RU2795457C2 - Anti-gdf15 antibodies, compositions and methods of use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antibody and its antigen-binding fragment that specifically bind to GDF15 are proposed. Also nucleic acids, an expression vector and a host cell for obtaining the said antibody or fragment thereof are proposed. The invention also relates to the use of antibodies against GDF15 to reduce the level of free GDF15 or reduce the activity of GDF15 in a patient, as well as in methods of treating cancer. Including the use of a combination of an anti-GDF15 antibody of the invention and a PD-1 axis binding antagonist or an anti-CD40 antagonist antibody for the treatment of cancer.

EFFECT: invention provides new potential therapeutic anti-GDF15 antibodies.

36 cl, 34 dwg, 24 tbl, 21 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind growth and differentiation factor 15 (GDF15) and compositions, methods and uses thereof, including the use of a combination of an anti-GDF15 antibody of the invention and a PD-1 axis binding antagonist and/or inhibitor CD40 for cancer treatment.

BACKGROUND OF THE INVENTION

GDF15, also known as macrophage inhibitory cytokine 1 (MIC-1), prostate-derived factor (PDF), placental bone morphogenetic protein (PLAB), NSAID-activated gene 1 (NAG-1), and placental activating growth factor β (PTGFB) , is a 12 kDa secreted protein that forms a 25 kDa disulfide-linked homodimer that is a member of the transforming growth factor beta (TGFβ) superfamily. Generally, GDF15 is poorly expressed or not expressed at all in tissues and plasma concentrations are low. GDF15 expression is upregulated during inflammation and malignancy, limiting inflammation and tumor growth. This increased expression results in markedly elevated circulating GDF15 concentrations (>1-100 ng/mL) in cancers (eg, prostate, pancreas, colon, and stomach), heart failure, chronic kidney disease (CKD), sarcopenia, and chronic obstructive pulmonary disease. lung disease (COPD).

GDF15-related weight loss has been shown in preclinical models. Exogenous administration of GDF15 reduces food intake and body weight under physiological and pathophysiological conditions. Elevated plasma GDF15 is associated with weight loss in cancer patients with cachexia (WO2005/099746; WO2009/021293, WO2014/100689, WO2016/049470). Although the evidence is strongest in cancer patients, an association between GDF15 and weight loss in heart failure-associated cachexia has also been reported (WO/2015/196142). In accordance with human data, elevated plasma levels of GDF15 are associated with cachexia in mouse tumor models. In addition, numerous studies using multivariate analysis have identified GDF15 as an independent predictive biomarker associated with poor survival in many types of cancer (eg, NSCLC, pancreatic, and sarcoma, among others), as well as in heart failure, CKD, and COPD.

More recently, in October 2017, it was reported that the activity of GDF15, such as its metabolic effects, is mediated by the binding of GDF15 to its cognate receptor, the GDNF family α-like receptor (GFRAL), an orphan member of the GFR-α family. See, for example, Hsu et al., 2017, Nature 550:255-259; Yang et al., 2017, Nature Med. 23(10): 1158; and Emmerson et al., 2017, Nature Med. 23(10):1215. These studies demonstrated that binding of GDF15 to GFRAL activates a GFRAL-mediated signaling pathway whereby the receptor tyrosine kinase, RET, is activated and acts as a co-receptor for GFRAL, and RET in turn mediates the subsequent phosphorylation of ERK (pERK), the S6 ribosomal protein (pS6 ), AKT, MAPK, and phospholipase C gamma 1 (PLC-γ1), among others. In addition, these studies demonstrated that GFRAL activation by GDF15 occurs in areas of the brainstem, posterior field and nucleus solitarius that contain chemosensory neurons and neuropeptide receptors that control appetite and vomiting. The posteriormost field senses chemical messengers in the blood and controls the autonomic physiological systems, including those that control metabolism and appetite. In addition, these brainstem regions are outside the blood-brain barrier (BBB), making them accessible to, among other things, large molecules, including antibodies that can bind GFRAL or GDF15 and prevent GFRAL-GDF15 interaction to modulate the metabolic effects of GDF15 associated with appetite, body weight, weight, fat mass and food intake. Thus, the GDF15-GFRAL pathway present in the brainstem is a potential target for the modulation of diseases, conditions and disorders mediated by GDF15 activity.

There continues to be a significant need for possible treatments for weight loss caused by or associated with cachexia that is mediated by or associated with elevated levels of GDF15. The present invention provides new potential therapeutic antibodies that meet this need.

PD-L1 (programmed death ligand 1; also known as CD274 and B7 homologue 1 [B7-H1]) is overexpressed in many cancers and is often associated with poor prognosis (Okazaki T et al., Intern. Immun. 2007 19(7) :813) (Thompson RH et al., Cancer Res 2006, 66(7):3381). Interestingly, most tumor-infiltrating T lymphocytes preferentially express PD-1, in contrast to T lymphocytes in normal tissues and peripheral blood. PD-1 (programmed cell death protein 1), a PD-L1 cognate receptor on tumor reactive T cells, may contribute to the disruption of antitumor immune responses (Ahmadzadeh et al, Blood 2009 1 14(8): 1537). This may be due to the use of PD-L1 signaling mediated by tumor cells expressing PD-L1 interacting with T cells expressing PD-1, resulting in attenuated T cell activation and evasion of immune surveillance (Sharpe et al., Nat Rev 2002) (Keir ME et al., 2008 Annu. Rev. Immunol. 26:677). Therefore, inhibition of the interaction of PD-L1/PD-1 and the signaling pathway (also referred to as the "PD-1 axis") may enhance CD8+ T cell-mediated tumor killing.

Inhibition of PD-1 axis signaling through its direct ligands (e.g., PD-L1, PD-L2 [programmed cell death ligand 2 1 and B7-DC]) has been proposed to enhance T cell immunity for cancer treatment (e.g., tumor immunity) . Moreover, a similar enhancement of T cell immunity was observed when PD-L1 was inhibited from binding to another binding partner, ie B7-1 (also known as CD80).

There are currently at least five PD-1 axis binding antagonists approved by the FDA for more than 10 cancer indications (A Ribas et al, Science, 359, 1350-1355, 2018), as well as others known in this the field of technology. These include nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), spartalizumab, pidilizumab, tisselizumab, AMP-224, AMP-514, cemiplimab, PF-06801591 (sasanlimab, RN888), each of which is an anti-PD-1 antibody, while while each of avelumab (BAVENCIO), atezolizumab (TECENTRIQ), durvalumab (IMFINZI), BMS-936559 (MDX-1105), MEDI4736, MPDL3280A (YW243.55.s70) is an anti-PD-L1 antibody.

Combination therapy with a PD-1 axis binding antagonist with one or more anticancer agents has been investigated, with the first clinical trial beginning in 2009. The number of new clinical trials targeting such combinations has increased dramatically; since then, 467 new studies have been registered in 2017. (C. Schmidt, Nature, Vol 552, 21/28 December 2017). Although combination therapy with nivolumab (anti-PD-1) and ipilimumab (anti-CTLA-4) for the treatment of melanoma, as well as combination therapy with pembrolizumab (anti-PD-1) with chemotherapy for the treatment of non-small cell lung cancer was approved by the FDA in 2015 and 2017 Accordingly, there has been an ongoing need to find an optimal therapeutic treatment that combines a PD-1 axis binding antagonist with one or more other anticancer agents to treat, stabilize, prevent and/or delay the development of various cancers.

