articles of manufacture (kits) comprising an antibody cytokine engrafted protein, as described herein, optionally contain one or more additional agent. In some embodiments, an article of manufacture (kit) contains antibody cytokine engrafted protein and a pharmaceutically acceptable diluent. In some embodiments an antibody cytokine engrafted protein is provided in an article of manufacture (kit) with one or more additional active agent in the same formulation (e.g., as mixtures). In some embodiments an antibody cytokine engrafted protein is provided in an article of manufacture (kit) with a second or third agent in separate formulations (e.g., in separate containers). In certain embodiments an article of manufacture (kit) contains aliquots of the antibody cytokine engrafted protein, wherein the aliquot provides for one or more doses. In some embodiments aliquots for multiple administrations are provided, wherein doses are uniform or varied. In particular embodiments varied dosing regimens are escalating or decreasing, as appropriate. In some embodiments dosages of an antibody cytokine engrafted protein and a second agent are independently uniform or independently varying. In certain embodiments, an article of manufacture (kit) comprises an additional agent selected from any of an antiinflammatory agent, immunomodulatory agent, aminosalicylate, and antibiotic. Selection of one or more additional agent will depend on the dosage, delivery, and disease condition to be treated. Methods of Treatment of and use of Antibody Cytokine Engrafted proteins in Immune Related Disorders

Conditions Subject to Treatment or Prevention

[00219] Antibody cytokine engrafted proteins find use in the treatment, amelioration or prophylaxis of immune related disorders. In one aspect, the disclosure provides methods of treatment of immune related disorders in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of an antibody cytokine engrafted protein as described herein. The disclosure also provides in one aspect, an antibody cytokine engrafted protein for use as a therapeutic agent. In some embodiment an antibody cytokine engrafted protein is provided for use as a therapeutic agent in the treatment or prophylaxis of an immune related disorder in an individual. In some embodiments use of an antibody cytokine engrafted protein is provided for manufacture of a medicament for treatment of an immune related disorder in an individual in need thereof. In a further aspect, the disclosure provides a composition comprising such an antibody cytokine engrafted protein for use in treating or ameliorating an immune related disorder in an individual in need thereof. [00220] For therapeutic purposes, an individual can have an immune related disorder.

For preventative or prophylactic purposes, an individual may be in remission from an active state of immune related disorder or may anticipate future onset. In some embodiments, the patient has an immune related disorder, is suspected of having an immune related disorder, or is in remission from an immune related disorder. Immune related disorders subject to treatment with the antibody cytokine engrafted protein usually derive benefit from activation of IL10 receptor signaling, as described herein. Immune related disorders subject to treatment include without limitation: inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, type I diabetes, acute pancreatitis, uveitis, Sjogren's disease, Behcet's disease, sarcoidosis, and graft versus host disease (GVHD).

Administration of Antibody Cytokine Engrafted Proteins

[00221] A physician or veterinarian can start doses of an antibody cytokine engrafted protein employed in a pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, effective doses of the compositions of the present disclosure vary depending upon many different factors, including the specific disease or condition to be treated, means of administration, target site, physiological state of the patient, whether a patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages typically require titration to optimize safety and efficacy. For administration with an antibody cytokine engrafted protein, dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.

Dosing can be daily, weekly, bi-weekly, monthly, or more or less often, as needed or desired. An exemplary treatment regime entails administration once weekly, once per every two weeks or once a month or once every 3 to 6 months.

[00222] The antibody cytokine engrafted protein can be administered in single or divided doses. An antibody cytokine engrafted protein is usually administered on multiple occasions. Intervals between single dosages can be weekly, bi-weekly, monthly or yearly, as needed or desired. Intervals can also be irregular as indicated by measuring blood levels of antibody cytokine engrafted protein in the patient. In some methods, dosage is adjusted to achieve a plasma antibody cytokine engrafted protein concentration of 1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively, antibody cytokine engrafted protein can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody cytokine engrafted protein in the patient. In general, antibody cytokine engrafted proteins show longer half-life than that of native cytokines (e.g. IL10). Dosage and frequency of administration can vary depending on whether treatment is prophylactic or therapeutic. In general for prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the duration of their lives. In general for therapeutic applications, a relatively high dosage in relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, a patient can be administered a prophylactic regime.

Co-Administration with a Second Agent

[00223] In some embodiments, an antibody cytokine engrafted protein is coadministered with one or more additional pharmacological agent(s). In some embodiments, an antibody cytokine engrafted protein and an additional one or more agent(s) are administered as a mixture. In some embodiments, an antibody cytokine engrafted protein and an additional one or more agent(s) are administered as separate formulations. In certain embodiments where separate formulations are utilized, administration is concurrent. In certain embodiments where separate formulations are utilized, administration is sequential. In certain embodiments where separate formulations are utilized, administration is via the same route. In certain embodiments where separate formulations are utilized, administration is via different routes. Exemplary additional agents for co-administration with an antibody cytokine engrafted protein include without limitation anti-inflammatory agents, immunomodulatory agents, aminosalicylates, and antibiotics. Appropriate selection may depend on preferred formulation, dosage and/or delivery method. The antibody cytokine engrafted proteins also find use in combination therapies with additional established procedures for treating immune related disorder conditions, e.g., surgery.

[00224] In some embodiments, an antibody cytokine engrafted protein is coadministered with an anti-inflammatory agent. In particular embodiments corticosteroid antiinflammatory agents may be used in conjunction with the antibody cytokine engrafted protein. Corticosteroids for use may be selected from any of methylprednisolone, hydrocortisone, prednisone, budenisonide, mesalamine, and dexamethasone. Appropriate selection will depend on formulation and delivery preferences. [00225] In some embodiments, an antibody cytokine engrafted protein is coadministered with an immunomodulatory agent. In certain embodiments, the

immunomodulatory agent is selected from any of 6-mercaptopurine, azathioprine, cyclosporine A, tacrolimus, and methotrexate. In particular embodiments, an immunomodulatory agent is selected from an anti-TNF agent (e.g., infliximab, adalimumab, certolizumab, golimumab), natalizumab, and vedolizumab.

[00226] In some embodiments an antibody cytokine engrafted protein is co-administered with an aminosalicylate agent. In particular embodiments the aminosalicylate agent is selected from sulfasalazine, mesalamine, balsalazide, olsalazine or other derivatives of 5-aminosalicylic acid.

[00227] In some embodiments an antibody cytokine engrafted protein is co-administered with an antibacterial agent. Exemplary antibacterial agents include without limitation sulfonamides (e.g., sulfanilamide, sulfadiazine, sulfamethoxazole, sulfisoxazole, sulfacetamide), trimethoprim, quinolones (e.g., nalidixic acid, cinoxacin, norfloxacin, ciprofloxacin, ofloxacin, sparfloxacin, fleroxacin, perloxacin, levofloxacin, garenoxacin and gemifloxacin), methenamine, nitrofurantoin, penicillins (e.g., penicillin G, penicillin V, methicilin oxacillin, cloxacillin, dicloxacillin, nafcilin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, and piperacillin), cephalosporins (e.g., cefazolin, cephalexin, cefadroxil, cefoxitin, cefaclor, cefprozil, cefuroxime, cefuroxime acetil, loracarbef, cefotetan, ceforanide, cefotaxime, cefpodoxime proxetil, cefibuten, cefdinir, cefditoren pivorxil, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, and cefepine), carbapenems (e.g., imipenem, aztreonam), and aminoglycosides (e.g., neomycin, kanamycin, streptomycin, gentamicin, toramycin, netilmicin, and amikacin).

EXAMPLES

Example 1: Creation of Immunoglobulin-ILIO engrafted constructs

[00228] Engrafted constructs IgGILl 0M1 , IgGILl 0M2, IgGILl 0M3 , IgGILl 0M4,

IgGIL10M5, IgGILl 0M6, IgGILl 0M7, IgGILl 0M8, IgGILl 0M9, IgGILlOMlO, IgGILl OMl l , IgGILl 0M12, IgGILl 0M13, IgGILl 0M14, and IgGIL10M15 were generated by engineering a monomeric IL10 sequence into CDR regions of various immunoglobulin scaffolds, then both heavy and light chain immunoglobulin chains were produced to generate final protein constructs. IgGILlOM engrafted constructs confer preferred therapeutic anti-inflammatory properties of IL10; however, IgGILlOM engrafted constructs have reduced proportional pro-inflammatory activity as compared with rhlLlO.

[00229] To create antibody cytokine engrafted proteins, monomeric IL10 (ILIOM), comprising residues 19-178 of full length IL10 with a six amino acid linker between residues 134 and 135 (SEQ ID NO: 209) was inserted into various CDR loops of immunoglobulin chain scaffold. Engrafted constructs were prepared using a variety of known immunoglobulin sequences which have been utilized in clinical settings as well as germline antibody sequences. Sequences of IL10M in two exemplary scaffolds, referred to as GFTX1 and GFTX3, are depicted in TABLE 1. Insertion points were selected to be the mid-point of the CDR loop based on available structural or homology model data. Antibody cytokine engrafted proteins were produced using standard molecular biology methodology utilizing recombinant DNA encoding the relevant sequences.

[00230] For example, a variable region of each antibody containing IL10M inserted into one of the six CDRs was synthesized. DNA encoding variable region was amplified via PCR and the resulting fragment was sub-cloned into vector containing either the light chain constant region or the heavy chain constant and Fc regions. In this manner IL10M antibody cytokine engrafted proteins were made corresponding to insertion of IL10M into each of the 6 CDRs (LI, L2, L3, HI, H2, H3). Resulting constructs are shown in TABLE 1. Transfections of the appropriate combination of heavy and light chain vectors results in the expression of a recombinant antibody with two grafted IL10M molecules (one IL10 monomer in each Fab arm). See Figure 2.

[00231] The selection of which CDR is chosen for cytokine engraftment is chosen on the parameters of: the required biology, the biophysical properties and a favorable development profile. At this time, modeling software is only partially useful in predicting which CDR and which location within the CDR will provide the desired parameters, so therefore all six possible antibody cytokine grafts are made and then evaluated in biological assays. If the required biological activity was achieved, then the biophysical properties such as structural resolution of the antibody cytokine engrafted molecule was resolved.

[00232] By virtue of the grafting of IL10 into a CDR, the antibody portion of the antibody cytokine engrafted protein presents the IL10 monomer with a unique structure which influences the binding to the IL10 receptor as discussed below. There are no off-target effects due to the antibody portion. In addition, the Fc portion of the antibody cytokine engrafted protein has been modified to be fully silent regarding ADCC (Antibody Dependent Cell- mediated Cytotoxicity) and CDC (Complement-Dependent Cytotoxicity).

[00233] In summary, the insertion point in each CDR was chosen on a structural basis, with the hypothesis that grafting into the CDR would provide some level of steric hindrance to individual subunits of the IL10 receptor. The final selection of which CDR graft is best for a particular cytokine is based on desired biology and biophysical properties. The nature of the cytokine receptor, the cytokine/receptor interactions and the mechanism of signaling also played a role and this was resolved by comparing each individual antibody cytokine engrafted molecule for their respective properties. For example, engrafting of IL10 into the light chain CDR1 (CDRLl) produced the desired biological activity of activating monocytes but not other cells such as NK cells. This was seen in the exemplary antibody cytokine engrafted proteins IgGIL10M7 and IgGIL10M13.

TABLE 1

SEQ ID

Description Comments NO:

CDRH1 of

1 IgGILlOMl GFTFSSY GFTX1

(Chothia)

CDRH2 of

2 IgGILlOMl SGSGGS GFTX1

(Chothia)

CDRH3 of

3 IgGILlOMl TRTKRF GFTX1

(Chothia)

SOSVSPGOGTOSENSCTHFPGNLPNML GFTX1

RDLRDAFSRVKTFFOMKDOLDNLLLK Monomeric

CDRLl of ESLLEDFKGYLGCOALSEMIOFYLEEV IL10 grafted

4 IgGILlOMl MPOAENODPDIKAHVNSLGENLKTLRL into

(Chothia) RLRRCHRFLPCENGGGSGGKSKAVEO CDRLl.

VKNAFNKLOEKGIYKAMSEFDIFINYIE IL10 is

AYMTMKIRNS S S Y bolded, SEQ ID

Description Comments NO:

underlined

CDRL2 of

5 IgGILlOMl GAS GFTX1

(Chothia)

CDRL3 of

6 IgGILlOMl YGSSPL GFTX1

(Chothia)

CDRH1 of

7 SYAMS GFTX1 IgGILlOMl (Kabat)

CDRH2 of

8 AISGSGGSTYYGDSVKG GFTX1 IgGILlOMl (Kabat)

CDRH3 of

9 TRTKRF GFTX1 IgGILlOMl (Kabat)

GFTX1

RASOSVSPGOGTOSENSCTHFPGNLPN

Monomeric

MLRDLRDAFSRVKTFFOMKDOLDNLL IL10 grafted

LKESLLEDFKGYLGCOALSEMIOFYLE

CDRL1 of into

10 EVMPOAENODPDIKAHVNSLGENLKTL

IgGILlOMl (Kabat) CDRLl.

RLRLRRCHRFLPCEGGGSGGNKSKAV IL10 is

EOVKNAFNKLOEKGIYKAMSEFDIFIN

bolded,

YIE AYMTMKIRNS S S YLA

underlined

CDRL2 of

11 GASSRAT GFTX1 IgGILlOMl (Kabat)

CDRL3 of

12 QQYGSSPLT GFTX1 IgGILlOMl (Kabat)

EVQLLESGGGLVQPGGSLRLSCAASGFTF S S YAM S WVRQ APGKGLEWVS AI S GS GGS

13 VH of IgGILlOMl TYYGDSVKGRFTISRDNSKNTLYLQMNS GFTX1

LRAEDTAVYYCARTRTKRFWGQGTLVT VSS

EIVLTQSPGTLSLSPGERATLSCRASQSVS GFTXIMon

14 VL of IgGILlOMl PGOGTOSENSCTHFPGNLPNMLRDLRD omeric IL10

AFSRVKTFFOMKDOLDNLLLKESLLED grafted into SEQ ID

Description Comments NO:

FKGYLGCOALSEMIOFYLEEVMPOAEN CDRL1. ODPDIKAHVNSLGENLKTLRLRLRRCH RFLP CENGGGS GGKSKA VE 0 VKNAFN

KLOEKGIYKAMSEFDIFINYIE

AYMTMKIRNSSSYLAWYOOKPGOAPRL LIYGASSRATGIPDRFSGSGSGTDFTLTISR LEPEDFAVYYCQQYGSSPLTFGGGTKVEI K

EVQLLESGGGLVQPGGSLRLSCAASGFTF

S S YAM S WVRQ APGKGLEWVS AI S GS GGS

TYYGDSVKGRFTISRDNSKNTLYLQMNS

LRAEDTAVYYCARTRTKRFWGQGTLVT

VSSASTKGPSVFPLAPSSKSTSGGTAALGC

L VKD YFPEP VT VS WNS GALT S GVHTFP A

VLQSSGLYSLSSVVTVPSSSLGTQTYICNV

Heavy chain of NHKPSNTKVDKRVEPKSCDKTHTCPPCP

15 GFTX1 IgGILlOMl APELLGGP S VFLFPPKPKDTLMI SRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLN

GKEYKCKVSNKALPAPIEKTISKAKGQPR

EPQVYTLPPSREEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDG

SFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

EIVLTQSPGTLSLSPGERATLSCRASQSVS

PGOGTOSENSCTHFPGNLPNMLRDLRD

AFSRVKTFFOMKDOLDNLLLKESLLED GFTX1

FKGYLGCOALSEMIOFYLEEVMPOAEN Monomeric

Light chain of

16 ODPDIKAHVNSLGENLKTLRLRLRRCH IL10 grafted IgGILlOMl

RFLP CENGGGS GGKSKA VE 0 VKNAFN into

KLOEKGIYKAMSEFDIFINYIEAYMTM CDRL1.

KIRNS SS YLAWYQQKPGQ APRLLI YGAS S

RATGIPDRFSGSGSGTDFTLTISRLEPEDFA SEQ ID

Description Comments NO:

VYYCQQYGSSPLTFGGGTKVEIKRTVAAP

SVFIFPPSDEQLKSGTASVVCLLNNFYPRE

AKVQWKVDNALQSGNSQESVTEQDSKD

STYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGEC

CDRH1 of

17 IgGIL10M2 GFTFSSY GFTX1

(Chothia)

CDRH2 of

18 IgGIL10M2 SGSGGS GFTX1

(Chothia)

CDRH3 of

19 IgGIL10M2 TRTKRF GFTX1

(Chothia)

CDRL1 of

20 SQSVSSSY GFTX1

IgGIL10M2(Chothia)

GFTX1

GASSSPGOGTOSENSCTHFPGNLPNML

Monomeric

RDLRDAFSRVKTFFOMKDOLDNLLLK IL10 grafted

CDRL2 of ESLLEDFKGYLGCOALSEMIOFYLEEV

into

21 IgGIL10M2 MPOAENODPDIKAHVNSLGENLKTLRL

CDRL2.