GDF15 has been shown to be induced by multiple pro-inflammatory factors and lipopolysaccharide (LPS) and is involved in a feedback mechanism to impair macrophage activation by suppressing tumor necrosis factor alpha (TNFα) production via inhibition of the NF-kB signaling pathway (Bootcov et al., 1997, PNAS 94(21): 11514-11519, Ratnam et al., 2017, J. Clin Invest 127(10): 3796-3809). Reduced TNFα expression is associated with a shift in the macrophage population towards the pro-oncogenic M2 phenotype (Kratochvill, 2015, Cell Reports 12(11): 1902-1914). Targeting the M2 phenotype in tumor-associated macrophages is a potential strategy to enhance response to cancer therapies focused on activating the host's immune response.

There continues to be a significant need for cancer treatment options, especially for solid tumors. The present invention provides new potential therapeutic GDF15 antibodies with or without one or more other anticancer agents that meet these needs. There also remains a need to find an optimal therapeutic treatment that combines a PD-1 axis binding antagonist with another therapeutic agent, such as a GDF15 inhibitor, with or without one or more other anti-cancer agents, to treat, stabilize, prevent and/or delay the development of various types of cancer. The present invention provides novel potentially useful therapeutic combinations of the GDF15 antibodies of the invention with a PD-1 axis binding antagonist that meet this need.

SUMMARY OF THE INVENTION

The invention provides antibodies and antigen-binding fragments thereof that specifically bind to GDF15, as well as uses and methods associated therewith. Those skilled in the art will understand, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following embodiments (E).

E1. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to GDF15.

E2. An antibody or antigen-binding fragment thereof, according to E1, containing HCDR-1, HCDR-2 and HCDR-3 sequences according to one of the group consisting of SEQ ID NO:21, 34, 44, 53, 60, 68, 73, 80, 86 , 93, 99, 106, 112, 120, 127, 136, 142, 148, 155, 161 and 166.

E3. An antibody or antigen-binding fragment thereof, according to any one of E1-E2, containing LCDR-1LCDR-1, LCDR-2 and LCDR-3 sequences according to one of the group consisting of SEQ ID NOS: 11, 30, 39, 49, 56, 64 , 71, 77, 83, 90, 96, 103, 109, 115, 123, 131, 139, 144, 151, 158 and 163.

E4. An antibody or antigen-binding fragment thereof, according to any one of E1-E3, containing one or more of (a)-(f)

(a) an LCDR-1LCDR-1 selected from the group consisting of SEQ ID NOs: 7, 27, 36, 46, 55, 62, 82, 88, 95, 101, 129, 138, 150 and 157,

(b) LCDR-2 selected from the group consisting of SEQ ID NOS: 8, 28, 37, 47, 70, 108, 114, 122 and 130,

(c) an LCDR-3 selected from the group consisting of SEQ ID NOS: 9, 29, 38, 48, 63, 76, 89 and 102,

(d) HCDR-1 selected from the group consisting of SEQ ID NOS: 17, 32, 41, 58, 66, 117, 125, 133 and 153,

(e) HCDR-2 selected from the group consisting of SEQ ID NOS: 18, 33, 42, 51, 59, 67, 85, 92, 98, 105, 118, 126, 134, 141, 146 and 165,

(f) HCDR-3 selected from the group consisting of SEQ ID NOS: 1, 19, 43, 52, 79, 111, 119, 135, 147, 154 and 160.

E5. An antibody or antigen-binding fragment thereof, at E1-E4 containing: i) HCDR-1 containing the amino acid sequence GYTFX ₁ X ₂ YNID, where X ₁ is S or T and X ₂ is S or D; ii) HCDR-2 containing the amino acid sequence X ₃ INPX ₄ X ₅ GX ₆ AX ₇ X ₈ X ₉ QKFQG, where X ₃ is G or Q; X ₄ is I or N; X ₅ is F or N; X ₆ is T or L; X ₇ is F or N; X ₈ is Y or F and X ₉ is N or A; and iii) HCDR-3 containing the amino acid sequence EX ₁₀ ITTX ₁₁ GAMDX ₁₂ where X ₁₀ is A or Q; X ₁₁ is V or I; and X ₁₂ is H or Y.

E6. An antibody or antigen-binding fragment thereof, according to E1-E5, containing: i) LCDR-1LCDR-1 containing the amino acid sequence RX ₁ SQX ₂ X ₃ X ₄ X ₅ YLA, where X ₁ is T or A, X ₂ is S or N , X ₃ is V or L, X ₄ is H or S and X ₅ is N or S; ii) LCDR-2 containing the amino acid sequence DAX ₆ X ₇ RAX ₈ where X ₆ is S or K; X ₇ is T or N; and X ₈ is D or T; and iii) LCDR-3 containing the amino acid sequence QQFX ₉ X ₁₀ X ₁₁ PX ₁₂ T, where X ₉ is W or S; X ₁₀ is S or N; X ₁₁ is W or D; and X ₁₂ is W or Y.

E7. An antibody or antigen-binding fragment thereof, according to any of E1-E6, containing one or more of the following:

(a) LCDR-1LCDR-1 containing the amino acid sequence of SEQ ID NO:174,

(b) LCDR-2 containing the amino acid sequence of SEQ ID NO:175,

(c) an LCDR-3 containing the amino acid sequence of SEQ ID NO:176,

(d) HCDR-1 containing the amino acid sequence of SEQ ID NO:171,

(e) HCDR-2 containing the amino acid sequence of SEQ ID NO:172,

(f) HCDR-3 containing the amino acid sequence of SEQ ID NO:173.

E8. An antibody or antigen-binding fragment thereof, according to any one of E1-E7, containing HCDR-1, HCDR-2 and HCDR-3 sequences from one of at least one sequence selected from the group consisting of SEQ ID NO:34, 106 , 148, 155 and 166.

E9. An antibody or antigen-binding fragment thereof, according to any one of E1-E8, containing LCDR-1LCDR-1, LCDR-2 and LCDR-3 sequences according to one of at least one sequence selected from the group consisting of SEQ ID NO:30 , 103, 144, 151 and 163.

E10. An antibody or antigen-binding fragment thereof, according to any one of E1-E9, containing one or more of (a)-(f):

(a) LCDR-1LCDR-1 selected from the group consisting of SEQ ID NOS: 27, 88, 95, 101 and 150.

(b) LCDR-2 selected from the group consisting of SEQ ID NOS: 8, 28 and 108.

(c) LCDR-3 selected from the group consisting of SEQ ID NOS: 9, 29, 38, 48 and 102.

(d) HCDR-1 selected from the group consisting of SEQ ID NOS: 32, 41 and 153.

(e) HCDR-2 selected from the group consisting of SEQ ID NOS: 33, 105, 146 and 165.

(f) HCDR-3 selected from the group consisting of SEQ ID NOS: 19, 52, 147 and 154.

E11. An antibody or antigen-binding fragment thereof, according to any one of E1-E10, containing the HCDR-1, HCDR-2 and HCDR-3 sequences of SEQ ID NO:166.

E12. An antibody or antigen-binding fragment thereof, according to any one of E1-E11, containing LCDR-1LCDR-1, LCDR-2 and LCDR-3 sequences of SEQ ID NO:163.

E13. An antibody or antigen-binding fragment thereof, according to any of E1-E12, containing one or more of the following:

(a) an LCDR-1 containing the sequence of SEQ ID NO:95,

(b) LCDR-2LCDR-2 containing the sequence of SEQ ID NO:28,

(c) an LCDR-3 containing the sequence of SEQ ID NO:9,

(d) HCDR-1 containing the sequence of SEQ ID NO:32,

(e) HCDR-2 containing the sequence of SEQ ID NO:165, and

(f) HCDR-3 containing the sequence of SEQ ID NO:52.

E14. An antibody or antigen-binding fragment thereof, according to any of E1-E13, containing LCDR-1, containing the amino acid sequence of SEQ ID NO:95, LCDR-2LCDR-2, containing the amino acid sequence of SEQ ID NO:28, LCDR-3, containing the amino acid sequence of SEQ ID NO:9, HCDR-1 containing the amino acid sequence of SEQ ID NO:32, HCDR-2 containing the amino acid sequence of SEQ ID NO:165 and HCDR-3 containing the amino acid sequence of SEQ ID NO:52.

E15. An antibody or antigen-binding fragment thereof, according to any one of E1-E14, containing one or more of the following substitutions:

a) 1, 2, 3, 4, 5, or 6 substitutions in LCDR-1 of the corresponding human germline VL sequence residue,

b) 1, 2, 3, 4, or 5 substitutions in LCDR-2 of the corresponding human germline VL sequence residue,

c) 1, 2, 3, 4, 5, or 6 substitutions in LCDR-3 of the corresponding human germline VL sequence residue,

d) 1 substitution in HCDR-1 of the corresponding residue of the human germline type VH sequence,

f) 1, 2, 3, 4, 5, 6, 7, or 8 substitutions in HCDR-2 of the corresponding human germline VH sequence residue,

where the VL human germline sequence is selected from the group consisting of IGKV1-12*01, IGKV1-13*02, IGKV1-33*01, IGKV1-39*01, IGKV1-5*01, IGKV3-11*01, IGKV3- 15*01, IGKV3-20*01, IGKV3D-20*02 and IGKV4-1*01, and a human germline type VH sequence selected from the group consisting of IGHV1-2*02, IGHV1-3*01, IGHV1-46* 01, IGHV1-69*01, IGHV1-69*02, IGHV1-8*01, IGHV3-13*01, IGHV3-23*01, IGHV3-23*04, IGHV3-30*01, IGHV3-30*18, IGHV5-10-1*01, IGHV5-10-1*04 and IGHV5-51*01.

E16. An antibody or antigen-binding fragment thereof, according to any one of E1-E15, comprising a VH framework sequence derived from a human germline VH sequence selected from the group consisting of IGHV1-2*02, IGHV1-3*01, IGHV1-46*01, IGHV1-69*01, IGHV1-69*02, IGHV1-8*01, IGHV3-13*01, IGHV3-23*01, IGHV3-23*04, IGHV3-30*01, IGHV3-30*18, IGHV5- 10-1*01, IGHV5-10-1*04 and IGHV5-51*01.

E17. An antibody or antigen-binding fragment thereof, according to any of E1-E16, containing the IGHV1-69*01VH framework sequence.

E18. An antibody or antigen-binding fragment thereof, according to any one of E1-E17, comprising a VL framework sequence derived from a human germline VL sequence selected from the group consisting of IGKV1-12*01, IGKV1-13*02, IGKV1-33*01, IGKV1-39*01, IGKV1-5*01, IGKV3-11*01, IGKV3-15*01, IGKV3-20*01, IGKV3D-20*02 and IGKV4-1*01.

E19. An antibody or antigen-binding fragment thereof, according to any of E1-E18, containing an IGKV3-11*01VL framework sequence.

E20. An antibody or antigen-binding fragment thereof, according to any one of E1-E19, comprising a VL framework sequence and a VH framework sequence, and wherein the VL framework sequence is at least 72% identical to the human germline sequence from which it is derived.

E21. An antibody or antigen-binding fragment thereof, according to any one of E1-E20, containing a VL framework sequence and a VH framework sequence, and where the VL framework sequence is at least 72%, 74%, 75%, 77%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the human germline sequence from which it was derived.

E22. An antibody or antigen-binding fragment thereof, according to any one of E1-E21, comprising a VL framework sequence and a VH framework sequence, and wherein the VH framework sequence is at least 53% identical to the human germline sequence from which it is derived.

E23. An antibody or antigen-binding fragment thereof, according to any one of E1-E22, containing a VL framework sequence and a VH framework sequence, and where the VH framework sequence is at least 53%, 58%, 60%, 63%, 71%, 72% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the human germline sequence from which it was derived.

E24. An antibody or antigen-binding fragment thereof, according to any one of E1-E23, containing a VH containing an amino acid sequence at least 90% identical to SEQ ID NO:166.

E25. An antibody or antigen-binding fragment thereof, according to any one of E1-E24, containing a VH containing an amino acid sequence of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % identical to SEQ ID NO:166.

E26. An antibody or antigen-binding fragment thereof, according to any one of E1-E25, containing a VH containing the amino acid sequence of SEQ ID NO:166.

E27. An antibody or antigen-binding fragment thereof, according to any one of E1-E26, containing a VL containing an amino acid sequence at least 90% identical to SEQ ID NO:163.

E28. An antibody or antigen-binding fragment thereof, according to any one of E1-E27, containing a VL containing an amino acid sequence of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% % identical to SEQ ID NO:163.

E29. An antibody or antigen-binding fragment thereof, according to any one of E1-E28, containing a VL containing the amino acid sequence of SEQ ID NO:163.

E30. An antibody or antigen-binding fragment thereof, according to any one of E1-E29, containing a VH containing the amino acid sequence of SEQ ID NO:166 and a VL amino acid sequence of SEQ ID NO:163.

E31. An antibody or antigen-binding fragment thereof, according to any one of E1-E30, containing an Fc domain.

E32. An antibody or antigen-binding fragment thereof, according to E31, where the Fc domain is the Fc domain of IgA (eg, IgA ₁ or IgA ₂ ), IgD, IgE, IgM, or IgG (eg, IgG ₁ , IgG ₂ , IgG ₃ , or IgG ₄ ).

E33. An antibody or antigen-binding fragment thereof, according to E32, where the Fc domain is the Fc domain of IgG.

E34. An antibody or antigen-binding fragment thereof, according to E33, wherein the IgG is selected from the group consisting of IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ .

E35. An antibody or antigen-binding fragment thereof, according to E34, where IgG is IgG ₁ .

E36. An antibody or antigen-binding fragment thereof, according to any one of E1-E35, containing a heavy chain containing an amino acid sequence at least 90% identical to SEQ ID NO:164.

E37. An antibody or antigen-binding fragment thereof, according to any one of E1-E36, containing a heavy chain containing an amino acid sequence of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:164.

E38. An antibody or antigen-binding fragment thereof, according to any one of E1-E37, containing a heavy chain containing the amino acid sequence of SEQ ID NO:164.

E39. An antibody or antigen-binding fragment thereof, according to any one of E1-E38, containing an LC containing an amino acid sequence at least 90% identical to SEQ ID NO:162.

E40. An antibody or antigen-binding fragment thereof, according to any one of E1-E39, containing an LC containing an amino acid sequence of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:162

E41. An antibody or antigen-binding fragment thereof, according to any one of E1-E40, containing an LC containing the amino acid sequence of SEQ ID NO:162.

E42. An antibody or antigen-binding fragment thereof, according to any one of E1-E41, containing a heavy chain containing the amino acid sequence of SEQ ID NO:164 and a light chain containing the amino acid sequence of SEQ ID NO:162.

E43. An antibody or antigen-binding fragment thereof, according to any one of E1-E42, containing CDR1, CDR2, and CDR3 encoded by an insert in a plasmid deposited with ATCC with ATCC accession number PTA-125038.