(Chothia) RLRRCHRFLPCENGGGSGGKSKAVEO

IL10 is

VKNAFNKLOEKGIYKAMSEFDIFINYIE

bolded,

AYMTMKIRN

underlined

CDRL3 of

22 IgGIL10M2 YGSSPL GFTX1

(Chothia)

CDRH1 of

23 SYAMS GFTX1 IgGIL10M2 (Kabat)

CDRH2 of

24 AISGSGGSTYYGDSVKG GFTX1 IgGIL10M2 (Kabat)

CDRH3 of

25 TRTKRF GFTX1 IgGIL10M2 (Kabat) SEQ ID

Description Comments NO:

CDRL1 of

26 RASQSVSSSYLA GFTX1 IgGIL10M2(Kabat)

GFTX1

GASSSPGOGTOSENSCTHFPGNLPNML

Monomeric

RDLRDAFSRVKTFFOMKDOLDNLLLK IL10 grafted

ESLLEDFKGYLGCOALSEMIOFYLEEV

CDRL2 of into

27 MPOAENODPDIKAHVNSLGENLKTLRL

IgGIL10M2 (Kabat) CDRL2.

RLRRCHRFLPCENGGGSGGKSKAVEO IL10 is

VKNAFNKLOEKGIYKAMSEFDIFINYIE

bolded,

AYMTMKIRNRAT

underlined

CDRL3 of

28 QQYGSSPLT GFTX1 IgGIL10M2 (Kabat)

EVQLLESGGGLVQPGGSLRLSCAASGFTF S S YAM S WVRQ APGKGLEWVS AI S GS GGS

29 VH of IgGIL10M2 TYYGDSVKGRFTISRDNSKNTLYLQMNS GFTX1

LRAEDTAVYYCARTRTKRFWGQGTLVT VSS

EIVLTQSPGTLSLSPGERATLSCRASQSVS

SSYLAWYQQKPGQAPRLLIYGASSSPGQ

GTOSENSCTHFPGNLPNMLRDLRDAFS

GFTX1

RVKTFFOMKDOLDNLLLKESLLEDFK

Monomeric

GYLGCOALSEMIOFYLEEVMPOAENO

30 VL of IgGIL10M2 IL10 grafted

DPDIKAHVNSLGENLKTLRLRLRRCHR

into

FLPCENGGGSGGKSKAVEOVKNAFNK

CDRL2.

LOEKGIYKAMSEFDIFINYIEAYMTMKI

RNRATGIPDRFSGSGSGTDFTLTISRLEPE

DFAVYYCQQYGSSPLTFGGGTKVEIK

EVQLLESGGGLVQPGGSLRLSCAASGFTF S S YAM S WVRQ APGKGLEWVS AI S GS GGS

Heavy chain of TYYGDSVKGRFTISRDNSKNTLYLQMNS

31 GFTX1 IgGIL10M2 LRAEDTAVYYCARTRTKRFWGQGTLVT

VSSASTKGPSVFPLAPSSKSTSGGTAALGC L VKD YFPEP VT VS WNS GALT S GVHTFP A SEQ ID

Description Comments NO:

VLQSSGLYSLSSVVTVPSSSLGTQTYICNV

NHKPSNTKVDKRVEPKSCDKTHTCPPCP

APELLGGP S VFLFPPKPKDTLMI SRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLN

GKEYKCKVSNKALPAPIEKTISKAKGQPR

EPQVYTLPPSREEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDG

SFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

EIVLTQSPGTLSLSPGERATLSCRASQSVS

SSYLAWYQQKPGQAPRLLIYGASSSPGQ

GTOSENSCTHFPGNLPNMLRDLRDAFS

RVKTFFOMKDOLDNLLLKESLLEDFK GFTX1

GYLGCOALSEMIOFYLEEVMPOAENO Monomeric

DPDIKAHVNSLGENLKTLRLRLRRCHR IL10 grafted

Light chain of FLPCENGGGSGGKSKAVEOVKNAFNK into

32

IgGIL10M2 LOEKGIYKAMSEFDIFINYIEAYMTMKI CDRL2.

RNRATGIPDRFSGSGSGTDFTLTISRLEPE IL10 is

DFAVYYCQQYGSSPLTFGGGTKVEIKRTV bolded,

AAPSVFIFPPSDEQLKSGTASVVCLLNNFY underlined

PREAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC

CDRH1 of

33 IgGIL10M3 GFTFSSY GFTX1

(Chothia)

CDRH2 of

34 IgGIL10M3 SGSGGS GFTX1

(Chothia)

CDRH3 of

35 IgGIL10M3 TRTKRF GFTX1

(Chothia) SEQ ID

Description Comments NO:

CDRL1 of

36 SQSVSSSY GFTX1

IgGIL10M3(Chothia)

CDRL2 of

37 IgGIL10M3 GAS GFTX1

(Chothia)

GFTX1

YGSSPGOGTOSENSCTHFPGNLPNMLR

Monomeric

DLRDAFSRVKTFFOMKDOLDNLLLKES IL10 grafted

CDRL3 of LLEDFKGYLGCOALSEMIOFYLEEVMP

into

38 IgGIL10M3 OAENODPDIKAHVNSLGENLKTLRLRL

CDRL3.

(Chothia) RRCHRFLP CENGGGS GGKSKA VE 0 VK

IL10 is

NAFNKLOEKGIYKAMSEFDIFINYIEAY

bolded,

MTMKIRNSPL

underlined

CDRH1 of

39 SYAMS GFTX1 IgGIL10M3 (Kabat)

CDRH2 of

40 AISGSGGSTYYGDSVKG GFTX1 IgGIL10M3 (Kabat)

CDRH3 of

41 TRTKRF GFTX1 IgGIL10M3 (Kabat)

CDRL1 of

42 RASQSVSSSYLA GFTX1 IgGIL10M3 (Kabat)

CDRL2 of

43 GASSRAT GFTX1 IgGIL10M3 (Kabat)

GFTX1

OOYGSSPGOGTOSENSCTHFPGNLPNM

Monomeric

LRDLRDAFSRVKTFFOMKDOLDNLLL IL10 grafted

KESLLEDFKGYLGCOALSEMIOFYLEE

CDRL3 of into

44 VMPOAENODPDIKAHVNSLGENLKTLR

IgGIL10M3 (Kabat) CDRL3.

LRLRRCHRFLPCENGGGSGGKSKAVE IL10 is

OVKNAFNKLOEKGIYKAMSEFDIFINYI

bolded,

E A YMTMKIRN SPLT

underlined

EVQLLESGGGLVQPGGSLRLSCAASGFTF

45 VH of !gGIL10M3 GFTX1

S S YAM S WVRQ APGKGLEWVS AI S GS GGS SEQ ID

Description Comments NO:

TYYGDSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCARTRTKRFWGQGTLVT VSS

EIVLTQSPGTLSLSPGERATLSCRASQSVS

SSYLAWYQQKPGQAPRLLIYGASSRATGI GFTX1

PDRF S GS GS GTDFTLTI SRLEPEDF AV Y YC Monomeric

OOYGSSPGOGTOSENSCTHFPGNLPNM IL10 grafted

LRDLRDAFSRVKTFFOMKDOLDNLLL into

46 VL of IgGIL10M3

KESLLEDFKGYLGCOALSEMIOFYLEE CDRL3.

VMPOAENODPDIKAHVNSLGENLKTLR IL10 is

LRLRRCHRFLPCENGGGSGGKSKAVE bolded,

OVKNAFNKLOEKGIYKAMSEFDIFINYI underlined

EAYMTMKIRNSPLTFGGGTKVEIK

EVQLLESGGGLVQPGGSLRLSCAASGFTF

S S YAM S WVRQ APGKGLEWVS AI S GS GGS

TYYGDSVKGRFTISRDNSKNTLYLQMNS

LRAEDTAVYYCARTRTKRFWGQGTLVT

VSSASTKGPSVFPLAPSSKSTSGGTAALGC

L VKD YFPEP VT VS WNS GALT S GVHTFP A

VLQSSGLYSLSSVVTVPSSSLGTQTYICNV

Heavy chain of NHKPSNTKVDKRVEPKSCDKTHTCPPCP

47 GFTX1 IgGIL10M3 APELLGGP S VFLFPPKPKDTLMI SRTPEVT

CVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLN

GKEYKCKVSNKALPAPIEKTISKAKGQPR

EPQVYTLPPSREEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDG

SFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

EIVLTQSPGTLSLSPGERATLSCRASQSVS GFTX1

Light chain of SSYLAWYQQKPGQAPRLLIYGASSRATGI Monomeric

48

IgGIL10M3 PDRF S GS GS GTDFTLTI SRLEPEDF AVYYC IL10 grafted

OOYGSSPGOGTOSENSCTHFPGNLPNM into SEQ ID

Description Comments NO:

LRDLRDAFSRVKTFFOMKDOLDNLLL CDRL3.

KESLLEDFKGYLGCOALSEMIOFYLEE

VMPOAENODPDIKAHVNSLGENLKTLR

LRLRRCHRFLPCENGGGSGGKSKAVE

OVKNAFNKLOEKGIYKAMSEFDIFINYI

EAYMTMKIRNSPLTFGGGTKVEIKRTVA

APSVFIFPPSDEQLKSGTASVVCLLNNFYP

REAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC

GFTX1

GFTFSPGOGTOSENSCTHFPGNLPNML

Monomeric

RDLRDAFSRVKTFFOMKDOLDNLLLK IL10 grafted

CDRH1 of ESLLEDFKGYLGCOALSEMIOFYLEEV

into

49 IgGIL10M4 MPOAENODPDIKAHVNSLGENLKTLRL

CDRH1.

(Chothia) RLRRCHRFLPCENGGGSGGKSKAVEO

IL10 is

VKNAFNKLOEKGIYKAMSEFDIFINYIE

bolded,

AYMTMKIRNSSY

underlined

CDRH2 of

50 IgGIL10M4 SGSGGS GFTX1

(Chothia)

CDRH3 of

51 IgGIL10M4 TRTKRF GFTX1

(Chothia)

CDRL1 of

52 IgGIL10M4 SQSVSSSY GFTX1

(Chothia)

CDRL2 of

53 IgGIL10M4 GAS GFTX1

(Chothia)

CDRL3 of

54 IgGIL10M4 YGSSPL GFTX1

(Chothia) SEQ ID

Description Comments NO:

GFTX1

SPGOGTOSENSCTHFPGNLPNMLRDLR

Monomeric

DAFSRVKTFFOMKDOLDNLLLKESLLE IL10 grafted

DFKGYLGCOALSEMIOFYLEEVMPOAE

CDRH1 of into

55 NODPDIKAHVNSLGENLKTLRLRLRRC

IgGIL10M4 (Kabat) CDRH1.

HRFLPCENGGGSGGKSKAVEOVKNAF IL10 is

NKLOEKGIYKAMSEFDIFINYIEAYMT

bolded,

MKIRNS S YAMS

underlined

CDRH2 of

56 AISGSGGSTYYGDSVKG GFTX1 IgGIL10M4 (Kabat)

CDRH3 of

57 TRTKRF GFTX1 IgGIL10M4 (Kabat)

CDRL1 of

58 RASQSVSSSYLA GFTX1 IgGIL10M4 (Kabat)

CDRL2 of

59 GASSRAT GFTX1 IgGIL10M4 (Kabat)

CDRL3 of

60 QQYGSSPLT GFTX1 IgGIL10M4 (Kabat)

EVQLLESGGGLVQPGGSLRLSCAASGFTF

SPGOGTOSENSCTHFPGNLPNMLRDLR

DAFSRVKTFFOMKDOLDNLLLKESLLE

DFKGYLGCOALSEMIOFYLEEVMPOAE

GFTX1

NODPDIKAHVNSLGENLKTLRLRLRRC

Monomeric

61 VH of IgGIL10M4 HRFLPCENGGGSGGKSKAVEOVKNAF

IL10 grafted

NKLOEKGIYKAMSEFDIFINYIEAYMT

into CDRH1

MKIRNS S YAMS WVROAPGKGLEWVSAI

SGSGGSTYYGDSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYYCARTRTKRFWGQ

GTLVTVSS

EIVLTQSPGTLSLSPGERATLSCRASQSVS SSYLAWYQQKPGQAPRLLIYGASSRATGI

62 VL of IgGIL10M4 GFTX1

PDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPLTFGGGTKVEIK SEQ ID

Description Comments NO:

EVQLLESGGGLVQPGGSLRLSCAASGFTF

SPGOGTOSENSCTHFPGNLPNMLRDLR

DAFSRVKTFFOMKDOLDNLLLKESLLE

DFKGYLGCOALSEMIOFYLEEVMPOAE

NODPDIKAHVNSLGENLKTLRLRLRRC

HRFLPCENGGGSGGKSKAVEOVKNAF

NKLOEKGIYKAMSEFDIFINYIEAYMT

MKIRNS S YAMS WVROAPGKGLEWVS AI

SGSGGSTYYGDSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYYCARTRTKRFWGQ GFTX1

Heavy chain of GTLVTVSSASTKGPSVFPLAPSSKSTSGGT Monomeric

63

IgGIL10M4 AALGCLVKDYFPEPVTVSWNSGALTSGV IL10 grafted

HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT into CDRH1

YICNVNHKPSNTKVDKRVEPKSCDKTHT

CPPCPAPELLGGPSVFLFPPKPKDTLMISR

TPEVTCVVVDVSHEDPEVKFNWYVDGVE

VHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKA

KGQPREPQVYTLPPSREEMTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSKLTVDKSRWQQGNVFSC

SVMHEALHNHYTQKSLSLSPGK

EIVLTQSPGTLSLSPGERATLSCRASQSVS

SSYLAWYQQKPGQAPRLLIYGASSRATGI

PDRF S GS GS GTDFTLTI SRLEPEDF AV Y YC

Light chain of QQYGSSPLTFGGGTKVEIKRTVAAPSVFIF

64 GFTX1 IgGIL10M4 PPSDEQLKSGTASVVCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSL

SSTLTLSKADYEKHKVYACEVTHQGLSSP

VTKSFNRGEC

CDRH1 of

65 GFTFSSY GFTX1 IgGIL10M5(Chothia)

66 CDRH2 of SGSGSPGOGTOSENSCTHFPGNLPNML GFTX1 SEQ ID

Description Comments NO:

IgGIL10M5 RDLRDAFSRVKTFFOMKDOLDNLLLK Monomeric (Chothia) ESLLEDFKGYLGCOALSEMIOFYLEEV IL10 grafted

MPOAENODPDIKAHVNSLGENLKTLRL into

RLRRCHRFLPCENGGGSGGKSKAVEO CDRH2.

VKNAFNKLOEKGIYKAMSEFDIFINYIE IL10 is

AYMTMKIRNGS bolded, underlined

CDRH3 of

67 IgGIL10M5 TRTKRF GFTX1

(Chothia)

CDRL1 of

68 IgGIL10M5 SQSVSSSY GFTX1

(Chothia)

CDRL2 of

69 IgGIL10M5 GAS GFTX1

(Chothia)

CDRL3 of

70 IgGIL10M5 YGSSPL GFTX1

(Chothia)

CDRH1 of

71 SYAMS GFTX1 IgGIL10M5 (Kabat)

GFTX1

AISGSGSPGOGTOSENSCTHFPGNLPNM

Monomeric

LRDLRDAFSRVKTFFOMKDOLDNLLL IL10 grafted

KESLLEDFKGYLGCOALSEMIOFYLEE

CDRH2 of into

72 VMPOAENODPDIKAHVNSLGENLKTLR

IgGIL10M5 (Kabat) CD RFC

LRLRRCHRFLPCENGGGSGGKSKAVE IL10 is

OVKNAFNKLOEKGIYKAMSEFDIFINYI

bolded,

EAYMTMKIRNGST YYGD SVKG

underlined

CDRH3 of

73 TRTKRF GFTX1 IgGIL10M5 (Kabat)

CDRL1 of

74 RASQSVSSSYLA GFTX1 IgGIL10M5 (Kabat) SEQ ID

Description Comments NO:

CDRL2 of

75 GASSRAT GFTX1 IgGIL10M5 (Kabat)

CDRL3 of

76 QQYGSSPLT GFTX1 IgGIL10M5 (Kabat)

EVQLLESGGGLVQPGGSLRLSCAASGFTF

SSYAMSWVRQAPGKGLEWVSAISGSGSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

FSRVKTFFOMKDOLDNLLLKESLLEDF

GFTX1

KGYLGCOALSEMIOFYLEEVMPOAEN

Monomeric

77 VH of IgGIL10M5 ODPDIKAHVNSLGENLKTLRLRLRRCH

IL10 grafted

RFLP CENGGGS GGKSKA VE 0 VKNAFN

into CDRH2

KLOEKGIYKAMSEFDIFINYIEAYMTM

KIRNGSTYYGDSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYYCARTRTKRFWGQ

GTLVTVSS

EIVLTQSPGTLSLSPGERATLSCRASQSVS SSYLAWYQQKPGQAPRLLIYGASSRATGI

78 VL of IgGIL10M5 GFTX1

PDRF S GS GS GTDFTLTI SRLEPEDF AV Y YC QQYGSSPLTFGGGTKVEIK

EVQLLESGGGLVQPGGSLRLSCAASGFTF

SSYAMSWVRQAPGKGLEWVSAISGSGSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

FSRVKTFFOMKDOLDNLLLKESLLEDF

KGYLGCOALSEMIOFYLEEVMPOAEN

ODPDIKAHVNSLGENLKTLRLRLRRCH GFTX1

Heavy chain of RFLP CENGGGS GGKSKA VE 0 VKNAFN Monomeric

79

IgGIL10M5 KLOEKGIYKAMSEFDIFINYIEAYMTM IL10 grafted

KIRNGSTYYGDSVKGRFTISRDNSKNTLY into CDRH2

LQMNSLRAEDTAVYYCARTRTKRFWGQ

GTLVTVSSASTKGPSVFPLAPSSKSTSGGT

AALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKRVEPKSCDKTHT SEQ ID

Description Comments NO:

CPPCPAPELLGGPSVFLFPPKPKDTLMISR

TPEVTCVVVDVSHEDPEVKFNWYVDGVE

VHNAKTKPREEQYNSTYRVVSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKA

KGQPREPQVYTLPPSREEMTKNQVSLTCL

VKGFYPSDIAVEWESNGQPENNYKTTPPV

LDSDGSFFLYSKLTVDKSRWQQGNVFSC

SVMHEALHNHYTQKSLSLSPGK

EIVLTQSPGTLSLSPGERATLSCRASQSVS

SSYLAWYQQKPGQAPRLLIYGASSRATGI

PDRF S GS GS GTDFTLTI SRLEPEDF AV Y YC

Light chain of QQYGSSPLTFGGGTKVEIKRTVAAPSVFIF

80 GFTX1 IgGIL10M5 PPSDEQLKSGTASVVCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSL

SSTLTLSKADYEKHKVYACEVTHQGLSSP

VTKSFNRGEC

CDRH1 of

81 IgGIL10M6 GFTFSSY GFTX1

(Chothia)

CDRH2 of

82 IgGIL10M6 SGSGGS GFTX1

(Chothia)

GFTX1

TRTSPGOGTOSENSCTHFPGNLPNMLR

Monomeric

DLRDAFSRVKTFFOMKDOLDNLLLKES IL10 grafted

CDRH3 of LLEDFKGYLGCOALSEMIOFYLEEVMP

into

83 IgGIL10M6 OAENODPDIKAHVNSLGENLKTLRLRL

CDRH3.

(Chothia) RRCHRFLP CENGGGS GGKSKA VE 0 VK

IL10 is

NAFNKLOEKGIYKAMSEFDIFINYIEAY

bolded,

MTMKIRNKRF

underlined

CDRL1 of

84 IgGIL10M6 SQSVSSSY GFTX1

(Chothia) SEQ ID

Description Comments NO:

CDRL2 of

85 IgGIL10M6 GAS GFTX1

(Chothia)

CDRL3 of

86 IgGIL10M6 YGSSPL GFTX1

(Chothia)

CDRH1 of

87 SYAMS GFTX1 IgGIL10M6 (Kabat)

CDRH2 of

88 AISGSGGSTYYGDSVKG GFTX1 IgGIL10M6 (Kabat)

GFTX1

TRTSPGOGTOSENSCTHFPGNLPNMLR

Monomeric

DLRDAFSRVKTFFOMKDOLDNLLLKES IL10 grafted

LLEDFKGYLGCOALSEMIOFYLEEVMP

CDRH3 of into

89 OAENODPDIKAHVNSLGENLKTLRLRL

IgGIL10M6 (Kabat) CDRH3.

RRCHRFLP CENGGGS GGKSKA VE 0 VK

IL10 is

NAFNKLOEKGIYKAMSEFDIFINYIEAY

bolded,

MTMKIRNKRF

underlined

CDRL1 of

90 RASQSVSSSYLA GFTX1 IgGIL10M6 (Kabat)

CDRL2 of

91 GASSRAT GFTX1 IgGIL10M6 (Kabat)

CDRL3 of

92 QQYGSSPLT GFTX1 IgGIL10M6 (Kabat)

EVQLLESGGGLVQPGGSLRLSCAASGFTF S S YAM S WVRQ APGKGLEWVS AI S GS GGS TYYGDSVKGRFTISRDNSKNTLYLQMNS

GFTX1 LRAEDTAVYYCARTRTSPGOGTOSENSC

Monomeric

93 VH of !gGIL10M6 THFPGNLPNMLRDLRDAFSRVKTFFOM

IL10 grafted KDOLDNLLLKESLLEDFKGYLGCOALS

into CDRH3 EMIOFYLEEVMPOAENODPDIKAHVNS LGENLKTLRLRLRRCHRFLPCENGGGS GGKSKA VEOVKNAFNKLOEKGIYKAM SEQ ID

Description Comments NO:

SEFDIFINYIEAYMTMKIRNKRFWGOGT

LVTVSS

EIVLTQSPGTLSLSPGERATLSCRASQSVS SSYLAWYQQKPGQAPRLLIYGASSRATGI

94 VL of IgGIL10M6 GFTX1

PDRF S GS GS GTDFTLTI SRLEPEDF AV Y YC QQYGSSPLTFGGGTKVEIK

EVQLLESGGGLVQPGGSLRLSCAASGFTF S S YAM S WVRQ APGKGLEWVS AI S GS GGS TYYGDSVKGRFTISRDNSKNTLYLQMNS LRAEDTAVYYCARTRTSPGOGTOSENSC

THFPGNLPNMLRDLRDAFSRVKTFFOM

KDOLDNLLLKESLLEDFKGYLGCOALS

EMIOFYLEEVMPOAENODPDIKAHVNS

LGENLKTLRLRLRRCHRFLPCENGGGS

GGKSKAVEOVKNAFNKLOEKGIYKAM

SEFDIFINYIEAYMTMKIRNKRFWGOGT GFTX1

Heavy chain of LVTVSSASTKGPSVFPLAPSSKSTSGGTAA Monomeric

95

IgGIL10M6 LGCLVKDYFPEPVTVSWNSGALTSGVHT IL10 grafted

FPAVLQSSGLYSLSSVVTVPSSSLGTQTYI into CDRH3

CNVNHKPSNTKVDKRVEPKSCDKTHTCP

PCPAPELLGGPSVFLFPPKPKDTLMISRTP

EVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNH YTQKSL SL SPGK

EIVLTQSPGTLSLSPGERATLSCRASQSVS SSYLAWYQQKPGQAPRLLIYGASSRATGI

Light chain of

96 PDRF S GS GS GTDFTLTI SRLEPEDF AVYYC GFTX1 IgGIL10M6

QQYGSSPLTFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQ SEQ ID

Description Comments NO:

WKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC

CDRH1 of

97 IgGIL10M7 GFSLSTSGM GFTX3

(Chothia)

CDRH2 of

98 IgGIL10M7 WWDDK GFTX3

(Chothia)

CDRH3 of

99 IgGIL10M7 SMITNWYFDV GFTX3

(Chothia)

OLSSPGOGTOSENSCTHFPGNLPNMLR Monomeric

DLRDAFSRVKTFFOMKDOLDNLLLKES IL10 grafted

CDRL1 of LLEDFKGYLGCOALSEMIOFYLEEVMP into

100 IgGIL10M7 OAENODPDIKAHVNSLGENLKTLRLRL CDRL1.

(Chothia) RRCHRFLP CENGGGS GGKSKA VE O VK IL10 is

NAFNKLOEKGIYKAMSEFDIFINYIEAY bolded,

MTMKIRNVGY underlined

CDRL2 of

101 IgGIL10M7 DTS GFTX3

(Chothia)

CDRL3 of

102 IgGIL10M7 GSGYPF GFTX3

(Chothia)

CDRH1 of

103 TSGMSVG GFTX3 IgGIL10M7 (Kabat)

CDRH2 of

104 DIWWDDKKDYNPSLKS GFTX3 IgGIL10M7 (Kabat)

CDRH3 of

105 SMITNWYFDV GFTX3 IgGIL10M7 (Kabat)

CDRL1 of KAOLSSPGOGTOSENSCTHFPGNLPNM Monomeric

106

IgGIL10M7 (Kabat) LRDLRDAFSRVKTFFOMKDOLDNLLL IL10 grafted SEQ ID

Description Comments NO:

KESLLEDFKGYLGCOALSEMIOFYLEE into

VMPOAENODPDIKAHVNSLGENLKTLR CDRLl.

LRLRRCHRFLPCENGGGSGGKSKAVE IL10 is

OVKNAFNKLOEKGIYKAMSEFDIFINYI bolded,

EAYMTMKIRNVGYMH underlined

CDRL2 of

107 DTSKLAS GFTX3 IgGIL10M7 (Kabat)

CDRL3 of

108 FQGSGYPFT GFTX3 IgGIL10M7 (Kabat)

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDD

109 VH of IgGIL10M7 KKDYNPSLKSRLTISKDTSANQVVLKVTN GFTX3

MDPADTATYYCARSMITNWYFDVWGAG

TTVTVSS

DIQMTQSPSTLSASVGDRVTITCKAQLSSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

FSRVKTFFOMKDOLDNLLLKESLLEDF

KGYLGCOALSEMIOFYLEEVMPOAEN Monomeric

ODPDIKAHVNSLGENLKTLRLRLRRCH IL10 grafted

110 VL of IgGIL10M7

RFLP CENGGGS GGKSKA VE O VKNAFN into

KLOEKGIYKAMSEFDIFINYIEAYMTM CDRLl.

KIRNVGYMHWYQQKPGKAPKLLIYDTS

KLASGVPSRFSGSGSGTAFTLTISSLQPDD

FATYYCFQGSGYPFTFGGGTKLEIK

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDD

KKDYNPSLKSRLTISKDTSANQVVLKVTN

MDPADTATYYCARSMITNWYFDVWGAG

Heavy chain of

111 TTVTVSSASTKGPSVFPLAPSSKSTSGGTA GFTX3 IgGIL10M7

ALGCLVKDYFPEPVTVSWNSGALTSGVH

TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

ICNVNHKPSNTKVDKRVEPKSCDKTHTCP

PCPAPELLGGPSVFLFPPKPKDTLMISRTP SEQ ID

Description Comments NO:

EVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNH YTQKSL SL SPGK

DIQMTQSPSTLSASVGDRVTITCKAQLSSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

FSRVKTFFOMKDOLDNLLLKESLLEDF

KGYLGCOALSEMIOFYLEEVMPOAEN

ODPDIKAHVNSLGENLKTLRLRLRRCH

RFLP CENGGGS GGKSKA VE 0 VKNAFN Monomeric

Light chain of KLOEKGIYKAMSEFDIFINYIEAYMTM IL10 grafted

112

IgGIL10M7 KIRNVGYMHWYQQKPGKAPKLLIYDTS into

KLASGVPSRFSGSGSGTAFTLTISSLQPDD CDRLl.

FATYYCFQGSGYPFTFGGGTKLEIKRTVA

APSVFIFPPSDEQLKSGTASVVCLLNNFYP

REAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC

CDRH1 of

113 IgGIL10M8 GFSLSTSGM GFTX3

(Chothia)

CDRH2 of

114 IgGIL10M8 WWDDK GFTX3

(Chothia)

CDRH3 of

115 IgGIL10M8 SMITNWYFDV GFTX3

(Chothia)

CDRL1 of

116 IgGIL10M8 QLSVGY GFTX3

(Chothia) SEQ ID

Description Comments NO:

GFTX3

DTSPGOGTOSENSCTHFPGNLPNMLRD

Monomeric

LRDAFSRVKTFFOMKDOLDNLLLKESL IL10 grafted

CDRL2 of LEDFKGYLGCOALSEMIOFYLEEVMPO

into

117 IgGIL10M8 AENODPDIKAHVNSLGENLKTLRLRLR

CDRL2.

(Chothia) RCHRFLPCENGGGSGGKSKAVEOVKN

IL10 is

AFNKLOEKGIYKAMSEFDIFINYIEAYM

bolded,

TMKIRNS

underlined

CDRL3 of

118 IgGIL10M8 GSGYPF GFTX3

(Chothia)

CDRH1 of

119 TSGMSVG GFTX3 IgGIL10M8 (Kabat)

CDRH2 of

120 DIWWDDKKDYNPSLKS GFTX3 IgGIL10M8 (Kabat)

CDRH3 of

121 SMITNWYFDV GFTX3 IgGIL10M8 (Kabat)

CDRL1 of

122 KAQLSVGYMH GFTX3 IgGIL10M8 (Kabat)

GFTX3

DTSPGOGTOSENSCTHFPGNLPNMLRD

Monomeric

LRDAFSRVKTFFOMKDOLDNLLLKESL IL10 grafted

LEDFKGYLGCOALSEMIOFYLEEVMPO

CDRL2 of into

123 AENODPDIKAHVNSLGENLKTLRLRLR

IgGIL10M8 (Kabat) CDRL2.

RCHRFLPCENGGGSGGKSKAVEOVKN IL10 is

AFNKLOEKGIYKAMSEFDIFINYIEAYM

bolded,

TMKIRNSKLAS

underlined

CDRL3 of

124 FQGSGYPFT GFTX3 IgGIL10M8 (Kabat)

QVTLRESGPALVKPTQTLTLTCTFSGFSLS TSGMSVGWIRQPPGKALEWLADIWWDD

125 VH of IgGIL10M8 GFTX3

KKDYNPSLKSRLTISKDTSANQVVLKVTN MDPADTATYYCARSMITNWYFDVWGAG SEQ ID

Description Comments NO:

TTVTVSS

DIQMTQSPSTLSASVGDRVTITCKAQLSV

GYMHWYOOKPGKAPKLLIYDTSPGOGT

OSENSCTHFPGNLPNMLRDLRDAFSRV

GFTX3

KTFFOMKDOLDNLLLKESLLEDFKGYL

Monomeric

GCOALSEMIOFYLEEVMPOAENODPDI

126 VL of IgGIL10M8 IL10 grafted

KAHVNSLGENLKTLRLRLRRCHRFLPC

into

ENGGGSGGKSKAVEOVKNAFNKLOEK

CDRL2.

GIYKAMSEFDIFINYIEAYMTMKIRNSK

LASGVPSRFSGSGSGTAFTLTISSLQPDDF

ATYYCFQGSGYPFTFGGGTKLEIK

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDD

KKDYNPSLKSRLTISKDTSANQVVLKVTN

MDPADTATYYCARSMITNWYFDVWGAG

TTVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVH

TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

Heavy chain of ICNVNHKPSNTKVDKRVEPKSCDKTHTCP

127 GFTX3 IgGIL10M8 PCPAPELLGGPSVFLFPPKPKDTLMISRTP

EVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNH YTQKSL SL SPGK

DIQMTQSPSTLSASVGDRVTITCKAQLSV

GFTX3

GYMHWYOOKPGKAPKLLIYDTSPGOGT

Monomeric

Light chain of OSENSCTHFPGNLPNMLRDLRDAFSRV

128 IL10 grafted IgGIL10M8 KTFFOMKDOLDNLLLKESLLEDFKGYL

into

GCOALSEMIOFYLEEVMPOAENODPDI

CDRL2.

KAHVNSLGENLKTLRLRLRRCHRFLPC SEQ ID

Description Comments NO:

ENGGGSGGKSKAVEOVKNAFNKLOEK GIYKAMSEFDIFINYIEAYMTMKIRNSK

LASGVPSRFSGSGSGTAFTLTISSLQPDDF

ATYYCFQGSGYPFTFGGGTKLEIKRTVAA

PSVFIFPPSDEQLKSGTASVVCLLNNFYPR

EAKVQWKVDNALQSGNSQESVTEQDSK

DSTYSLSSTLTLSKADYEKHKVYACE

VTHQGLSSPVTKSFNRGEC

CDRH1 of

129 IgGIL10M9 GFSLSTSGM GFTX3

(Chothia)

CDRH2 of

130 IgGIL10M9 WWDDK GFTX3

(Chothia)

CDRH3 of

131 IgGIL10M9 SMITNWYFDV GFTX3

(Chothia)

CDRL1 of

132 IgGIL10M9 QLSVGY GFTX3

(Chothia)

CDRL2 of

133 IgGIL10M9 DTS GFTX3

(Chothia)

GFTX3

GSGSPGOGTOSENSCTHFPGNLPNMLR

Monomeric

DLRDAFSRVKTFFOMKDOLDNLLLKES IL10 grafted

CDRL3 of LLEDFKGYLGCOALSEMIOFYLEEVMP

into

134 IgGIL10M9 OAENODPDIKAHVNSLGENLKTLRLRL

CDRL3.