E44. An antibody or antigen-binding fragment thereof, according to any one of E1-E43, containing CDR1, CDR2, and CDR3 encoded by an insert in a plasmid deposited with ATCC with ATCC accession number PTA-125039.

E45. An antibody or antigen-binding fragment thereof, according to any of E1-E44, encoded by an insert in a plasmid deposited with ATCC and bearing the ATCC accession number PTA-125038.

E46. An antibody or antigen-binding fragment thereof, according to any of E1-E45, encoded by an insert in a plasmid deposited with ATCC and bearing the ATCC accession number PTA-125039.

E47. An antibody or antigen-binding fragment thereof, according to any one of E1-E46, containing the amino acid sequence encoded by the insert in the plasmid deposited with ATCC and having the accession number ATCC PTA-125038, and the amino acid sequence encoded by the insert in the plasmid deposited with the ATCC and having the accession number ATCC PTA-125039.

E48. An antibody or antigen-binding fragment thereof, according to any one of E1-E47, wherein the antibody or antigen-binding fragment is an Fc fusion protein, monobody, maxibody, bifunctional antibody, scFab, scFv, peptibody.

E49. An antibody or antigen-binding fragment thereof, according to E1-E48, wherein the antibody or antigen-binding fragment thereof binds human GDF15 with a K _D of about or less than a value selected from the group consisting of about 10 nM, 5 nM, 2 nM, 1 nM, 900 pM , 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM and 10 pM.

E50. An antibody or antigen-binding fragment thereof, according to E1-E49, wherein the antibody or antigen-binding fragment thereof binds cynomolgus monkey GDF15 with a K _D of about or less than a value selected from the group consisting of about 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM, 15 pM, 13 pM, 10 pM and 9 pM.

E51. An antibody or antigen-binding fragment thereof, at E1-E50, wherein the antibody or antigen-binding fragment thereof binds cynomolgus monkey GDF15 with a K _D of about 8 pM or 9 pM.

E52. An antibody or antigen-binding fragment thereof, according to E1-E51, wherein the antibody or antigen-binding fragment thereof binds cynomolgus monkey GDF15 with a K _D of about 8.28 pM.

E53. An antibody or antigen-binding fragment thereof, according to any one of E1-E52, wherein the terminal half-life in humans is at least about 16 days.

E54. An antibody or antigen-binding fragment thereof, according to any one of E1-E53, wherein the terminal half-life in humans is at least 17 days.

E55. An antibody or antigen-binding fragment thereof, at E1-E54, wherein the predicted immunogenic potential of the antibody, as indicated by the corrected t-regitope score (tReg), is less than about -24.

E56. An antibody or antigen-binding fragment thereof, according to E1-E55, wherein the predicted immunogenic potential of the antibody, as indicated by the adjusted tReg score, is less than the adjusted tReg score selected from the group consisting of about -24, -26, -27, -30, - 32, -33, -34, -35, -36, -37, -38, -39, -40, -41, -42, -43, -50 and -51.

E57. An antibody, or antigen-binding fragment thereof, at E1-E56, wherein the predicted immunogenic potential of the antibody, as indicated by the corrected tReg score, is selected from the group consisting of about -26, -34, -36, -41, and -42.

E58. An antibody, or antigen-binding fragment thereof, at E1-E57, wherein the predicted immunogenic potential of the antibody, as indicated by the corrected tReg score, is about -41 or -42.

E59. An antibody or antigen-binding fragment thereof according to E1-E58, wherein the antibody or antigen-binding fragment has a viscosity selected from the group consisting of at least about 10 centipoise (cP), at least about 15 cP, at least about 20 cP of at least about 40 cP and at least about 70 cP, measured at 25°C.

E60. An antibody or antigen-binding fragment thereof according to E1-E59, wherein the antibody or antigen-binding fragment has a viscosity of about 20 centipoise measured at 25°C.

E61. An antibody or antigen-binding fragment thereof according to E1-E60, wherein the antibody or antigen-binding fragment has a viscosity of 20 centipoise measured at 25°C.

E62. An antibody or antigen-binding fragment thereof, according to E1-E61, wherein the ratio of K _D binding of the antibody or antigen-binding fragment to human GDF15 compared to binding to mouse GDF15 is between about 0.05 and about 0.10.

E63. An antibody or antigen-binding fragment thereof, according to E1-E62, wherein the K _D ratio of antibody or antigen-binding fragment binding to human GDF15 compared to binding to mouse GDF15 is about 0.07.

E64. An antibody or antigen-binding fragment thereof, according to E1-E63, wherein the K _D ratio of antibody or antigen-binding fragment binding to human GDF15 compared to binding to mouse GDF15 is 0.07.

E65. An antibody or antigen-binding fragment thereof, according to E1-E64, wherein the ratio of K _D binding of the antibody or antigen-binding fragment to human GDF15 compared to binding to cynomolgus monkey GDF15 is from about 1.0 to about 1.5.

E66. An antibody or antigen-binding fragment thereof, according to E1-E65, wherein the ratio of K _D binding of the antibody or antigen-binding fragment to human GDF15 compared to binding to cynomolgus monkey GDF15 is about 1.2.

E67. An antibody or antigen-binding fragment thereof, according to E1-E66, wherein the K _D ratio of antibody or antigen-binding fragment binding to human GDF15 compared to binding to cynomolgus monkey GDF15 is 1.21.

E68. An antibody or antigen-binding fragment thereof, according to E1-E67, wherein the _KD ratio of antibody or antigen-binding fragment binding to cynomolgus monkey GDF15 compared to binding to murine GDF15 is from about 0.03 to about 0.09.

E69. An antibody or antigen-binding fragment thereof, according to E1-E68, wherein the K _D ratio of antibody or antigen-binding fragment binding to cynomolgus monkey GDF15 compared to binding to mouse GDF15 is from about 0.04 to 0.08.

E70. An antibody or antigen-binding fragment thereof, according to E1-E69, wherein the K _D ratio of antibody or antigen-binding fragment binding to cynomolgus monkey GDF15 compared to binding to mouse GDF15 is from about 0.05 to 0.06.

E71. An antibody or antigen-binding fragment thereof, according to E1-E70, wherein the K _D ratio of antibody or antigen-binding fragment binding to cynomolgus monkey GDF15 compared to binding to mouse GDF15 is 0.05.

E72. An antibody according to E1-E71, wherein the antibody has thermal stability with a melting point (T _m 1) or a temperature at which the C _H 2 of the antibody is 50% unfolded, about 71° C. or more, as measured by differential scanning calorimetry.

E73. An antibody according to E1-E72, wherein the antibody has thermal stability with a melting point (T _m 1) or a temperature at which C _H 2 of the antibody is 50% unfolded, from 71°C to 72°C, as measured by differential scanning calorimetry.

E74. An antibody according to E1-E73, wherein the antibody has thermal stability with a melting point (T _m 1) or a temperature at which the C _H 2 of the antibody is 50% unfolded between 71°C and 72°C as measured by differential scanning calorimetry.

E75. The antibody of E1-E74, wherein the antibody has thermal stability with a melting point (T _m 2), or the temperature at which the Fab of the antibody is 50% unfolded, of about 80° C. or greater, as measured by differential scanning calorimetry.

E76. An antibody according to E1-E75, wherein the antibody has thermal stability with a melting point (T _m 2), or the temperature at which the Fab of the antibody is 50% unfolded, from 80°C to 86°C, as measured by differential scanning calorimetry.

E77. An antibody according to E1-E76, wherein the antibody has thermal stability with a melting point (T _m 2), or the temperature at which the Fab of the antibody is 50% unfolded, from 84°C to 85°C, as measured by differential scanning calorimetry.