(Chothia) RRCHRFLP CENGGGS GGKSKA VE 0 VK

IL10 is

NAFNKLOEKGIYKAMSEFDIFINYIEAY

bolded,

MTMKIRNYPF

underlined

CDRH1 of

135 TSGMSVG GFTX3 IgGIL10M9 (Kabat) SEQ ID

Description Comments NO:

CDRH2 of

136 DIWWDDKKDYNPSLKS GFTX3 IgGIL10M9 (Kabat)

CDRH3 of

137 SMITNWYFDV GFTX3 IgGIL10M9 (Kabat)

CDRL1 of

138 KAQLSVGYMH GFTX3 IgGIL10M9 (Kabat)

CDRL2 of

139 DTSKLAS GFTX3 IgGIL10M9 (Kabat)

GFTX3

FOGSGSPGOGTOSENSCTHFPGNLPNM

Monomeric

LRDLRDAFSRVKTFFOMKDOLDNLLL IL10 grafted

KESLLEDFKGYLGCOALSEMIOFYLEE

CDRL3 of into

140 VMPOAENODPDIKAHVNSLGENLKTLR

IgGIL10M9 (Kabat) CDRL3.

LRLRRCHRFLPCENGGGSGGKSKAVE IL10 is

OVKNAFNKLOEKGIYKAMSEFDIFINYI

bolded,

EAYMTMKIRNYPFT

underlined

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDD

141 VH of IgGIL10M9 KKDYNPSLKSRLTISKDTSANQVVLKVTN GFTX3

MDPADTATYYCARSMITNWYFDVWGAG

TTVTVSS

DIQMTQSPSTLSASVGDRVTITCKAQLSV

GYMHWYQQKPGKAPKLLIYDTSKLASG

VPSRFSGSGSGTAFTLTISSLQPDDFATYY

GFTX3

CFOGSGSPGOGTOSENSCTHFPGNLPN

Monomeric

MLRDLRDAFSRVKTFFOMKDOLDNLL

142 VL of IgGIL10M9 IL10 grafted

LKESLLEDFKGYLGCOALSEMIOFYLE

into

EVMPOAENODPDIKAHVNSLGENLKTL

CDRL3.

RLRLRRCHRFLPCENGGGSGGKSKAV

EOVKNAFNKLOEKGIYKAMSEFDIFIN

YIEAYMTMKIRNYPFTFGGGTKLEIK

Heavy chain of QVTLRESGPALVKPTQTLTLTCTFSGFSLS

143 GFTX3 IgGIL10M9 TSGMSVGWIRQPPGKALEWLADIWWDD SEQ ID

Description Comments NO:

KKDYNPSLKSRLTISKDTSANQVVLKVTN

MDPADTATYYCARSMITNWYFDVWGAG

TTVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVH

TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

ICNVNHKPSNTKVDKRVEPKSCDKTHTCP

PCPAPELLGGPSVFLFPPKPKDTLMISRTP

EVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPEN YKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNH YTQKSL SL SPGK

DIQMTQSPSTLSASVGDRVTITCKAQLSV

GYMHWYQQKPGKAPKLLIYDTSKLASG

VPSRFSGSGSGTAFTLTISSLQPDDFATYY

CFOGSGSPGOGTOSENSCTHFPGNLPN

MLRDLRDAFSRVKTFFOMKDOLDNLL

GFTX3

LKESLLEDFKGYLGCOALSEMIOFYLE

Monomeric

Light chain of EVMPOAENODPDIKAHVNSLGENLKTL

144 IL10 grafted IgGIL10M9 RLRLRRCHRFLPCENGGGSGGKSKAV

into

EOVKNAFNKLOEKGIYKAMSEFDIFIN

CDRL3.

YIEAYMTMKIRNYPFTFGGGTKLEIKRT

VAAPSVFIFPPSDEQLKSGTASVVCLLNNF

YPREAKVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHKVYACE

VTHQGLSSPVTKSFNRGEC

GFSLSPGOGTOSENSCTHFPGNLPNML GFTX3

CDRH1 of RDLRDAFSRVKTFFOMKDOLDNLLLK Monomeric

145 IgGILlOMlO ESLLEDFKGYLGCOALSEMIOFYLEEV IL10 grafted

(Chothia) MPOAENODPDIKAHVNSLGENLKTLRL into

RLRRCHRFLPCENGGGSGGKSKAVEO CDRH1. SEQ ID

Description Comments NO:

VKNAFNKLOEKGIYKAMSEFDIFINYIE IL10 is AYMTMKIRNSTSGM bolded, underlined

CDRH2 of

146 IgGILlOMlO WWDDK GFTX3

(Chothia)

CDRH3 of

147 IgGILlOMlO SMITNWYFDV GFTX3

(Chothia)

CDRL1 of

148 IgGILlOMlO QLSVGY GFTX3

(Chothia)

CDRL2 of

149 IgGILlOMlO DTS GFTX3

(Chothia)

CDRL3 of

150 IgGILlOMlO GSGYPF GFTX3

(Chothia)

GFTX3

SPGOGTOSENSCTHFPGNLPNMLRDLR

Monomeric

DAFSRVKTFFOMKDOLDNLLLKESLLE IL10 grafted

DFKGYLGCOALSEMIOFYLEEVMPOAE

CDRH1 of into

151 NODPDIKAHVNSLGENLKTLRLRLRRC

IgGILlOMlO (Kabat) CDRH1.

HRFLPCENGGGSGGKSKAVEOVKNAF IL10 is

NKLOEKGIYKAMSEFDIFINYIEAYMT

bolded,

MKIRNSTSGMSVG

underlined

CDRH2 of

152 DIWWDDKKDYNPSLKS GFTX3 IgGILlOMlO (Kabat)

CDRH3 of

153 SMITNWYFDV GFTX3 IgGILlOMlO (Kabat)

CDRL1 of

154 KAQLSVGYMH GFTX3

IgGILlOMlO (Kabat)

155 CDRL2 of DTSKLAS GFTX3 SEQ ID

Description Comments NO:

IgGILlOMlO (Kabat)

CDRL3 of

156 FQGSGYPFT GFTX3

IgGILlOMlO (Kabat)

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

PGOGTOSENSCTHFPGNLPNMLRDLRD

AFSRVKTFFOMKDOLDNLLLKESLLED

FKGYLGCOALSEMIOFYLEEVMPOAEN

GFTX3

ODPDIKAHVNSLGENLKTLRLRLRRCH

Monomeric

157 VH of lgGILlOMlO RFLP CENGGGS GGKSKA VE 0 VKNAFN

IL10 grafted

KLOEKGIYKAMSEFDIFINYIEAYMTM

into CDRH1

KIRNSTSGMSVGWIRQPPGKALEWLADI

WWDDKKDYNPSLKSRLTISKDTSANQVV

LKVTNMDPADTATYYCARSMITNWYFD

VWGAGTTVTVSS

DIQMTQSPSTLSASVGDRVTITCKAQLSV GYMHWYQQKPGKAPKLLIYDTSKLASG

158 VL of lgGILlOMlO GFTX3

VPSRFSGSGSGTAFTLTISSLQPDDFATYY CFQGSGYPFTFGGGTKLEIK

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

PGOGTOSENSCTHFPGNLPNMLRDLRD

AFSRVKTFFOMKDOLDNLLLKESLLED

FKGYLGCOALSEMIOFYLEEVMPOAEN

ODPDIKAHVNSLGENLKTLRLRLRRCH

RFLP CENGGGS GGKSKA VE 0 VKNAFN

GFTX3

KLOEKGIYKAMSEFDIFINYIEAYMTM

Heavy chain of Monomeric

159 KIRNSTSGMSVGWIRQPPGKALEWLADI

IgGILlOMlO IL10 grafted

WWDDKKDYNPSLKSRLTISKDTSANQVV

into CDRH1

LKVTNMDPADTATYYCARSMITNWYFD

VWGAGTTVTVSSASTKGPSVFPLAPSSKS

TSGGTAALGCLVKDYFPEPVTVSWNSGA

LTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKRVEPKSC

DKTHTCPPCPAPELLGGPSVFLFPPKPKDT SEQ ID

Description Comments NO:

LMISRTPEVTCVVVDVSHEDPEVKFNWY

VDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEK

TISKAKGQPREPQVYTLPPSREEMTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYK

TTPPVLDSDGSFFLYSKLTVDKSRWQQG

NVFSCSVMHEALHNHYTQKSLSLSPGK

DIQMTQSPSTLSASVGDRVTITCKAQLSV

GYMHWYQQKPGKAPKLLIYDTSKLASG

VPSRFSGSGSGTAFTLTISSLQPDDFATYY

Light chain of CFQGSGYPFTFGGGTKLEIKRTVAAPSVFI

160 GFTX3 IgGILlOMlO FPPSDEQLKSGTASVVCLLN FYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYS

LSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC

CDRH1 of

161 IgGILlOMl l GFSLSTSGM GFTX3

(Chothia)

GFTX3

WWDSPGOGTOSENSCTHFPGNLPNML

Monomeric

RDLRDAFSRVKTFFOMKDOLDNLLLK IL10 grafted

CDRH2 of ESLLEDFKGYLGCOALSEMIOFYLEEV

into

162 IgGILlOMl l MPOAENODPDIKAHVNSLGENLKTLRL

CDRH2.

(Chothia) RLRRCHRFLPCENGGGSGGKSKAVEO

IL10 is

VKNAFNKLOEKGIYKAMSEFDIFINYIE

bolded,

AYMTMKIRNDK

underlined

CDRH3 of

163 IgGILlOMl l SMITNWYFDV GFTX3

(Chothia)

CDRL1 of

164 IgGILlOMl l QLSVGY GFTX3

(Chothia)

165 CDRL2 of DTS GFTX3 SEQ ID

Description Comments NO:

IgGILlOMl l

(Chothia)

CDRL3 of

166 IgGILlOMl l GSGYPF GFTX3

(Chothia)

CDRH1 of

167 TSGMSVG GFTX3 IgGILlOMl l (Kabat)

GFTX3

DIWWDSPGOGTOSENSCTHFPGNLPNM

Monomeric

LRDLRDAFSRVKTFFOMKDOLDNLLL IL10 grafted

KESLLEDFKGYLGCOALSEMIOFYLEE

CDRH2 of into

168 VMPOAENODPDIKAHVNSLGENLKTLR

IgGILlOMl l (Kabat) CDRH2.

LRLRRCHRFLPCENGGGSGGKSKAVE IL10 is

OVKNAFNKLOEKGIYKAMSEFDIFINYI

bolded,

EAYMTMKIRNDKKDYNPSLKS

underlined

CDRH3 of

169 SMITNWYFDV GFTX3 IgGILlOMl l (Kabat)

CDRL1 of

170 KAQLSVGYMH GFTX3

IgGILlOMl l (Kabat)

CDRL2 of

171 DTSKLAS GFTX3

IgGILlOMl l (Kabat)

CDRL3 of

172 FQGSGYPFT GFTX3

IgGILlOMl l (Kabat)

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

FSRVKTFFOMKDOLDNLLLKESLLEDF GFTX3

KGYLGCOALSEMIOFYLEEVMPOAEN Monomeric

173 VH of IgGILlOMl l

ODPDIKAHVNSLGENLKTLRLRLRRCH IL10 grafted

RFLP CENGGGS GGKSKA VE 0 VKNAFN into CDRH2

KLOEKGIYKAMSEFDIFINYIEAYMTM

KIRNDKKDYNPSLKSRLTISKDTSANQVV

LKVTNMDPADTATYYCARSMITNWYFD SEQ ID

Description Comments NO:

VWGAGTTVTVSS

DIQMTQSPSTLSASVGDRVTITCKAQLSV GYMHWYQQKPGKAPKLLIYDTSKLASG

174 VL of lgGILlOMl l GFTX3

VPSRFSGSGSGTAFTLTISSLQPDDFATYY CFQGSGYPFTFGGGTKLEIK

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

FSRVKTFFOMKDOLDNLLLKESLLEDF

KGYLGCOALSEMIOFYLEEVMPOAEN

ODPDIKAHVNSLGENLKTLRLRLRRCH

RFLP CENGGGS GGKSKA VE 0 VKNAFN

KLOEKGIYKAMSEFDIFINYIEAYMTM

KIRNDKKDYNPSLKSRLTISKDTSANQVV

LKVTNMDPADTATYYCARSMITNWYFD GFTX3

Heavy chain of VWGAGTTVTVSSASTKGPSVFPLAPSSKS Monomeric

175

IgGILlOMl l TSGGTAALGCLVKDYFPEPVTVSWNSGA IL10 grafted

LTSGVHTFPAVLQSSGLYSLSSVVTVPSSS into CDRH2

LGTQTYICNVNHKPSNTKVDKRVEPKSC

DKTHTCPPCPAPELLGGPSVFLFPPKPKDT

LMISRTPEVTCVVVDVSHEDPEVKFNWY

VDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEK

TISKAKGQPREPQVYTLPPSREEMTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYK

TTPPVLDSDGSFFLYSKLTVDKSRWQQG

NVFSCSVMHEALHNHYTQKSLSLSPGK

DIQMTQSPSTLSASVGDRVTITCKAQLSV

GYMHWYQQKPGKAPKLLIYDTSKLASG

Light chain of VPSRFSGSGSGTAFTLTISSLQPDDFATYY

176 GFTX3 IgGILlOMl l CFQGSGYPFTFGGGTKLEIKRTVAAPSVFI

FPPSDEQLKSGTASVVCLLNNFYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYS SEQ ID

Description Comments NO:

LSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

CDRH1 of

177 IgGIL10M12 GFSLSTSGM GFTX3

(Chothia)

CDRH2 of

178 IgGIL10M12 WWDDK GFTX3

(Chothia)

GFTX3

SMITSPGOGTOSENSCTHFPGNLPNMLR

Monomeric

DLRDAFSRVKTFFOMKDOLDNLLLKES IL10 grafted

CDRH3 of LLEDFKGYLGCOALSEMIOFYLEEVMP

into

179 IgGIL10M12 OAENODPDIKAHVNSLGENLKTLRLRL

CDRH3.

(Chothia) RRCHRFLP CENGGGS GGKSKA VE 0 VK

IL10 is

NAFNKLOEKGIYKAMSEFDIFINYIEAY

bolded,

MTMKIRNNWYFDV

underlined

CDRL1 of

180 IgGIL10M12 QLSVGY GFTX3

(Chothia)

CDRL2 of

181 IgGIL10M12 DTS GFTX3

(Chothia)

CDRL3 of

182 IgGIL10M12 GSGYPF GFTX3

(Chothia)

CDRH1 of

183 TSGMSVG GFTX3 IgGIL10M12 (Kabat)

CDRH2 of

184 DIWWDDKKDYNPSLKS GFTX3 IgGIL10M12 (Kabat)

SMITSPGOGTOSENSCTHFPGNLPNMLR GFTX3

CDRH3 of DLRDAFSRVKTFFOMKDOLDNLLLKES Monomeric

185

IgGIL10M12 (Kabat) LLEDFKGYLGCOALSEMIOFYLEEVMP IL10 grafted

OAENODPDIKAHVNSLGENLKTLRLRL into SEQ ID

Description Comments NO:

RRCHRFLP CENGGGS GGKSKA VE 0 VK CDRH3. NAFNKLOEKGIYKAMSEFDIFINYIEAY IL10 is MTMKIRNNWYFDV bolded, underlined

CDRL1 of

186 KAQLSVGYMH GFTX3

IgGIL10M12 (Kabat)

CDRL2 of

187 DTSKLAS GFTX3

IgGIL10M12 (Kabat)

CDRL3 of

188 FQGSGYPFT GFTX3

IgGIL10M12 (Kabat)

QVTLRESGPALVKPTQTLTLTCTFSGFSLS TSGMSVGWIRQPPGKALEWLADIWWDD KKDYNPSLKSRLTISKDTSANQVVLKVTN MDPADTATYYCARSMITSPGOGTOSENS

GFTX3

CTHFPGNLPNMLRDLRDAFSRVKTFFO

Monomeric

189 VH of IgGIL10M12 MKDOLDNLLLKESLLEDFKGYLGCOA

IL10 grafted

LSEMIOFYLEEVMPOAENODPDIKAHV

into CDRH3

NSLGENLKTLRLRLRRCHRFLPCENGG

GSGGKSKAVEOVKNAFNKLOEKGIYK

AMSEFDIFINYIEAYMTMKIRNNWYFD

VWGAGTTVTVSS

DIQMTQSPSTLSASVGDRVTITCKAQLSV GYMHWYQQKPGKAPKLLIYDTSKLASG

190 VL of IgGIL10M12 GFTX3

VPSRFSGSGSGTAFTLTISSLQPDDFATYY CFQGSGYPFTFGGGTKLEIK

QVTLRESGPALVKPTQTLTLTCTFSGFSLS TSGMSVGWIRQPPGKALEWLADIWWDD KKDYNPSLKSRLTISKDTSANQVVLKVTN GFTX3

Heavy chain of MDPADTATYYCARSMITSPGOGTOSENS Monomeric

191

IgGIL10M12 CTHFPGNLPNMLRDLRDAFSRVKTFFO IL10 grafted

MKDOLDNLLLKESLLEDFKGYLGCOA into CDRH3 LSEMIOFYLEEVMPOAENODPDIKAHV NSLGENLKTLRLRLRRCHRFLPCENGG SEQ ID

Description Comments NO:

GSGGKSKAVEOVKNAFNKLOEKGIYK AMSEFDIFINYIEAYMTMKIRNNWYFD

VWGAGTTVTVSSASTKGPSVFPLAPSSKS

TSGGTAALGCLVKDYFPEPVTVSWNSGA

LTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKRVEPKSC

DKTHTCPPCPAPELLGGPSVFLFPPKPKDT

LMISRTPEVTCVVVDVSHEDPEVKFNWY

VDGVEVHNAKTKPREEQYNSTYRVVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEK

TISKAKGQPREPQVYTLPPSREEMTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYK

TTPPVLDSDGSFFLYSKLTVDKSRWQQG

NVFSCSVMHEALHNHYTQKSLSLSPGK

DIQMTQSPSTLSASVGDRVTITCKAQLSV

GYMHWYQQKPGKAPKLLIYDTSKLASG

VPSRFSGSGSGTAFTLTISSLQPDDFATYY

Light chain of CFQGSGYPFTFGGGTKLEIKRTVAAPSVFI

192 GFTX3 IgGIL10M12 FPPSDEQLKSGTASVVCLLNNFYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYS

LSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC

CDRH1 of

193 IgGILlOMB GFSLSTSGM GFTX3

(Chothia)

CDRH2 of

194 IgGILlOMB WWDDK GFTX3

(Chothia)

CDRH3 of

195 IgGILlOMB SMITNWYFDV GFTX3

(Chothia)

CDRL1 of OLSSPGOGTOSENSCTHFPGNLPNMLR Monomeric

196

IgGILlOMB DLRDAFSRVKTFFOMKDOLDNLLLKES IL10 grafted SEQ ID

Description Comments NO:

(Chothia) LLEDFKGYLGCOALSEMIOFYLEEVMP into

OAENODPDIKAHVNSLGENLKTLRLRL CDRL1. RRCHRFLP CENGGGS GGKSKA V E O VK IL10 is NAFNKLOEKGIYKAMSEFDIFINYIEAY bolded, MTMKIRNVGY underlined

CDRL2 of

197 IgGILlOMO DTS GFTX3

(Chothia)

CDRL3 of

198 IgGILlOMO GSGYPF GFTX3

(Chothia)

CDRH1 of

199 TSGMSVG GFTX3 IgGILlOMO (Kabat)

CDRH2 of

200 DIWWDDKKDYNPSLKS GFTX3

IgGILlOMO (Kabat)

CDRH3 of

201 SMITNWYFDV GFTX3

IgGILlOMO (Kabat)

KAOLSSPGOGTOSENSCTHFPGNLPNM Monomeric

LRDLRDAFSRVKTFFOMKDOLDNLLL IL10 grafted

KESLLEDFKGYLGCOALSEMIOFYLEE into CDRL1

CDRL1 of

202 VMPOAENODPDIKAHVNSLGENLKTLR IL10 is

IgGILlOMO (Kabat)

LRLRRCHRFLPCENGGGSGGKSKAVE bolded,

OVKNAFNKLOEKGIYKAMSEFDIFINYI underlined

EAYMTMKIRNVGYMH GFTX3

CDRL2 of

203 DTSKLAS GFTX3

IgGILlOMO (Kabat)

CDRL3 of

204 FQGSGYPFT GFTX3

IgGILlOMO (Kabat)

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDD

205 VH of IgGILlOM KKDYNPSLKSRLTISKDTSANQVVLKVTN GFTX3

MDPADTATYYCARSMITNWYFDVWGAG

TTVTVSS SEQ ID

Description Comments NO:

DIQMTQSPSTLSASVGDRVTITCKAQLSSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

FSRVKTFFOMKDOLDNLLLKESLLEDF

KGYLGCOALSEMIOFYLEEVMPOAEN Monomeric

ODPDIKAHVNSLGENLKTLRLRLRRCH IL10 grafted

206 VL of IgGILlOMO

RFLP CENGGGS GGKSKA V E O VKNAFN into CDRL1

KLOEKGIYKAMSEFDIFINYIEAYMTM GFTX3

KIRNVGYMHWYQQKPGKAPKLLIYDTS

KLASGVPSRFSGSGSGTAFTLTISSLQPDD

FATYYCFQGSGYPFTFGGGTKLEIK

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDD

KKDYNPSLKSRLTISKDTSANQVVLKVTN

MDPADTATYYCARSMITNWYFDVWGAG

TTVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVH

TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

Heavy chain of ICNVNHKPSNTKVDKRVEPKSCDKTHTCP

207 GFTX3

IgGILlOMO PCPAPELLGGPSVFLFPPKPKDTLMISRTP

EVTCVVVAVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALAAPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNH YTQKSL SL SPGK

DIQMTQSPSTLSASVGDRVTITCKAQLSSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

Monomeric

FSRVKTFFOMKDOLDNLLLKESLLEDF

Light chain of IL10 grafted

208 KGYLGCOALSEMIOFYLEEVMPOAEN

IgGILlOMO into CDRL1

ODPDIKAHVNSLGENLKTLRLRLRRCH GFTX3

RFLP CENGGGS GGKSKA VE O VKNAFN

KLOEKGIYKAMSEFDIFINYIEAYMTM SEQ ID

Description Comments NO:

KIRNVGYMHWYQQKPGKAPKLLIYDTS

KLASGVPSRFSGSGSGTAFTLTISSLQPDD

FATYYCFQGSGYPFTFGGGTKLEIKRTVA

APSVFIFPPSDEQLKSGTASVVCLLNNFYP

REAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC

SPGQGTQSENSCTHFPGNLPNMLRDLRD Mature form

AFSRVKTFFQMKDQLDNLLLKESLLEDFK of lLlO,

Monomeric IL10 GYLGCQAL SEMIQF YLEEVMPQAENQDP with an

209

(IL10M) DIKAHVNSLGENLKTLRLRLRRCHRFLPC internal

ENGGGSGGKSKAVEQVKNAFNKLQEKGI G3SG2

YKAMSEFDIFINYIEAYMTMKIRN spacer

CDRH1 of

210 IgGIL10M14 GFSLSTSGM GFTX3

(Chothia)

CDRH2 of

211 IgGILlOMM WWDDK GFTX3

(Chothia)

CDRH3 of

212 IgGIL10M14 SMITNWYFDV GFTX3

(Chothia)

Monomeric

OLSSPGOGTOSENSCTHFPGNLPNMLR IL10 grafted

DLRDAFSRVKTFFOMKDOLDNLLLKES

into

CDRL1 of LLEDFKGYLGCOALSEMIOFYLEEVMP

CDRL1.

213 IgGILlOMM OAENODPDIKAHVNSLGENLKTLRLRL

IL10 is

(Chothia) RRCHRFLP CENGGGS GGKSKA VE O VK

bolded,

NAFNKLOEKGIYKAMSEFDIFINYIEAY

underlined

MTMKIRNVGY GFTX3

CDRL2 of

214 IgGIL10M14 DTS GFTX3

(Chothia) SEQ ID

Description Comments NO:

CDRL3 of

215 IgGIL10M14 GSGYPF GFTX3

(Chothia)

CDRH1 of

216 TSGMSVG GFTX3

IgGIL10M14 (Kabat)

CDRH2 of

217 DIWWDDKKDYNPSLKS GFTX3

IgGIL10M14 (Kabat)

CDRH3 of

218 SMITNWYFDV GFTX3

IgGIL10M14 (Kabat)

Monomeric

KAOLSSPGOGTOSENSCTHFPGNLPNM IL10 grafted

LRDLRDAFSRVKTFFOMKDOLDNLLL

into

KESLLEDFKGYLGCOALSEMIOFYLEE

CDRL1 of CDRL1.

219 VMPOAENODPDIKAHVNSLGENLKTLR

IgGIL10M14 (Kabat) IL10 is

LRLRRCHRFLPCENGGGSGGKSKAVE

bolded,

OVKNAFNKLOEKGIYKAMSEFDIFINYI

underlined

EAYMTMKIRNVGYMH GFTX3

CDRL2 of

220 DTSKLAS GFTX3

IgGIL10M14 (Kabat)

CDRL3 of

221 FQGSGYPFT GFTX3

IgGIL10M14 (Kabat)

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDD

222 VH of IgGIL10M14 KKDYNPSLKSRLTISKDTSANQVVLKVTN GFTX3

MDPADTATYYCARSMITNWYFDVWGAG

TTVTVSS

DIQMTQSPSTLSASVGDRVTITCKAQLSSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

Monomeric

FSRVKTFFOMKDOLDNLLLKESLLEDF IL10 grafted

223 VL of IgGIL10M14 KGYLGCOALSEMIOFYLEEVMPOAEN

into CDRL1

ODPDIKAHVNSLGENLKTLRLRLRRCH GFTX3

RFLP CENGGGS GGKSKA VE 0 VKNAFN

KLOEKGIYKAMSEFDIFINYIEAYMTM SEQ ID

Description Comments NO:

KIRNVGYMHWYQQKPGKAPKLLIYDTS

KLASGVPSRFSGSGSGTAFTLTISSLQPDD

FATYYCFQGSGYPFTFGGGTKLEIK

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDD

KKDYNPSLKSRLTISKDTSANQVVLKVTN

MDPADTATYYCARSMITNWYFDVWGAG

TTVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVH

TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY

Heavy chain of ICNVNHKPSNTKVDKRVEPKSCDKTHTCP GFTX3

224

IgGIL10M14 PCP APEAAGGP S VFLFPPKPKDTLMI SRTP LALA

EVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPEN YKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFSCS

VMHEALHNH YTQKSL SL SPGK

DIQMTQSPSTLSASVGDRVTITCKAQLSSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

FSRVKTFFOMKDOLDNLLLKESLLEDF

KGYLGCOALSEMIOFYLEEVMPOAEN

ODPDIKAHVNSLGENLKTLRLRLRRCH GFTX3

RFLP CENGGGS GGKSKA VE 0 VKNAFN LALA

Light chain of KLOEKGIYKAMSEFDIFINYIEAYMTM Monomeric

225

IgGIL10M14 KIRNVGYMHWYQQKPGKAPKLLIYDTS IL10 grafted

KLASGVPSRFSGSGSGTAFTLTISSLQPDD into

FATYYCFQGSGYPFTFGGGTKLEIKRTVA CDRLl.

APSVFIFPPSDEQLKSGTASVVCLLNNFYP

REAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC SEQ ID

Description Comments NO:

CDRH1 of

226 IgGIL10M15 GFSLSTSGM GFTX3

(Chothia)

CDRH2 of

227 IgGIL10M15 WWDDK GFTX3

(Chothia)

CDRH3 of

228 IgGIL10M15 SMITNWYFDV GFTX3

(Chothia)

OLSSPGOGTOSENSCTHFPGNLPNMLR Monomeric

DLRDAFSRVKTFFOMKDOLDNLLLKES IL10 grafted

CDRL1 of LLEDFKGYLGCOALSEMIOFYLEEVMP into

229 IgGIL10M15 OAENODPDIKAHVNSLGENLKTLRLRL CDRL1.

(Chothia) RRCHRFLP CENGGGS GGKSKA VE 0 VK IL10 is

NAFNKLOEKGIYKAMSEFDIFINYIEAY bolded,

MTMKIRNVGY underlined

CDRL2 of

230 IgGIL10M15 DTS GFTX3

(Chothia)

CDRL3 of

231 IgGIL10M15 GSGYPF GFTX3

(Chothia)

CDRH1 of

232 TSGMSVG GFTX3

IgGIL10M15 (Kabat)

CDRH2 of

233 DIWWDDKKDYNPSLKS GFTX3

IgGIL10M15 (Kabat)

CDRH3 of

234 SMITNWYFDV GFTX3

IgGIL10M15 (Kabat)

KAOLSSPGOGTOSENSCTHFPGNLPNM Monomeric

LRDLRDAFSRVKTFFOMKDOLDNLLL IL10 grafted

CDRL1 of

235 KESLLEDFKGYLGCOALSEMIOFYLEE into CDRL1

IgGIL10M15 (Kabat)

VMPOAENODPDIKAHVNSLGENLKTLR IL10 is

LRLRRCHRFLPCENGGGSGGKSKAVE bolded, SEQ ID

Description Comments NO:

OVKNAFNKLOEKGIYKAMSEFDIFINYI underlined EAYMTMKIRNVGYMH GFTX3

CDRL2 of

236 DTSKLAS GFTX3

IgGIL10M15 (Kabat)

CDRL3 of

237 FQGSGYPFT GFTX3

IgGIL10M15 (Kabat)

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDD

238 VH of IgGIL10M15 KKDYNPSLKSRLTISKDTSANQVVLKVTN GFTX3

MDPADTATYYCARSMITNWYFDVWGAG

TTVTVSS

DIQMTQSPSTLSASVGDRVTITCKAQLSSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

Monomeric

FSRVKTFFOMKDOLDNLLLKESLLEDF IL10 grafted

KGYLGCOALSEMIOFYLEEVMPOAEN

into CDRL1

ODPDIKAHVNSLGENLKTLRLRLRRCH

239 VL of IgGIL10M15 IL10 is

RFLP CENGGGS GGKSKA VE 0 VKNAFN

bolded,

KLOEKGIYKAMSEFDIFINYIEAYMTM

underlined

KIRNVGYMHWYQQKPGKAPKLLIYDTS GFTX3

KLASGVPSRFSGSGSGTAFTLTISSLQPDD

FATYYCFQGSGYPFTFGGGTKLEIK

QVTLRESGPALVKPTQTLTLTCTFSGFSLS

TSGMSVGWIRQPPGKALEWLADIWWDD

KKDYNPSLKSRLTISKDTSANQVVLKVTN

MDPADTATYYCARSMITNWYFDVWGAG

TTVTVSSASTKGPSVFPLAPSSKSTSGGTA

Heavy chain of ALGCLVKDYFPEPVTVSWNSGALTSGVH GFTX3

240

IgGIL10M15 TFPAVLQSSGLYSLSSVVTVPSSSLGTQTY NEM

ICNVNHKPSNTKVDKRVEPKSCDKTHTCP

PCPAPELLGGPSVFLFPPKPKDTLMISRTP

EVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYASTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAK SEQ ID

Description Comments NO:

GQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWVSNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCS VIHEALHNHYTQKSL SL SPGK

DIQMTQSPSTLSASVGDRVTITCKAQLSSP

GOGTOSENSCTHFPGNLPNMLRDLRDA

FSRVKTFFOMKDOLDNLLLKESLLEDF

GFTX3

KGYLGCOALSEMIOFYLEEVMPOAEN NEM

ODPDIKAHVNSLGENLKTLRLRLRRCH

Monomeric

RFLP CENGGGS GGKSKA VE 0 VKNAFN

IL10 grafted

Light chain of KLOEKGIYKAMSEFDIFINYIEAYMTM

241 into

IgGIL10M15 KIRNVGYMHWYQQKPGKAPKLLIYDTS

CDRLl.