E78. An antibody according to E1-E77, wherein the antibody has thermal stability with a melting point (T _m 3), or the temperature at which the C _H 3 of the antibody is 50% unfolded, of about 82° C. or greater, as measured by differential scanning calorimetry.

E79. An antibody according to E1-E78, wherein the antibody has thermal stability with a melting point (T _m 3), or the temperature at which the C _H 3 of the antibody is 50% unfolded, from 83°C to 91°C, as measured by differential scanning calorimetry.

E80. An antibody according to E1-79 wherein the antibody has thermal stability with a melting point (T _m 3), or the temperature at which the C _H 3 of the antibody is 50% unfolded, from 87°C to 89°C, as measured by differential scanning calorimetry.

E81. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof according to any one of E1-E80.

E82. An isolated nucleic acid molecule comprising at least one nucleic acid sequence encoding an antibody or antigen-binding fragment thereof, according to any one of E1-E81.

E83. An isolated nucleic acid molecule encoding VL, VH, or both, an antibody, or an antigen-binding portion thereof, that specifically binds to human GDF15, wherein said nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:167, the nucleic acid sequence of SEQ ID NO:168, or both .

E84. An isolated nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:167, the nucleic acid sequence of SEQ ID NO:168, or both.

E85. An isolated nucleic acid molecule encoding a light chain, a heavy chain, or both, an antibody, or an antigen-binding portion thereof, that specifically binds to human GDF15, wherein said nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:169, the nucleic acid sequence of SEQ ID NO:170 or both.

E86. An isolated nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:169, the nucleic acid sequence of SEQ ID NO:170, or both.

E87. An isolated nucleic acid molecule comprising at least one nucleic acid sequence selected from the group consisting of a plurality of sequences indicated as SEQ ID NO:167, 168, 169, or 170.

E88. An isolated nucleic acid molecule containing the nucleic acid sequence shown as SEQ ID NO:167.

E89. An isolated nucleic acid molecule containing the nucleic acid sequence indicated as SEQ ID NO:168.

E90. An isolated nucleic acid molecule containing the nucleic acid sequence indicated as SEQ ID NO:169.

E91. An isolated nucleic acid molecule containing the nucleic acid sequence indicated as SEQ ID NO:170.

E92. An isolated nucleic acid molecule encoding an antibody or an antigen-binding portion thereof that specifically binds to human GDF15, wherein said nucleic acid molecule contains the nucleic acid sequence of an insert plasmid deposited with ATCC and having accession number PTA-125038.

E93. An isolated nucleic acid molecule encoding an antibody or antigen-binding portion thereof that specifically binds to human GDF15, wherein said nucleic acid molecule contains the nucleic acid sequence of an insert plasmid deposited with ATCC and having accession number PTA-125039.

E94. An isolated nucleic acid molecule encoding an antibody, or an antigen-binding portion thereof, that specifically binds to human GDF15, wherein said nucleic acid molecule contains the nucleic acid sequence of a plasmid insert deposited with ATCC and has accession number PTA-125038 and the nucleic acid sequence of a plasmid insert deposited with ATCC and having the accession number PTA-125039.

E95. An isolated nucleic acid molecule containing the nucleic acid sequence of the plasmid insert deposited with the ATCC with accession number PTA-125038.

E96. An isolated nucleic acid molecule containing the nucleic acid sequence of the plasmid insert deposited with the ATCC and assigned accession number PTA-125039.

E97. An isolated nucleic acid molecule containing the nucleic acid sequence of the plasmid insert deposited with ATCC and having accession number PTA-125038 and the nucleic acid sequence of the plasmid insert deposited with ATCC and having accession number PTA-125039.

E98. A vector containing a nucleic acid molecule according to any of E81-E97.

E99. A host cell containing a nucleic acid molecule according to any of E81-E98, or a vector according to E98.

E100. E99 host cell, wherein said cell is a mammalian cell.

E101. An E100 host cell, wherein said host cell is a CHO cell, a HEK-293 cell, an NS0 cell, a PER.C6® cell, or an Sp2.0 cell.

E102. A method for producing an antibody or antigen-binding fragment thereof, comprising culturing a host cell according to any of E99-E101, under conditions where said antibody or antigen-binding fragment is expressed by said host cell.

E103. The method of E102 further comprising isolating said antibody or antigen-binding fragment thereof.

E104. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any of E1-E103, and a pharmaceutically acceptable carrier or excipient.

E105. Composition according to E104 containing thiotepa, cyclophosphamide (CYTOXAN), busulfan, improsulfan, piposulfan, benzodopa, carbokwon, meturedopa, uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamin; bullatacin, bullatacinone), delta-9-tetrahydrocannabinol (dronabinol, MARINOL), beta-lapachone, lapachol, colchicines, betulinic acid, topotecan (HYCAMTIN), CPT-11 (irinotecan, CAMPTOSAR), acetylcamptothecin, scopolelectin, 9-aminocamptothecin, bryostatin , pemetrexed, callistatin, CC-1065 (including its adoselesin, carcelesin and bicelesin synthetic analogs), podophyllotoxin, podophyllic acid, teniposide, cryptophycins, dolastatin, duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1), eleutherobin, pancratistatin, TLK-286, CDP323, oral alpha-4 integrin inhibitor, sarcodictin, spongistatin, chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembikhin, phenesterine, prednimustine, trofosfamide, uramustine, carmustine, chlorozotocin, fotemustine , lomustine, nimustine, ranimnustin, endediine antibiotics (including calicheamicin, calicheamicin gamma and calicheamicin omega), dynemycin, dynemycin A, esperamycin, neocarcistatin chromophore and related chromoprotein non-diine antibiotics chromophores, aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carsinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (incl. (DOXIL ) and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, kelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, gemcitabine (GEMZAR ), tegafur (UFTORAL), capecitabine (XELODA), epothilone, 5-fluorouracil (5-FU), denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur , cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine, imatinib, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglaton, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraksat, defofamine, de mecolcin, diaziquone, elfornithine, elliptin acetate , etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerin, pentostatin, fenamet, pyrarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxan, rizoxin, sisophy ran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidin, urethane, vindesine (ELDISINE, FILDESIN), dacarbazine, mannomustine, mitobronite, mitolactol, pipobroman, hacytosine, arabinoside ("Ara-C"), thiotepa, paclitaxel (TAXOL), albumin-engineered nanoparticulate formulation of paclitaxel (ABRAXANE), doxetaxel (TAXOTERE), chlorambucil, 6-thioguanine, mercaptopurine, methotrexate, cisplatin, carboplatin, vinblastine (VELBAN), platinum , etoposide (VP-16), ifosfamide, mitoxantrone, vincristine (ONCOVIN), oxaliplatin, leucovovin, vinorelbine (NAVELBINE), novantrone, edatraxate, daunomycin, aminopterin, ibandronate, topoisomerase inhibitor RFS 2000, difluoromethylchylomitine (DMFO), antiestrogen and selective modulators estrogen receptor receptor (SERM) (including, for example, tamoxifen (including NOLVADEX tamoxifen), raloxifene (EVISTA), droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY 1 1 7018, onapristone and toremifene (FARESTON), antiprogesterones, estrogen receptor suppressors ( ERD), fulvestrant (FASLODEX), lulibirin (LHRF1) agonists (including leuprolide acetate (LUPRON and ELIGARD), goserelin acetate, buserelin acetate and tripterelin), antiandrogens (including fiutamide, nilutamide and bicalutamide); aromatase inhibitors (including 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE), exemestane (AROMASIN), formestani, fadrozole, vorozol (RJVISOR), letrozole (FEMARA) and anastrozole (ARIMIDEX), bisphosphonates (including clodronate (BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid/zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate (SKELID) and risedronate (ACTONEL), troxcitabine, antisense oligonucleotides (including PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R)), THERATOPE vaccine, gene therapy vaccine (including ALLOVECTIN vaccine, LEUVECTIN vaccine and VAXID® vaccine; topoisomerase 1 inhibitor (e.g. LURTOTECAN)), fulvestrant; imatinib, EXEL-0862, erlotinib, cetuximab, bevacizumab, arinotecan, rmRH (e.g. ABARELIX), lapatinib, lapatinib ditosylate (also known as GW572016), 17AAG, inotuzumab ozogamicin (BESPONSA), bosutinib (BOSULIF), palbociclib (IBRANCE), axitinib ( INLYTA), sunitinib malate (SUTENT), crizotinib (XALKORI), enzalutamide (XTANDI), and combinations of two or more pharmaceutically acceptable salts and/or acids or derivatives of any of the above.