KLASGVPSRFSGSGSGTAFTLTISSLQPDD IL10 is

FATYYCFQGSGYPFTFGGGTKLEIKRTVA

bolded,

APSVFIFPPSDEQLKSGTASVVCLLNNFYP

underlined

REAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC

CAGGTCACACTGAGAGAGTCAGGCCCT

GCCCTGGTCAAGCCTACTCAGACCCTGA

CCCTGACCTGCACCTTTAGCGGCTTTAG

CCTGAGCACTAGCGGAATGAGCGTGGG

CTGGATTAGACAGCCCCCTGGTAAAGC

CCTGGAGTGGCTGGCCGATATTTGGTGG

GACGATAAGAAGGACTATAACCCTAGC

242 VH of IgGIL10M7

CTGAAGTCTAGGCTGACTATCTCTAAGG

ACACTAGCGCTAATCAGGTGGTGCTGA

AAGTGACTAATATGGACCCCGCCGACA

CCGCTACCTACTACTGCGCTAGATCTAT

GATCACTAACTGGTACTTCGACGTGTGG

GGCGCTGGCACTACCGTGACCGTGTCTA

GC

243 VL of IgGIL10M7 GATATTCAGATGACTCAGTCACCTAGCA SEQ ID

Description Comments NO:

CCCTGAGCGCTAGTGTGGGCGATAGAG

TGACTATCACCTGTAAAGCTCAGCTGTC

TAGCCCAGGTCAGGGAACTCAGTCAGA

GAATAGCTGCACTCACTTCCCCGGTAAC

CTGCCTAATATGCTGAGAGATCTGAGG

GACGCCTTCTCTAGGGTCAAGACCTTCT

TTCAGATGAAGGATCAGCTGGATAACC

TGCTGCTGAAAGAGTCACTGCTGGAGG

ACTTTAAGGGCTACCTGGGCTGTCAGGC

CCTGAGCGAGATGATTCAGTTCTACCTG

GAAGAAGTGATGCCCCAGGCCGAGAAT

CAGGACCCCGATATTAAGGCTCACGTG

AACTCACTGGGCGAGAACCTGAAAACC

CTGAGACTGAGGCTGAGGCGGTGTCAC

CGGTTTCTGCCCTGCGAGAACGGCGGA

GGTAGCGGCGGTAAATCTAAGGCCGTG

GAACAGGTCAAAAACGCCTTTAACAAG

CTGCAGGAAAAGGGAATCTATAAGGCT

ATGAGCGAGTTCGACATCTTTATTAACT

ATATCGAGGCCTATATGACTATGAAGA

TTAGGAACGTGGGCTATATGCACTGGT

ATCAGCAGAAGCCCGGTAAAGCCCCTA

AGCTGCTGATCTACGACACCTCTAAGCT

GGCTAGTGGCGTGCCCTCTAGGTTTAGC

GGTAGCGGTAGTGGCACCGCCTTCACC

CTGACTATCTCTAGCCTGCAGCCCGACG

ACTTCGCTACCTACTACTGTTTTCAGGG

TAGCGGCTACCCCTTCACCTTCGGCGGA

GGCACTAAGCTGGAGATTAAG

CAGGTCACACTGAGAGAGTCAGGCCCT

Heavy Chain of GCCCTGGTCAAGCCTACTCAGACCCTGA

244

IgGIL10M7 CCCTGACCTGCACCTTTAGCGGCTTTAG

CCTGAGCACTAGCGGAATGAGCGTGGG SEQ ID

Description Comments NO:

CTGGATTAGACAGCCCCCTGGTAAAGC

CCTGGAGTGGCTGGCCGATATTTGGTGG

GACGATAAGAAGGACTATAACCCTAGC

CTGAAGTCTAGGCTGACTATCTCTAAGG

ACACTAGCGCTAATCAGGTGGTGCTGA

AAGTGACTAATATGGACCCCGCCGACA

CCGCTACCTACTACTGCGCTAGATCTAT

GATCACTAACTGGTACTTCGACGTGTGG

GGCGCTGGCACTACCGTGACCGTGTCTA

GCGCTAGCACTAAGGGCCCAAGTGTGT

TTCCCCTGGCCCCCAGCAGCAAGTCTAC

TTCCGGCGGAACTGCTGCCCTGGGTTGC

CTGGTGAAGGACTACTTCCCCGAGCCC

GTGACAGTGTCCTGGAACTCTGGGGCTC

TGACTTCCGGCGTGCACACCTTCCCCGC

CGTGCTGCAGAGCAGCGGCCTGTACAG

CCTGAGCAGCGTGGTGACAGTGCCCTC

CAGCTCTCTGGGAACCCAGACCTATATC

TGCAACGTGAACCACAAGCCCAGCAAC

ACCAAGGTGGACAAGAGAGTGGAGCCC

AAGAGCTGCGACAAGACCCACACCTGC

CCCCCCTGCCCAGCTCCAGAACTGCTGG

GAGGGCCTTCCGTGTTCCTGTTCCCCCC

CAAGCCCAAGGACACCCTGATGATCAG

CAGGACCCCCGAGGTGACCTGCGTGGT

GGTGGACGTGTCCCACGAGGACCCAGA

GGTGAAGTTCAACTGGTACGTGGACGG

CGTGGAGGTGCACAACGCCAAGACCAA

GCCCAGAGAGGAGCAGTACAACAGCAC

CTACAGGGTGGTGTCCGTGCTGACCGTG

CTGCACCAGGACTGGCTGAACGGCAAA

GAATACAAGTGCAAAGTCTCCAACAAG

GCCCTGCCAGCCCCAATCGAAAAGACA SEQ ID

Description Comments NO:

ATCAGCAAGGCCAAGGGCCAGCCACGG

GAGCCCCAGGTGTACACCCTGCCCCCC

AGCCGGGAGGAGATGACCAAGAACCAG

GTGTCCCTGACCTGTCTGGTGAAGGGCT

TCTACCCCAGCGATATCGCCGTGGAGTG

GGAGAGCAACGGCCAGCCCGAGAACAA

CTACAAGACCACCCCCCCAGTGCTGGA

CAGCGACGGCAGCTTCTTCCTGTACAGC

AAGCTGACCGTGGACAAGTCCAGGTGG

CAGCAGGGCAACGTGTTCAGCTGCAGC

GTGATGCACGAGGCCCTGCACAACCAC

TACACCCAGAAGTCCCTGAGCCTGAGC

CCCGGCAAG

GATATTCAGATGACTCAGTCACCTAGCA

CCCTGAGCGCTAGTGTGGGCGATAGAG

TGACTATCACCTGTAAAGCTCAGCTGTC

TAGCCCAGGTCAGGGAACTCAGTCAGA

GAAT AGCT GCACT CACTT CCCCGGT AAC

CTGCCTAATATGCTGAGAGATCTGAGG

GACGCCTTCTCTAGGGTCAAGACCTTCT

TTCAGATGAAGGATCAGCTGGATAACC

TGCTGCTGAAAGAGTCACTGCTGGAGG

Light Chain of ACTTTAAGGGCTACCTGGGCTGTCAGGC

245

IgGIL10M7 CCTGAGCGAGATGATTCAGTTCTACCTG

GAAGAAGTGATGCCCCAGGCCGAGAAT

CAGGACCCCGATATTAAGGCTCACGTG

AACTCACTGGGCGAGAACCTGAAAACC

CTGAGACTGAGGCTGAGGCGGTGTCAC

CGGTTTCTGCCCTGCGAGAACGGCGGA

GGTAGCGGCGGTAAATCTAAGGCCGTG

GAACAGGTCAAAAACGCCTTTAACAAG

CTGCAGGAAAAGGGAATCTATAAGGCT

ATGAGCGAGTTCGACATCTTTATTAACT SEQ ID

Description Comments NO:

ATATCGAGGCCTATATGACTATGAAGA

TTAGGAACGTGGGCTATATGCACTGGT

ATCAGCAGAAGCCCGGTAAAGCCCCTA

AGCTGCTGATCTACGACACCTCTAAGCT

GGCTAGTGGCGTGCCCTCTAGGTTTAGC

GGTAGCGGTAGTGGCACCGCCTTCACC

CTGACTATCTCTAGCCTGCAGCCCGACG

ACTTCGCTACCTACTACTGTTTTCAGGG

TAGCGGCTACCCCTTCACCTTCGGCGGA

GGCACTAAGCTGGAGATTAAGCGTACG

GTGGCCGCTCCCAGCGTGTTCATCTTCC

CCCCCAGCGACGAGCAGCTGAAGAGCG

GCACCGCCAGCGTGGTGTGCCTGCTGA

ACAACTTCTACCCCCGGGAGGCCAAGG

TGCAGTGGAAGGTGGACAACGCCCTGC

AGAGCGGCAACAGCCAGGAGAGCGTCA

CCGAGCAGGACAGCAAGGACTCCACCT

ACAGCCTGAGCAGCACCCTGACCCTGA

GCAAGGCCGACTACGAGAAGCATAAGG

TGTACGCCTGCGAGGTGACCCACCAGG

GCCTGTCCAGCCCCGTGACCAAGAGCTT

CAACAGGGGCGAGTGC

CAGGTCACACTGAGAGAGTCAGGCCCT

GCCCTGGTCAAGCCTACTCAGACCCTGA

CCCTGACCTGCACCTTTAGCGGCTTTAG

CCTGAGCACTAGCGGAATGAGCGTGGG

CTGGATTAGACAGCCCCCTGGTAAAGC

246 VH of IgGIL10M13 CCTGGAGTGGCTGGCCGATATTTGGTGG

GACGATAAGAAGGACTATAACCCTAGC

CTGAAGTCTAGGCTGACTATCTCTAAGG

ACACTAGCGCTAATCAGGTGGTGCTGA

AAGTGACTAATATGGACCCCGCCGACA

CCGCTACCTACTACTGCGCTAGATCTAT SEQ ID

Description Comments NO:

GATCACTAACTGGTACTTCGACGTGTGG GGCGCTGGCACTACCGTGACCGTGTCTA GC

GATATTCAGATGACTCAGTCACCTAGCA

CCCTGAGCGCTAGTGTGGGCGATAGAG

TGACTATCACCTGTAAAGCTCAGCTGTC

TAGCCCAGGTCAGGGAACTCAGTCAGA

GAAT AGCT GCACT CACTT CCCCGGT AAC

CTGCCTAATATGCTGAGAGATCTGAGG

GACGCCTTCTCTAGGGTCAAGACCTTCT

TTCAGATGAAGGATCAGCTGGATAACC

TGCTGCTGAAAGAGTCACTGCTGGAGG

ACTTTAAGGGCTACCTGGGCTGTCAGGC

CCTGAGCGAGATGATTCAGTTCTACCTG

GAAGAAGTGATGCCCCAGGCCGAGAAT

CAGGACCCCGATATTAAGGCTCACGTG

AACTCACTGGGCGAGAACCTGAAAACC

CTGAGACTGAGGCTGAGGCGGTGTCAC

247 VL of lgGILlOMB

CGGTTTCTGCCCTGCGAGAACGGCGGA

GGTAGCGGCGGTAAATCTAAGGCCGTG

GAACAGGTCAAAAACGCCTTTAACAAG

CTGCAGGAAAAGGGAATCTATAAGGCT

ATGAGCGAGTTCGACATCTTTATTAACT

ATATCGAGGCCTATATGACTATGAAGA

TTAGGAACGTGGGCTATATGCACTGGT

ATCAGCAGAAGCCCGGTAAAGCCCCTA

AGCTGCTGATCTACGACACCTCTAAGCT

GGCTAGTGGCGTGCCCTCTAGGTTTAGC

GGTAGCGGTAGTGGCACCGCCTTCACC

CTGACTATCTCTAGCCTGCAGCCCGACG

ACTTCGCTACCTACTACTGTTTTCAGGG

TAGCGGCTACCCCTTCACCTTCGGCGGA

GGCACTAAGCTGGAGATTAAG SEQ ID

Description Comments NO:

CAGGTCACACTGAGAGAGTCAGGCCCT

GCCCTGGTCAAGCCTACTCAGACCCTGA

CCCTGACCTGCACCTTTAGCGGCTTTAG

CCTGAGCACTAGCGGAATGAGCGTGGG

CTGGATTAGACAGCCCCCTGGTAAAGC

CCTGGAGTGGCTGGCCGATATTTGGTGG

GACGATAAGAAGGACTATAACCCTAGC

CTGAAGTCTAGGCTGACTATCTCTAAGG

ACACTAGCGCTAATCAGGTGGTGCTGA

AAGTGACTAATATGGACCCCGCCGACA

CCGCTACCTACTACTGCGCTAGATCTAT

GATCACTAACTGGTACTTCGACGTGTGG

GGCGCTGGCACTACCGTGACCGTGTCTA

GCGCTAGCACTAAGGGCCCCTCCGTGTT

CCCTCTGGCCCCTTCCAGCAAGTCTACC

TCCGGCGGCACAGCTGCTCTGGGCTGCC

Heavy Chain of

248 TGGTCAAGGACTACTTCCCTGAGCCTGT

IgGILlOMB

GACAGTGTCCTGGAACTCTGGCGCCCTG

ACCTCTGGCGTGCACACCTTCCCTGCCG

TGCTGCAGTCCTCCGGCCTGTACTCCCT

GTCCTCCGTGGTCACAGTGCCTTCAAGC

AGCCTGGGCACCCAGACCTATATCTGC

AACGTGAACCACAAGCCTTCCAACACC

AAGGTGGACAAGCGGGTGGAGCCTAAG

TCCTGCGACAAGACCCACACCTGTCCTC

CCTGCCCTGCTCCTGAACTGCTGGGCGG

CCCTTCTGTGTTCCTGTTCCCTCCAAAG

CCCAAGGACACCCTGATGATCTCCCGG

ACCCCTGAAGTGACCTGCGTGGTGGTG

GCCGTGTCCCACGAGGATCCTGAAGTG

AAGTTCAATTGGTACGTGGACGGCGTG

GAGGTGCACAACGCCAAGACCAAGCCT

CGGGAGGAACAGTACAACTCCACCTAC SEQ ID

Description Comments NO:

CGGGTGGTGTCCGTGCTGACCGTGCTGC

ACCAGGACTGGCTGAACGGCAAAGAGT

ACAAGTGCAAAGTCTCCAACAAGGCCC

TGGCCGCCCCTATCGAAAAGACAATCT

CCAAGGCCAAGGGCCAGCCTAGGGAAC

CCCAGGTGTACACCCTGCCACCCAGCC

GGGAGGAAATGACCAAGAACCAGGTGT

CCCTGACCTGTCTGGTCAAGGGCTTCTA

CCCTTCCGATATCGCCGTGGAGTGGGA

GTCTAACGGCCAGCCTGAGAACAACTA

CAAGACCACCCCTCCTGTGCTGGACTCC

GACGGCTCCTTCTTCCTGTACTCCAAAC

TGACCGTGGACAAGTCCCGGTGGCAGC

AGGGCAACGTGTTCTCCTGCTCCGTGAT

GCACGAGGCCCTGCACAACCACTACAC

CCAGAAGTCCCTGTCCCTGTCTCCCGGC

AAG

GATATTCAGATGACTCAGTCACCTAGCA

CCCTGAGCGCTAGTGTGGGCGATAGAG

TGACTATCACCTGTAAAGCTCAGCTGTC

TAGCCCAGGTCAGGGAACTCAGTCAGA

GAATAGCTGCACTCACTTCCCCGGTAAC

CTGCCTAATATGCTGAGAGATCTGAGG

GACGCCTTCTCTAGGGTCAAGACCTTCT

Light Chain of TTCAGATGAAGGATCAGCTGGATAACC

249

IgGILlOMB TGCTGCTGAAAGAGTCACTGCTGGAGG

ACTTTAAGGGCTACCTGGGCTGTCAGGC

CCTGAGCGAGATGATTCAGTTCTACCTG

GAAGAAGTGATGCCCCAGGCCGAGAAT

CAGGACCCCGATATTAAGGCTCACGTG

AACTCACTGGGCGAGAACCTGAAAACC

CTGAGACTGAGGCTGAGGCGGTGTCAC

CGGTTTCTGCCCTGCGAGAACGGCGGA SEQ ID

Description Comments NO:

GGTAGCGGCGGTAAATCTAAGGCCGTG

GAACAGGTCAAAAACGCCTTTAACAAG

CTGCAGGAAAAGGGAATCTATAAGGCT

ATGAGCGAGTTCGACATCTTTATTAACT

ATATCGAGGCCTATATGACTATGAAGA

TTAGGAACGTGGGCTATATGCACTGGT

ATCAGCAGAAGCCCGGTAAAGCCCCTA

AGCTGCTGATCTACGACACCTCTAAGCT

GGCTAGTGGCGTGCCCTCTAGGTTTAGC

GGTAGCGGTAGTGGCACCGCCTTCACC

CTGACTATCTCTAGCCTGCAGCCCGACG

ACTTCGCTACCTACTACTGTTTTCAGGG

TAGCGGCTACCCCTTCACCTTCGGCGGA

GGCACTAAGCTGGAGATTAAGCGTACG

GTGGCCGCTCCCAGCGTGTTCATCTTCC

CCCCCAGCGACGAGCAGCTGAAGAGCG

GCACCGCCAGCGTGGTGTGCCTGCTGA

ACAACTTCTACCCCCGGGAGGCCAAGG

TGCAGTGGAAGGTGGACAACGCCCTGC

AGAGCGGCAACAGCCAGGAGAGCGTCA

CCGAGCAGGACAGCAAGGACTCCACCT

ACAGCCTGAGCAGCACCCTGACCCTGA

GCAAGGCCGACTACGAGAAGCATAAGG

TGTACGCCTGCGAGGTGACCCACCAGG

GCCTGTCCAGCCCCGTGACCAAGAGCTT

CAACAGGGGCGAGTGC

AGTCCCGGTCAGGGAACTCAGTCAGAG

AATAGCTGCACTCACTTCCCCGGTAACC

TGCCTAATATGCTGAGAGATCTGAGGG

250 Monomeric IL10 ACGCCTTCTCTAGGGTCAAGACCTTCTT

TCAGATGAAGGATCAGCTGGATAACCT

GCTGCTGAAAGAGTCACTGCTGGAGGA

CTTTAAGGGCTACCTGGGCTGTCAGGCC SEQ ID

Description Comments NO:

CTGAGCGAGATGATTCAGTTCTACCTGG

AAGAAGTGATGCCCCAGGCCGAGAATC

AGGACCCCGATATTAAGGCTCACGTCA

ACTCACTGGGCGAGAACCTGAAAACCC

TGAGACTGAGGCTGAGGCGGTGTCACC

GGTTTCTGCCCTGCGAGAACGGCGGAG

GTAGCGGCGGTAAATCTAAGGCCGTGG

AACAGGTCAAAAACGCCTTTAACAAGC

TGCAGGAAAAGGGAATCTATAAGGCTA

TGAGCGAGTTCGACATCTTTATTAACTA

TATCGAGGCCTATATGACTATGAAGATT

AGGAAC

251 linker GGGSGG

252 linker GGGGS

253 linker GGGGA

Example 2: Antibody cytokine engrafted proteins have anti-inflammatory activity

[00234] Using an assay developed in support of rhILlO's pro- inflammatory activity in the clinic (Lauw et al, J Immunol. 2000; 165(5):2783-9), the pro-inflammatory activity of IgGIL10M13 in human whole blood was assessed. In order to assess pro-inflammatory activity, antibody cytokine engrafted proteins were profiled for their ability to induce interferon gamma (IFNy) or granzyme B in activated primary human CD8 T cells. It was found that antibody cytokine engrafted proteins such as IgGIL10M13 demonstrated significantly less proinflammatory activity than recombinant human IL10 (rhILlO) as measured by IFNy production. This data is shown in Figure 3A. Similar results were found in assays measuring granzyme B (data not shown), as well as with other exemplary antibody cytokine engrafted proteins

(IgGIL10M7). The significantly decreased pro-inflammatory activity demonstrated by

IgGIL10M13 as compared to rhILlO indicates it would be superior to rhILlO for treating immune related disorders, as IgGIL10M13 could be administered over a broader dose range. [00235] To examine anti-inflammatory activity, antibody cytokine engrafted proteins and rhILlO were tested for their ability to inhibit LPS-induced TNFa in human whole blood. This data is shown in Figure 3B, wherein increasing concentrations of either rhILlO or

IgGIL10M13 reduced TNFa production. Note that the rhILlO and IgGIL10M13 curves are similar, indicating that both molecules had potent anti-inflammatory activity.

[00236] In summary, these results show that antibody cytokine engrafted proteins have the desired properties of having anti-inflammatory properties similar to IL10, but without the dose limiting, and unwanted pro-inflammatory properties.

Example 3: IL10 dependent signaling

[00237] In vitro signaling studies in human PBMCs and whole blood indicate that antibody cytokine engrafted proteins such as IgGIL10M13 had a more specific signaling profile when compared to rhILlO. Using CyTOF, a FACS based method that utilizes mass

spectrometry, antibody cytokine engrafted protein signaling in multiple different cell populations in whole blood was assessed by pSTAT3 detection (Figure 4). Antibody cytokine engrafted proteins such IgGIL10M13 induced a pSTAT3 signal only on monocytes, macrophages and plasmacytoid dendritic cells above μΜ concentrations (up to 1.8μΜ). All of these cell types are known to have increased expression of IL10 receptor. rhILlO induced a pSTAT3 signal on monocytes, but also on additional cell types such as T cells, B cells, and NK cells. This was seen even at low nM concentrations of rhlLl 0. In whole blood treated with rhILl 0 at a concentration of lOOnM, the strongest pSTAT3 signal was seen on monocytes and myeloid dendritic cells with additional moderate activation of T, NK, B cells, and Granulocytes. The functional

consequences of pSTAT3 signaling leads to increased production of IFNy and Granzyme B from CD8 T cells and NK cells. There is also proliferation of B cells in response to rhILlO signaling. This pro-inflammatory activity of rhILlO in human whole blood is observed at exposures less than 5-fold above the anti- inflammatory IC90. The more selective cellular profile of antibody cytokine engrafted proteins such as IgGIL10M13 resulted in reduced pro- inflammatory activity leading to better anti-inflammatory efficacy.

Example 4: Antibody cytokine engrafted protein signaling in various species

[00238] rhILlO potently inhibits LPS-induced pro- inflammatory cytokine production in human monocytes, PBMCs, and whole blood. The antibody cytokine engrafted protein IgGIL10M13 exhibits pM potency on target cells, although 10-fold less potent than rhILlO. Table 2 is a potency comparison for IL10 or IgGIL10M13 activity in human whole blood as well as whole blood of selected toxicity species.

[00239] Potency calculations are based on ex vivo whole blood assays from either mouse, cynomolgus monkey or human. For each species tested, IgGIL10M13 or rhILlO were titrated and assessed for ability to inhibit LPS-induced TNFa production. IC50s were calculated as the level of molecule that gave rise to 50% inhibition of total TNFa signal. IC90s and IC30s were calculated taking into account Hill slope value for each assay with the following equation: logEC50=logECF - (l/HillSlope)*lob(F/100-F)), where ECF is the concentration that gives a response of F percent of total TNFa signal.

TABLE 2

IgGIL10M13 (CV%) IL10 (CV%)

Mouse IC30 2.2 pM (pooled blood) 0.57 pM (pooled blood)

IC50 12 pM 1.7 pM

IC90 108 pM 15 pM

Cyno IC30 4.13 pM (48% n=3) 0.44 pM (28% n=3)

IC50 6.67 pM (53%) 0.65 pM (31%)

IC90 24 pM (73%) 1.9 pM (43%)

Human IC30 10.8 pM (76% n=48) 1.3 pM (96% n=48)

IC50 25.2 pM (76%) 2.8 pM (98%)

IC90 262 pM (94%) 22.8 pM (79%)

Example 5: Evaluation of antibody cytokine engrafted protein pharmacokinetics

[00240] rhILlO has a short half-life, limiting its target tissue exposure and requiring the patient to undergo multiple dosing. The half-life of antibody cytokine engrafted proteins was assessed in C57B1/6 mice. Antibody cytokine engrafted proteins (e.g. IgGIL10M13) were injected at 0.2mg/kg subcutaneously and blood was sampled beginning at 5 minutes post- injection up to 144 hours post-injection. IgGIL10M13 had a significant half-life extension of approximately 4.4 days (Figure 5B) compared to rhILlO which had a half-life of approximately lhr (Figure 5 A).

Example 6: Evaluation of antibody cytokine engrafted protein Pharmacodynamics

[00241] Consistent with extended half-life, antibody cytokine engrafted proteins also demonstrated improved pharmacodynamics. Phospho-STAT3 (pSTAT3), a marker of IL10 receptor activation and signaling was monitored in mouse colon after subcutaneous dosing of IgGIL10M13. Enhanced pSTAT3 signal was detected in colon at least up to 72 hours post-dose, and absent by 144 hours post-dose. See Figure 5C. This profile is a dramatic improvement over rhlLlO, whose signal is absent by 24 hours post-dose. Figure 5D depicts improved duration of in vivo response of IgGIL10M13 as compared to rhlLlO as measured by inhibition of TNFa in blood in response to LPS challenge following antibody cytokine engrafted protein dosing.

Example 7: Efficacy of antibody cytokine engrafted proteins in a mouse model

[00242] A direct comparison of efficacy for TNFa inhibition after LPS challenge was performed. C57/BL6 mice were dosed subcutaneously with vehicle, or equimolar levels of IL10 at 11 Onmol/mouse, calculated for both recombinant IL10 and IgGIL10M13. Mice were then challenged with LPS delivered intraperitoneally to assess IL10 dependent inhibition of TNFa levels. IgGIL10M13 demonstrated comparable efficacy to rhlLlO at the initial assessment time period of 0.5 hour, however, up to at least forty-eight hours post dosing, IgGIL10M13 sustained superior efficacy to rhlLlO as measured by TNFa production. This data is shown in Figure 6.

Example 8: Antibody cytokine engrafted proteins have improved exposure

[00243] The peak serum concentration (Cmax) of antibody cytokine engrafted proteins was assessed in C57B1/6 mice. Antibody cytokine engrafted proteins were injected at 0.2mg/kg (lOml/kg dose volume) in 0.9% saline subcutaneously and blood was sampled beginning at 1 hour post-injection and up to 144 hours post-injection. Whole blood was collected into heparin- treated tubes at each time point and centrifuged at 12,500rpm for 10 minutes at 4°C. Plasma supernatant was collected and stored at -80°C until all time points were collected. Antibody cytokine engrafted proteins levels in plasma were measured using two different immunoassay methods to enable detection of both the IL10 and antibody domains of the antibody cytokine engrafted protein. As shown in Figure 7, the antibody cytokine engrafted protein (e.g.

IgGIL10M13) maintained greater than 60% Cmax past 100 hours. In contrast, rhlLlO levels dropped below 20% Cmax within 3.5 hours.

Example 9: Antibody cytokine engrafted proteins act only on certain cell types in human patients [00244] CyTOF was run as previously described (see Example 3 and Materials and

Methods below) on immune cells from human healthy donors and patients with Crohn's disease. As shown in the graphs in Figure 8, IgGIL10M13 stimulated only monocytes, and the stimulation as measured by pSTAT3 levels is comparable to rhlLlO. Monocytes are the target cells for inflammatory related disorders such as Crohn's disease and Ulcerative Colitis and express very high levels of IL10 receptor. However, Figure 9 also shows the unwanted proinflammatory effects of rhlLlO, for example, the increased pSTAT3 signaling on CD4 T cells, CD8 T cells and NK cells. It is noteworthy that IgGIL10M13, does not display this unwanted pro-inflammatory effect either on normal human cells or in cells taken Crohn's disease patients. This demonstrates that IgGIL10M13 has a larger, safer therapeutic index as administration of the antibody cytokine engrafted protein will act only on the desired cell type and not on other cell types such as CD8 T cells which would only worsen immune related disorders such as Crohn's disease and Ulcerative Colitis.

Example 10: IgGIL10M13 has reduced pro-inflammatory activity in PHA stimulated human whole blood compared to rhlLlO

[00245] Despite extensive clinical data linking genetic IL10 deficiency to IBD susceptibility, rhlLlO showed only mild efficacy in IBD clinical trials (Herfarth et al, Gut 2002: 50(2): 146-147). Retrospective analyses of trial data suggest that rhILlO's efficacy was limited by its intrinsic pro-inflammatory activity such as enhanced production of IFNy. As discussed previously, in human functional cell-based assays, rhlLlO signaling leads to production of IFNy and Granzyme B from T cells and NK cells.

[00246] Whole blood was taken from patients with Crohn's Disease and the levels of

IFNy were measured after stimulation with rhlLlO, IgGIL10M13 and PHA alone. This data is shown in Figures 9A-9C. Increasing doses of rhlLlO causes a sharp increase in IFNy production, which then plateaus. In contrast, in treatment of these cells with IgGIL10M13 little to no production of IFNy was seen, indicating that IgGIL10M13 did not induce, or induced only very low levels of IFNy production from T cells or NK cells.

[00247] An additional titration experiment was performed with these patient donor samples. In this experiment, IL10 levels from the donor patient sera was measured and found to be in the range of 1.5 to 5 femtomolar (fM), although the scientific literature has reported that patient IL10 levels could be as high as 20fM (Szkaradkiewicz et al., Arch. Immunol. Ther Exp 2009: 57(4):291-294). rhILlO was administered to the donor patient cells at the fixed concentrations of 2 femtomolar (fM), 2 pM, 2nM and 200nM. To these fixed concentrations of rhILlO, increasing concentrations of the antibody cytokine engrafted protein IgGIL10M13 was administered, and IFNy production assayed. The data is shown in Figure 9D. At the fixed concentrations of 2fM and 2pM, IgGIL10M13 competes with rhILlO and reduced the production of IFNy to baseline levels. At the fixed concentration of 2nM, IFNy production was reduced by nanomolar concentrations of IgGIL10M13. Finally, at the fixed excess concentration of 200nM rhILlO, only very little reduction of IFNy production by IgGIL10M13 was seen. This indicates that at physiological levels of IL10, IgGIL10M13 competed out IL10, reducing the production of IFNy, and the unwanted pro-inflammatory effects.

Example 11: Aggregation properties of antibody cytokine engrafted proteins

[00248] In clinical trials for IBD, rhILlO was observed to have a very short half-life; however simple Fc fusions to the IL10 dimer to extend half-life were not pursued given aggregation properties of such a molecule. Figure 10A shows aggregation of both an IL10 wild type linked to an Fc and IL10 monomer linked to an Fc. However, as shown in Figure 10B, the antibody structure of the antibody cytokine engrafted protein prevents IL10 aggregation, thus promoting ease of administration. In addition, reducing aggregation has the benefit of reducing an immune reaction to the therapeutic, and the generation of anti-drug antibodies.

Example 12: Retained binding of antibody cytokine engrafted proteins

[00249] Palivizumab is an anti-RSV antibody, and was chosen as the antibody structure for cytokine engrafting. This antibody had the advantages of a known structure, and as its target was RSV, a non-human target. The choice of a non-human target was to insure that there would be no toxicity associated with the antibody cytokine engrafted protein binding to an off target human antigen. It was uncertain after engrafting IL10M into palivizumab, whether the final IL10 antibody cytokine engrafted protein would still bind the RSV target protein. As assayed by ELISA, the IL10 antibody cytokine engrafted protein still bound to RSV target protein, despite the presence of the IL10M. This data is shown in Figure 11.

Example 13: Structural conformation of the antibody cytokine engrafted protein results in differential activity across cell types [00250] Antibody cytokine engrafted proteins (e.g. IgGIL10M13) incorporates monomeric IL10 (SEQ ID NO:209) into the Light Chain CDR 1 of an antibody (Figure 2).

Insertion of a 6 amino acid glycine-serine linker between helices D and E of IL10 renders the normally heterodimeric molecule incapable of domain swapping dimerization. As such, engrafting IL10M into an antibody results in an antibody cytokine engrafted protein with 2 monomeric IL10 molecules. However, due to flexibility of the antibody Fab arms, the angle and distance between the IL10 monomers is not fixed, as in the wild-type IL10 dimer, thus affecting its interaction with the IL10R1/R2 receptor complex. This is shown graphically in Figure 12. Specifically, due to antibody engraftment, the angle of the engrafted IL10 dimer is larger and variable, rendering signal transduction less efficient on cells with lower expression levels of ILlORland R2 as found on the pro-inflammatory cell types such as CD4 and CD8 T cells, B cells and NK cells. In contrast, antibody cytokine engrafted proteins signal more efficiently on cells with high IL-10R1 and R2 expression such as monocytes. A class average negative stain EM study of IgGIL10M13 highlighted the additional flexibility and wider angle between monomers, confirming that the geometry is altered compared to rhILlO. The less restricted geometry of the IL10 dimer in IgGIL10M13 alters its interaction with IL10R complex. As a consequence, the structure of the IgGIL10M13 antibody cytokine engrafted protein results in the biological effect of only producing a productive signal on cell types with high levels of IL10R1 and R2 expression.

Example 14: Crystal structure of IgGIL10M13

[00251] The IgGILl 0M13 Fab was concentrated to 16.2 mg/ml in 20 mM HEPES pH

8.0, 150 mM NaCl and used directly in hanging drop vapor diffusion crystallization trials.

Crystallization screens were setup by mixing 0.2 μΐ of protein solution with 0.2 μΐ of reservoir solution and equilibrated against 50 μΐ of the same reservoir solutions. Crystals for data collection appeared after 3-4 weeks at 20°C from a reservoir solution of 20% PEG3350, 200 mM magnesium acetate, pH 7.9. Prior to data collection, the crystals were soaked in reservoir solution supplemented with 20% ethylene glycol and flash cooled in liquid nitrogen.

Diffraction data were collected at the ALS beamline 5.0.3 with an ADSC Quantum 315R detector. Data was indexed and scaled using the HKL2000 software package (Otwinowski and Minor. (1997) Methods in Enzymology, Volume 276: Macromolecular Crystallography, part A, p.307-326). The data for the IgGIL10M13 Fab was processed to 2.40 A in space group P2i with cell dimensions a = 80.6 A, b = 104.7 A, c = 82.8 A, alpha = 90°, beta = 115.3°, gamma = 90°. The structure was solved by molecular replacement using PHASER (McCoy et al, (2007) J. Appl. Cryst. 40:658-674) with the palivizumab Fab structure (PDB code: 2HWZ) and monomeric IL10 structure (PDB Code: 1LK3 chain A) as search models. The top molecular replacement solution contained 2 molecules of the IgGIL10M13 Fab in the asymmetric unit. The final model was built in COOT (Emsley & Cowtan (2004) Acta Cryst. D60:2126-2132) and refined with PHENIX (Adams et al, (2010) Acta Cryst. D66, 213-221). The R _work and R _free values are 18.8% and 23.9% respectively with root-mean- square (r.m.s) deviation values from ideal bond lengths and bond angles were 0.005 A and 0.882° respectively.

Overall Structure

[00252] The IgGIL10M13 Fab crystallized with 2 molecules in the asymmetric unit, both with similar conformations. The electron density maps were similar for both molecules. The overall structure (Figure 13 A) shows that the Fab and grafted monomeric IL10 (IL10M) can adopt a collinear arrangement (Fab light chain in white, Fab heavy chain in black, IL10M in dark grey). Figure 13B shows a closer view of the grafting point in CDR-L1. The three flanking CDR residues are show with dark grey sticks. Dashed lines illustrate portions of the structure which could not be fit in the model due to missing electron density, presumably due to structural flexibility in these areas. The two areas include 6 residues at N-terminus of IL10M just after the grafting point and 8 residues between helices 4 and 5 in IL10M which encompass the inserted 6 residue linker (GGGSGG) (SEQ ID NO:251). There are also 3 pairs of hydrogen bonding interactions between the grafted IL10M molecule and portions of the Fab heavy chain (Figure 13C). These include R138 and N104 (sidechain), R135 and D56 (sidechain), and N38 and K58 (backbone/sidechain).