E106. A method for reducing GDF15 activity, comprising administering to a patient a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any of embodiments E1-E80, or a pharmaceutical composition according to E104 or E105, and comparing the activity of GDF15 before administration with the level of GDF15 activity after administration of the antibody, thereby reducing the activity GDF15.

E107. The method of E106, wherein the GDF15 activity is selected from the group consisting of:

(a) GFRAL binding;

(b) reducing food intake;

(c) weight loss;

(d) decrease in muscle mass;

(e) fat mass reduction;

(f) RET activation;

(g) increasing ERK (pERK) phosphorylation; And

(h) increasing phosphorylation of ribosomal protein S6 (S6)

(i) increasing AKT phosphorylation;

(j) increasing MAPK phosphorylation; And

(k) increasing PLC-γ1 phosphorylation.

E108. A method of reducing free GDF15 levels in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof, according to any one of embodiments E1-E80, or a pharmaceutical composition according to E104 or E105.

E109. The method according to E108, wherein the level of free GDF15 before administration is compared with the level of free GDF15 after administration, and the dose and dosing regimen is adjusted to reduce the level of free GDF15 below 1 ng/ml.

E110. The method of E108 wherein the pre-administration free GDF15 level is compared with the post-administration free GDF15 level and the dose and dosing regimen is adjusted to reduce the free GDF15 level below 0.9 ng/mL.

E111. The method of E108 wherein the pre-administration free GDF15 level is compared with the post-administration free GDF15 level and the dose and dosing regimen is adjusted to reduce the free GDF15 level below 0.8 ng/mL.

E112. The method according to E108, wherein the level of free GDF15 before administration is compared with the level of free GDF15 after administration, and the dose and dosing regimen is adjusted to reduce the level of free GDF15 below 0.7 ng/ml.

E113. The method of E108 wherein the pre-administration free GDF15 level is compared with the post-administration free GDF15 level and the dose and dosing regimen is adjusted to reduce the free GDF15 level below 0.6 ng/mL.

E114. The method according to E108, wherein the level of free GDF15 before administration is compared with the level of free GDF15 after administration, and the dose and dosing regimen is adjusted to reduce the level of free GDF15 below 0.5 ng/ml.

E115. The method of E108 wherein the pre-administration free GDF15 level is compared with the post-administration free GDF15 level and the dose and dosing regimen is adjusted to reduce the free GDF15 level below 0.4 ng/mL.

E116. A method for treating cachexia, comprising administering to a patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any of E1-E80, or a pharmaceutical composition according to E104 or E105.

E117. The method according to E116, wherein the cachexia is associated with cancer, chemotherapy, chemotherapy in combination with immuno-oncological therapy, chronic obstructive pulmonary disease, chronic kidney disease, chronic heart failure, congestive heart failure or sarcopenia.

E118. The method of E117, wherein the cancer is a solid tumor, pancreatic cancer, lung cancer, non-small cell lung cancer, colorectal cancer, prostate cancer, ovarian cancer, cervical cancer, or testicular cancer.

E119. The method according to E117 or E118, wherein the chemotherapy is thiotepa, cyclophosphamide (CYTOXAN), busulfan, improsulfan, piposulfan, benzodopa, carbokwon, meturedopa, uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamin; bullatacin, bullatacinone), delta-9-tetrahydrocannabinol (dronabinol, MARINOL), beta-lapachone, lapachol, colchicines, betulinic acid, topotecan (HYCAMTIN), CPT-11 (irinotecan, CAMPTOSAR), acetylcamptothecin, scopolelectin, 9-aminocamptothecin, bryostatin , pemetrexed, callistatin, CC-1065 (including its adoselesin, carcelesin and bicelesin synthetic analogs), podophyllotoxin, podophyllic acid, teniposide, cryptophycins, dolastatin, duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1), eleutherobin, pancratistatin, TLK-286, CDP323, oral alpha-4 integrin inhibitor, sarcodictin, spongistatin, chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembikhin, phenesterine, prednimustine, trofosfamide, uramustine, carmustine, chlorozotocin, fotemustine , lomustine, nimustine, ranimnustin, endediine antibiotics (including calicheamicin, calicheamicin gamma and calicheamicin omega), dynemycin, dynemycin A, esperamycin, neocarcistatin chromophore and related chromoprotein non-diine antibiotics chromophores, aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carsinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (incl. (DOXIL ) and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, kelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, gemcitabine (GEMZAR ), tegafur (UFTORAL), capecitabine (XELODA), epothilone, 5-fluorouracil (5-FU), denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur , cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine, imatinib, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglaton, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraksat, defofamine, de mecolcin, diaziquone, elfornithine, elliptin acetate , etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerin, pentostatin, fenamet, pyrarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxan, rizoxin, sisophy ran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2'-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidin, urethane, vindesine (ELDISINE, FILDESIN), dacarbazine, mannomustine, mitobronite, mitolactol, pipobroman, hacytosine, arabinoside ("Ara-C"), thiotepa, paclitaxel (TAXOL), albumin-engineered nanoparticulate paclitaxel (ABRAXANE), doxetaxel (TAXOTERE), chlorambucil, 6-thioguanine, mercaptopurine, methotrexate, cisplatin, carboplatin, vinblastine (VELBAN), platinum , etoposide (VP-16), ifosfamide, mitoxantrone, vincristine (ONCOVIN), oxaliplatin, leucovovin, vinorelbine (NAVELBINE), novantrone, edatraxate, daunomycin, aminopterin, ibandronate, topoisomerase inhibitor RFS 2000, difluoromethylchylomitine (DMFO), antiestrogen and selective modulators estrogen receptor receptor (SERM) (including, for example, tamoxifen (including NOLVADEX tamoxifen), raloxifene (EVISTA), droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY 1 1 7018, onapristone and toremifene (FARESTON), antiprogesterones, estrogen receptor suppressors ( ERD), fulvestrant (FASLODEX), lulibirin (LHRF1) agonists (including leuprolide acetate (LUPRON and ELIGARD), goserelin acetate, buserelin acetate and tripterelin), antiandrogens (including fiutamide, nilutamide and bicalutamide); aromatase inhibitors (including 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE), exemestane (AROMASIN), formestani, fadrozole, vorozol (RJVISOR), letrozole (FEMARA) and anastrozole (ARIMIDEX), bisphosphonates (including clodronate (BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid/zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate (SKELID) and risedronate (ACTONEL), troxcitabine, antisense oligonucleotides (including PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R)), THERATOPE vaccine, gene therapy vaccine (including ALLOVECTIN vaccine, LEUVECTIN vaccine and VAXID® vaccine; topoisomerase 1 inhibitor (e.g. LURTOTECAN)), fulvestrant; imatinib, EXEL-0862, erlotinib, cetuximab, bevacizumab, arinotecan, rmRH (e.g. ABARELIX), lapatinib, lapatinib ditosylate (also known as GW572016), 17AAG, inotuzumab ozogamicin (BESPONSA), bosutinib (BOSULIF), palbociclib (IBRANCE), axitinib ( INLYTA), sunitinib malate (SUTENT), crizotinib (XALKORI), enzalutamide (XTANDI), and combinations of two or more pharmaceutically acceptable salts and/or acids or derivatives of any of the above.