MATERIALS AND METHODS

Anti-inflammatory assays (LPS-challenge human whole blood)

[00253] IL10 antibody cytokine engrafted proteins were prepared at lOx at lOOOng/ml in assay medium (RPMI 1640 with glutamine (Hyclone), 10% heat inactivated FBS (Omega Scientific), 1% Penicillin/Streptomycin (Gibco), 50uM 2-mercaptoethonal (Gibco), lOmM Hepes ph 7.4 (Hyclone), O. lmM Non-essential Amino Acids (Hyclone), ImM Sodium Pyruvate (Hyclone) and lx human insulin/transferrin/selenium (Gibco). Each lOx stock of IL10 antibody cytokine engrafted protein in assay medium was serially diluted 1 :3 to create an 11 -point dose titration.

[00254] Human whole blood was diluted to 90% in assay medium and gently mixed.

Unstimulated diluted whole blood was plated in n=3 wells (45μ1Λνβ11), and assay medium added (5ul/well) to bring final well volume to 50μ1. Lipopolysaccharide (LPS, Invivogen stock 100μg/ml) was spiked into diluted whole blood at 220ng/ml and gently mixed. LPS spiked whole blood was then plated, IL10 proteins added to each respective well, then gently mixed and incubated for -20 hours in a 37C incubator, 5% C02. Plates were then gently mixed and centrifuged at 1400rpm for 5 minutes at room temperature and supernatant harvested by transferring ΙΟμΙ/well from assay plate to a proxy plate for TNFa detection using the Human TNF-alpha HTRF kit (Cisbio®) according to manufacturer's instructions, and results compared to a standard curve generated using manufacturer provided control.

Pro-inflammatory assays (human primary CD8 T-cell activation)

[00255] Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from the Blutspende Zentrum Basel. Eight Leucosep tubes (Greiner, 227290) per buffy coat were each filled with 15 ml Ficoll-Paque PLUS® (GE Healthcare, 17-1440-03) and centrifuged (lminute, 1000 g, 20°C). Buffy coats were diluted 1 :4 in phosphate buffered saline (PBS) pH 7.4 (Gibco, 10010-015) and 35 ml were overlaid onto each Ficoll gradient. After centrifugation (800 g, 20 min, 20°C) the lymphocytes separate into a white cell layer. This cell layer was transferred to fresh tubes and washed with PBS. Erythrocytes were lysed by resuspending cell pellets in 2 ml Gey's red blood lysing buffer (155 mM NH ₄ C1, 10 mM KHC0 ₃ , 0.1 mM EDTA) per tube. After incubation for 5 min, the cells were washed twice with PBS. After the last wash, the PBMCs were resuspended in T cell medium (TCM). TCM contains RPMI 1640 (Gibco, 21875-034), 10% fetal bovine serum (Gibco, 10082147), 1% Penicillin-Streptomycin (Gibco, 15070063) 2 mM GlutaMax (Gibco, 35050-038) and 50 U/ml penicillin, 50 μg/ml streptomycin. PBMCs were filtered through 40 μπι cell strainers (BD Falcon, 734-0002) to obtain single cell suspensions, and counted. CD8 ⁺ T cells were purified from PBMCs using the human CD8 ⁺ T cell enrichment kit (StemCell, 19053) according to the manufacturer's protocol for the Big Easy Magnet® (StemCell, 18001). After isolation, the cells were washed, counted and resuspended in TCM at a concentration of 1.8x10 ⁶ cells/ml. The anti-CD3/CD28 coated plate (see section 2.1.1) was washed once with 2 ml TCM per well followed by the addition of 1.8x10 ⁶ CD8 ⁺ T cells in 1 ml per well. Plates were centrifuged (5 min, 520 g, 20°C) and incubated for 3 days in a cell incubator (Binder) at 37° C, 5% C0 ₂ , 95% humidity

Stimulation with IL10 antibody cytokine engrafted proteins

[00256] After three days of incubation, activated CD8+ T cells were pooled from the 24 well plates and washed once with TCM. The cells were counted, resuspended in TCM at 3x10 ⁶ cells per ml and 300,000 cells per well in 100 μΐ were added into Nunclon Delta Surface® 96 well round bottom plate (Thermo Scientific, 163320). IL10 antibody cytokine engrafted protein pre-dilutions at double the final concentration were prepared in TCM in a separate plate at the following concentrations: 40, 4, 0.4, 0.04, 0.004 and 0 nM. 100 μΐ of these pre-dilutions were added in duplicates to the 100 μΐ cell suspension resulting in the final concentration of the antibody cytokine engrafted proteins of 20, 2, 0.2, 0.02, 0.002 and 0 nM. Plates were incubated at 37° C for 48 h

PMA and anti-CD3 stimulation and Golgistop

[00257] After the incubation period, the plates were centrifuged (2 min, 970 g, 20°C) and the supernatant was discarded. Cell pellets were resuspended in 200 μΐ TCM containing 2 ng/ml Phorbol-myristate-acetate (PMA, Sigma Aldrich, 79346), 2.5 μ /ηι1 anti-CD3 (BD, 555336) and 1 : 1500 GolgiStop (BD, 554724). Plates were incubated for 5 h at 37°C to restimulate the cells. Afterwards, plates were centrifuged (2 minutes, 970 g, 20°C), supernatants discarded and 200 μΐ ice cold PBS was added to wash the cell pellet. After centrifugation (2 minutes, 970 g, 20°C), PBS was removed and the cells were reuspended in 200 μΐ ice cold 200 mM Tris-HCl buffer (pH 8.1) containing 2% Triton X-100 (Serva, 39795) to lyse the cells. The plates were left for 15 min on ice and centrifuged (10 min, 970 g, 4°C) to eliminate debris. The supernatant was carefully transferred into a new 96 well plate and frozen for storage. To detect interferon-T (IFN-τ in the supernatant, the human IFN-τ DuoSet® ELISA (R&D systems, DY285) was used according to the manufacturer's protocol. The supernatants obtained as described in section 2.1.5 were diluted 1 :2 in the recommended assay diluent. To detect Granzyme B (GrzB) in the supernatant, the human Granzyme B® ELISA (Mabtech, 3485-1H- 20) was used according to the manufacturer's protocol. The supernatants were diluted 1 :50 in the recommended assay diluent. The optical density (OD) of the ELISA plates were acquired by SpectraMax® 340PC plate reader (Molecular Devices) and converted to concentrations by SoftMax® Pro software according to the automatically generated standard curve. The data were exported to Microsoft Excel where the concentrations were back calculated regarding their dilution factor.

IL10 dependent signaling

[00258] CyTOF is a FACS/Mass spectrometry technique to assess the activation of multiple cell populations by a single agent simultaneously. Antibody cytokine engrafted protein was incubated with human whole blood 20 minutes. Post-stimulation, PBMCs were treated with metal-conjugated antibodies against cell specific surface receptors CD14, HLA-DR, CD4, CD8, CD 19, CD56 and the signaling marker pSTAT3 and analyzed by CyTOF. Results indicate that IgGIL10M13 at all doses primarily activated monocyte and macrophage cell populations with little activation of T cell, B cell, NK cell and dendritic cell populations. Conversely, rhILlO activated all cell populations tested to some extent.

Pharmacokinetics Evaluation

[00259] Half-life of the antibody cytokine engrafted proteins was assessed in C57B1/6 mice. Antibody cytokine engrafted proteins were injected at 0.2mg/kg (lOml/kg dose volume) in 0.9% saline subcutaneously and blood was sampled beginning at lhour post- injection and up to 144 hours post-injection. Whole blood was collected into heparin-treated tubes at each time point and centrifuged at 12,500rpm for 10 minutes at 4°C. Plasma supernatant was collected and stored at -80°C until all time points were collected. Antibody cytokine engrafted proteins levels in plasma were measured using two different immunoassay methods to enable detection of both the IL10 and antibody domains of the antibody cytokine engrafted protein. The first method utilized a commercially available lL-10 ELISA kit employed as recommended (BD OptEIA® Human IL10 ELISA Set, Capture: rhlL 10/ Detection: biotin-rhlLlO). The second consisted of an IL10 based capture and Fc-based detect immunoassay run on a GyroLab® xP Workstation (Gyros AB Uppsala, Sweden). Specifically, the reagents employed consisted of Biotinylated Human IL10 capture (R&D Systems BAF217) and Alexafluor 647 goat anti-human IgG, Fey specific detection (Jackson ImmunoResearch #109-605-098). The assay was run on 200nL CDs (Gyros #P0004180) using a Gyros-approved wizard method. The buffers used were Rexxip A® (Gyros #P0004820) for standard and sample dilution and Rexxip F® (Gyros #P0004825) for detection preparation. Analysis of results was done using the Gyrolab® data analysis software. Pharmacodynamics Evaluation

[00260] Consistent with the extended half-life, antibody cytokine engrafted proteins also demonstrated improved pharmacodynamics. Phospho-stat3 (pSTAT3), a marker of IL10R activation was monitored in target tissues (blood and colon) after subcutaneous dosing.

Antibody cytokine engrafted proteins were injected at 0.2mg/kg (lOml/kg dose volume) in 0.9% saline subcutaneously. Terminal whole blood and colon tissue (2cm at ileo-cecal junction) were harvested beginning at 1 hour post- injection and up to 144 hours post- injection. Whole blood was collected into heparin-treated tubes containing lx phosphatase inhibitors (Pierce Halt Phosphatase Inhibitor Cocktail) and kept on ice until phospho-Stat3 assay. Colon tissue was collected in tubes containing cold PBS and lx phosphatase inhibitors. Once all tissue was collected for the time point, colon tissue was transferred into tubes containing a steel bead and 700 μΐ of Complete Cell Lysis Buffer containing PBS with 10 μΜ DTT, lx protease inhibitor cocktail, lOx phosphatase inhibitor cocktail and lOx cell lysis buffer (Active Motif Nuclear Extraction Kit). Colon tissue was homogenized by tissue-lyser at 30rps for 5 minutes at room temperature. Lysed tissue was centrifuged for 10 minutes at 14,000xg at 4°C. Supernatant was collected and stored on ice until phospho-STAT3 assay.

[00261] A phospho-Stat3 assay plate (Meso Scale Discovery® pSTAT3(Tyr705) Assay) was run on the same day as whole blood and colon tissue collection and processing. Ice-cold whole blood was lysed using the provided lx MSD Lysis Buffer containing lx Phosphatase Inhibitor 2, lx Phosphatase Inhibitor 3 and lx Protease Inhibitor. Each tube of whole blood was centrifuged at 12,500rpm for 10 minutes at 4°C. Plasma was collected and discarded. Pelleted whole blood was resuspended in 220 μΐ of MSD Lysis Buffer + Inhibitors and vortexed thoroughly. Lysed whole blood was plated into respective wells of the phospho-STAT3 assay plate at 50 μΐ/well. Colon tissue supernatant protein detection was performed using the Bradford Assay (Pierce). Colon protein was then plated on the phospho- STAT3 assay plate at 50 μΐ/well. Plates were incubated at room temperature for 2 hours, washed, and treated with phospho- STAT3 or Total STAT3 antibody (Meso Scale Discovery). Plates were analyzed for relative fluorescence units (RFU) on the MSD Sector Imager 2400 (Meso Scale Discovery). Whole blood phospho-STAT3 RFU was normalized to total STAT3 RFU. Colon protein phospho- STAT3 RFU was normalized to loaded protein concentration. Enhanced pSTAT3 signal is detected in both tissues at least up to 72 hours post-dose, and absent by 144 hours post-dose. See Figure 5, not shown. This profile is a dramatic improvement over rhILlO, whose signal is absent by 24 hours post-dose.

Ex vivo efficacy [00262] A direct comparison of efficacy for TNFa inhibition after LPS challenge was performed. In the assay, C57/B16 mice were dosed subcutaneously with vehicle, 0.2mg/kg of IgGIL10M13 (lOml/kg dose volume), or equimolar levels of rhlLlO. Whole blood was collected prior to, at 1.5 hrs post-dose and up to 144 hrs post-dose. Whole blood was collected into heparin-treated tubes. Prior to blood collection, assay medium was prepared. Assay medium contained RPMI 1640 with glutamine (Hy clone) with 10% heat inactivated FBS (Omega Scientific), 1% Penicillin/Streptomycin (Gibco), 50 μΜ 2-mercaptoethonal (Gibco), lOmM Hepes ph 7.4 (Hyclone), O. lmM Non-essential Amino Acids (Hyclone) and ImM Sodium Pyruvate (Hyclone). Mouse whole blood was plated in 25 μΐ/well/replicate/mouse on a 384 well plate. Assay medium was added to unstimulated control wells (25μ1Λνβ11) to bring final well volume to 50 μΐ. For LPS challenge, LPS (Invivogen, stock 100 μg/ml) was spiked into assay medium at 200 ng/ml [100 ng/ml final in assay] and gently mixed. LPS spiked BCM was then plated at 25 μΐ/well for each required well containing mouse whole blood. The plate was gently mixed and incubated for 21 hours in a 37°C incubator, 5% C02. The next day, the assay plate was centrifuged at 1400 rpm for 5 minutes at room temperature. Supernatants from each well were collected and frozen at -80°C until all time points had been assayed. Once all time points could be analysed, supernatants were plated onto an MSD V-plex® Mouse Pro-Inflammatory Cytokine Assay Plate (Meso Scale Discovery). Plates were analysed for TNFa (pg/ml) on the MSD Sector Imager 2400® (Meso Scale Discovery)

Immunostimulatory assays

MC/9 cell proliferation

[00263] The immunostimulatory activity of IL10 antibody cytokine engrafted proteins were assessed by examining the ability to stimulate MC/9 (a mast cell line derived from mouse fetal liver) cell proliferation. First, IL10 antibody cytokine engrafted proteins were diluted in a separate 384 well plate at 7X in growth media with no T-STIM (7X = 700 ng/ml). IL-4 was included as a positive control (7X = 1,000 ng/ml, [final] = 143 ng/ml). Second, 30μ1 of

1.67X10 ⁴ cells/ml MC/9 cells were plated in a white 384- well TC treated plate (500 cells/well) in media with no T-STIM (washing cells prior to assay was not necessary). Third, 5 μΐ of diluted IL10 antibody cytokine engrafted protein was added to the cells. Plate was briefly mixed using a plate mixer, and then pulse spun at 1,000 rpm. Plate was covered with custom porous lid to aerate cells. Fourth, plate was incubated at 37°C for 72 hours. Fifth, 30 μΐ of Promega Cell Titer Glo® was added to the cells, incubated for 10 min at room temp and read on Envision® (0.1 sec read).

B cell proliferation

[00264] PBMCs were isolated from human buffy coats as described above. For the isolation of B cells, the human B cell enrichment kit® (Stemcell, 19054) was used according to manufacturer's protocol for the Big Easy Magnet® (Stemcell, 18001). After isolation, the B cells were washed with B cell medium (BCM), counted and resuspended at a concentration of 4x10 ⁵ cells/ml. BCM contains RPMI 1640, 10% fetal bovine serum (Gibco, 10082147), 1% Penicillin- Streptomycin (Gibco, 15070063) and 2 mM GlutaMax (Gibco, 35050-038), 1% Non-essential amino acids (Gibco, 11140035) and lmM sodium pyruvate (Gibco, 11360-039), 50 U/ml penicillin and 50 μg/ml streptomycin. Isolated B cells were counted and resuspended at a concentration of 4xl0 ⁵ cells/ml in BCM. To stimulate the B cells, 8.4 μg/ml anti-CD40 and 40 U/ml recombinant human IL-2 (R&D systems, 202-IL-50) were added to the suspension. B cells were then added at a concentration of 4x10 ⁴ cells in 100 μΐ into wells of Nunclon Delta Surface® 96 well round bottom plates (Thermo Scientific, 163320). IL10 antibody engrafted protein predictions at the double the final concentration were prepared in a separate plate. A titration of the compounds was performed obtaining the following concentrations: 40, 4, 0.4, 0.04, 0.004 and 0 nM. 100 μΐ of these pre-dilutions were added in duplicates to the ΙΟΟμΙ cell suspension resulting in the final concentration of the compounds of 20, 2, 0.2, 0.02, 0.002 and 0 nM and a final concentration of 4.2 mg/ml anti-CD40 and 20 U/ml recombinant IL-2. The plate was incubated for 5 days in a cell culture incubator (37° C, 5% C0 ₂ , 95% humidity). After 5 days of incubation, proliferation of B cells was determined by the thymidine incorporation assay. Cells were pulsed with 0.5 μθΐ 3H-thymidine (ANAWA, ART- 178) per well in 20 μΐ BCM for the final 16 h of the culture period at 37° C. Using the TomTec 9600® harvester, cells were harvested onto a membrane according to manufacturer's protocol. Membranes were sealed in a bag, scintillation liquid was added and radioactivity was measured on scintillation counter. For each of the antibody engrafted proteins, the percentage of proliferation at 20 nM ("top") was calculated relative to the proliferation at an equimolar concentration of rhlLlO, in relation to the respective background cytokine production ("bottom"). The resulting % of max values were calculated using the following formula:

% of max = (top _C o _m pound-bottom _compound )/(top _r hiL-io-bottom _rh iL-io) * 100 Means of different experimental series were determined, and the standard error of the mean (SEM) was calculated if the compound was measured on more than two donors.

[00265] It is understood that the examples and embodiments described herein are for illustrative purposes and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.