E120. The method of E119 wherein the chemotherapy is platinum-based chemotherapy.

E121. The method of any one of E106-E120, wherein said subject is a human.

E122. The method of any one of E106-E121, comprising subcutaneously administering said antibody, or antigen-binding fragment thereof, or pharmaceutical composition.

E123. The method of any one of E106-E122, comprising administering said antibody, or antigen-binding fragment thereof, or pharmaceutical composition intravenously.

E124. The method of any one of E106-E123, wherein said antibody, or antigen-binding fragment thereof, or pharmaceutical composition is administered about twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months , once every three months or once every four months once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once at eleven months or once every twelve months.

E125. The method of any one of E106-E124, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once a week at a dose of about 0.1 mg to about 60 mg.

E126. The method of any of E106-E125, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once a week at a dose of about 2 mg to about 50 mg.

E127. The method of any one of E106-E126, wherein said antibody or antigen-binding fragment thereof or pharmaceutical composition is administered once a week at a dose selected from the group consisting of about 2 mg, about 5 mg, about 7 mg, about 10 mg, about 12 mg, about 15 mg, about 25 mg, about 40 mg and about 50 mg.

E128. The method of any one of E106-E124, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once every two weeks at a dose of about 0.1 mg to about 130 mg.

E129. The method of any of E106-E124 or E128, wherein said antibody, or antigen-binding fragment thereof, or pharmaceutical composition is administered once every two weeks at a dose of about 5 mg to about 125 mg.

E130. The method of any one of E106-E124 or E128-E129, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once every two weeks at a dose selected from the group consisting of about 5 mg, about 12 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 60 mg, about 90 mg and about 125 mg.

E131. The method of any of E106-E124, wherein said antibody or antigen-binding fragment or pharmaceutical composition is administered once every four weeks at a dose of about 0.1 mg to about 400 mg.

E132. The method of any of E106-E124 or E131, wherein said antibody or antigen-binding fragment or pharmaceutical composition is administered once every four weeks at a dose of about 15 mg to about 385 mg.

E133. The method of any one of E106-E124 or E131-E132, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once every four weeks at a dose selected from the group consisting of about 15 mg, about 40 mg, about 60 mg, about 75 mg, about 100 mg, about 115 mg, about 200 mg, about 300 mg and about 385 mg.

E134. An antibody or antigen-binding fragment thereof, according to any of E1-E80, or a pharmaceutical composition according to E104 or E105, for use as a medicine.

E135. An antibody or antigen-binding fragment thereof, according to any of E1-80, or a pharmaceutical composition according to E104 or E105, for use in reducing GDF15 activity in a subject.

E136. A method for reducing GDF15 activity, comprising administering to a patient a therapeutically effective amount of an antibody or its antigen-binding fragment containing

a) HCDR-1, HCDR-2 and HCDR-3 of SEQ ID NO:177 and LCDR-1, LCDR-2LCDR-2 and LCDR-3 of SEQ ID NO:178, or

b) VH containing the amino acid sequence of SEQ ID NO:177 and VL containing the amino acid sequence of SEQ ID NO:178

and comparing GDF15 activity before administration with the level of GDF15 activity after administration of the antibody, thereby reducing GDF15 activity.

E137. A method for reducing GDF15 activity, comprising administering to a patient a therapeutically effective amount of an antibody or its antigen-binding fragment containing

a) a pharmaceutical composition containing an antibody or antigen-binding fragment containing HCDR-1, HCDR-2 and HCDR-3 sequences of SEQ ID NO:177; LCDR-1, LCDR-2 and LCDR-3 sequence SEQ ID NO:178; and a pharmaceutically acceptable carrier or excipient, or

b) a pharmaceutical composition containing an antibody or antigen-binding fragment containing VH containing the amino acid sequence of SEQ ID NO:177; VL containing the amino acid sequence of SEQ ID NO:178; and a pharmaceutically acceptable carrier or excipient, thereby reducing the activity of GDF15.

E138. E137 method, GDF15 activity is selected from the group consisting of:

(a) GFRAL binding;

(b) reducing food intake;

(c) weight loss;

(d) decrease in muscle mass;

(e) fat mass reduction;

(f) RET activation;

(g) increasing ERK (pERK) phosphorylation; And

(h) increasing phosphorylation of ribosomal protein S6 (S6)

(i) increasing AKT phosphorylation;

(j) increasing MAPK phosphorylation; And

(k) increasing PLC-γ1 phosphorylation.

E139. A method for treating cachexia, comprising administering to a patient a therapeutically effective amount of an antibody or antigen-binding fragment containing

a) HCDR-1, HCDR-2 and HCDR-3 of SEQ ID NO:177 and LCDR-1, LCDR-2 and LCDR-3 of SEQ ID NO:178, or

b) VH containing the amino acid sequence of SEQ ID NO:177 and VL containing the amino acid sequence of SEQ ID NO:178.

E140. A method for treating cachexia, comprising administering to a patient a therapeutically effective amount of an antibody or antigen-binding fragment containing

a) a pharmaceutical composition containing an antibody or antigen-binding fragment containing HCDR-1, HCDR-2 and HCDR-3 sequences of SEQ ID NO:177; LCDR-1, LCDR-2 and LCDR-3 sequence SEQ ID NO:178; and a pharmaceutically acceptable carrier or excipient, or

b) a pharmaceutical composition containing an antibody or antigen-binding fragment containing VH containing the amino acid sequence of SEQ ID NO:177; VL containing the amino acid sequence of SEQ ID NO:178; and a pharmaceutically acceptable carrier or excipient.

E141. The method according to E139 or E140, wherein the cachexia is associated with cancer, chemotherapy, chemotherapy in combination with immuno-oncological therapy, chronic obstructive pulmonary disease, chronic kidney disease, chronic heart failure, congestive heart failure or sarcopenia.

E142. The method according to E141, wherein the cancer is a solid tumor, pancreatic cancer, lung cancer, non-small cell lung cancer, colorectal cancer, prostate cancer, ovarian cancer, cervical cancer or testicular cancer.

E143. The method according to E141 or E142, wherein the chemotherapy is thiotepa, cyclophosphamide (CYTOXAN), busulfan, improsulfan, piposulfan, benzodopa, carbokwon, meturedopa, uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamin; bullatacin, bullatacinone), delta-9-tetrahydrocannabinol (dronabinol, MARINOL), beta-lapachone, lapachol, colchicines, betulinic acid, topotecan (HYCAMTIN), CPT-11 (irinotecan, CAMPTOSAR), acetylcamptothecin, scopolelectin, 9-aminocamptothecin, bryostatin , pemetrexed, callistatin, CC-1065 (including its adoselesin, carcelesin and bicelesin synthetic analogs), podophyllotoxin, podophyllic acid, teniposide, cryptophycins, dolastatin, duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1), eleutherobin, pancratistatin, TLK-286, CDP323, oral alpha-4 integrin inhibitor, sarcodictin, spongistatin, chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembikhin, phenesterine, prednimustine, trofosfamide, uramustine, carmustine, chlorozotocin, fotemustine , lomustine, nimustine, ranimnustin, endediine antibiotics (including calicheamicin, calicheamicin gamma and calicheamicin omega), dynemycin, dynemycin A, esperamycin, neocarcistatin chromophore and related chromoprotein non-diine antibiotics chromophores, aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carsinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (incl. (DOXIL ) and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, kelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, gemcitabine (GEMZAR ), tegafur (UFTORAL), capecitabine (XELODA), epothilone, 5-fluorouracil (5-FU), denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur , cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine, imatinib, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglaton, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraksat, defofamine, de mecolcin, diaziquone, elfornithine, elliptin acetate , etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerin, pentostatin, fenamet, pyrarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxan, rizoxin, sisophy ran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2'-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidin, urethane, vindesine (ELDISINE, FILDESIN), dacarbazine, mannomustine, mitobronite, mitolactol, pipobroman, hacytosine, arabinoside ("Ara-C"), thiotepa, paclitaxel (TAXOL), albumin-engineered nanoparticulate paclitaxel (ABRAXANE), doxetaxel (TAXOTERE), chlorambucil, 6-thioguanine, mercaptopurine, methotrexate, cisplatin, carboplatin, vinblastine (VELBAN), platinum , etoposide (VP-16), ifosfamide, mitoxantrone, vincristine (ONCOVIN), oxaliplatin, leucovovin, vinorelbine (NAVELBINE), novantrone, edatraxate, daunomycin, aminopterin, ibandronate, topoisomerase inhibitor RFS 2000, difluoromethylchylomitine (DMFO), antiestrogen and selective modulators estrogen receptor (SERM) (including, for example, tamoxifen (including NOLVADEX tamoxifen), raloxifene (EVISTA), droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY11 7018, onapristone and toremifene (FARESTON), antiprogesterones, estrogen receptor suppressors (ERD) , fulvestrant (FASLODEX), lulibirin (LHRF1) agonists (including leuprolide acetate (LUPRON and ELIGARD), goserelin acetate, buserelin acetate, and tripterelin), antiandrogens (including fiutamide, nilutamide, and bicalutamide); aromatase inhibitors (including 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE), exemestane (AROMASIN), formestani, fadrozole, vorozol (RJVISOR), letrozole (FEMARA) and anastrozole (ARIMIDEX), bisphosphonates (including clodronate (BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid/zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate (SKELID) and risedronate (ACTONEL), troxcitabine, antisense oligonucleotides (including PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R)), THERATOPE vaccine, gene therapy vaccine (including ALLOVECTIN vaccine, LEUVECTIN vaccine and VAXID® vaccine; topoisomerase 1 inhibitor (e.g. LURTOTECAN)), fulvestrant; imatinib, EXEL-0862, erlotinib, cetuximab, bevacizumab, arinotecan, rmRH (e.g. ABARELIX), lapatinib, lapatinib ditosylate (also known as GW572016), 17AAG, inotuzumab ozogamicin (BESPONSA), bosutinib (BOSULIF), palbociclib (IBRANCE), axitinib ( INLYTA), sunitinib malate (SUTENT), crizotinib (XALKORI), enzalutamide (XTANDI), and combinations of two or more pharmaceutically acceptable salts and/or acids or derivatives of any of the above.

E144. The method of E141 wherein the chemotherapy is platinum-based chemotherapy.

E145. The method of any one of E136-E144, wherein said subject is a human.

E146. The method of any one of E136-E145, comprising subcutaneously administering said antibody, or antigen-binding fragment thereof, or pharmaceutical composition.

E147. The method according to any of E136-E146, which includes the introduction of the specified antibody or its antigennegative fragment, or pharmaceutical composition intravenously.

E148. The method of any one of E136-E147, wherein said antibody, or antigen-binding fragment thereof, or pharmaceutical composition is administered about twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months , once every three months or once every four months.

E149. The method of any of E136-E148, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once a week at a dose of about 0.1 mg to about 60 mg.

E150. The method of any one of E136-E149, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once a week at a dose of about 2 mg to about 50 mg.

E151. The method of any one of E136-E150, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once a week at a dose selected from the group consisting of about 2 mg, about 5 mg, about 7 mg, about 10 mg, about 12 mg, about 15 mg, about 25 mg, about 40 mg and about 50 mg.

E152. The method of any of E136-E148, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once every two weeks at a dose of about 0.1 mg to about 130 mg.

E153. The method of any of E136-E148 or E152, wherein said antibody or antigen-binding fragment or pharmaceutical composition is administered once every two weeks at a dose of about 5 mg to about 125 mg.

E154. The method of any one of E136-E148 or E152-E153, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once every two weeks at a dose selected from the group consisting of about 5 mg, about 12 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 60 mg, about 90 mg and about 125 mg.

E155. The method of any of E136-E148, wherein said antibody or antigen-binding fragment or pharmaceutical composition is administered once every four weeks at a dose of about 0.1 mg to about 400 mg.

E156. The method of any of E136-E148 or E155, wherein said antibody or antigen-binding fragment or pharmaceutical composition is administered once every four weeks at a dose of about 15 mg to about 385 mg.

E157. The method of any one of E136-E148 or E155-E156, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once every four weeks at a dose selected from the group consisting of about 15 mg, about 40 mg, about 60 mg, about 75 mg, about 100 mg, about 115 mg, about 200 mg, about 300 mg and about 385 mg.

E158. A method for reducing the level of free GDF15 in a patient's plasma, wherein said method comprises administering an antibody according to any one of E1-E80 or a composition according to any one of E104-E105.

E159. The method of any one of E106-E108 or E116-E158, wherein the subject's free GDF15 plasma level is reduced from about 0.05 ng/mL to about 3 ng/mL.

E160. The method of any one of E106-E108 or E116-E159, wherein the subject's free GDF15 plasma level is reduced from about 0.1 ng/mL to about 1 ng/mL.

E161. The method of any one of E106-E108 or E116-E160, wherein the subject's free GDF15 plasma level is reduced from about 0.4 ng/mL to about 0.8 ng/mL.

E162. The method of any one of E106-E114 or E116-E158, wherein the subject's free GDF15 plasma level is reduced to below 0.5 ng/mL.

E163. The method of any of E106-E158 or E162, wherein the subject's free GDF15 plasma level is reduced to below 0.4 ng/mL.

E164. The method of any one of E106-E108 or E116-E158, wherein the subject's free GDF15 plasma level is reduced to a range whose lower value is selected from the group consisting of 0.05, 0.06, 0.07, 0.08, 0, 09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2, 5, 2.6, 2.7, 2.8, 2.9 ng/ml and the upper value of which is selected from the group consisting of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1, 4, 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and 3.0 ng/ml.

E165. The method of E106-E114 or E116-E158, wherein the subject's free GDF15 plasma level is reduced to less than about 0.5 ng/mL.

E166. The method of any of E106-E165, wherein the subject's plasma free GDF15 level is reduced below the lower detection level using an assay known in the art to determine plasma free GDF15.

E167. A pharmaceutical composition containing an antibody or antigen-binding fragment thereof from E1-E80, a PD-1 binding antagonist of the axis, and a pharmaceutically acceptable carrier or excipient.

E168. A pharmaceutical composition comprising an antibody or antigen-binding fragment at E1-E80, a PD-1 axis binding antagonist, and a pharmaceutically acceptable carrier or excipient, provided that the PD-1 axis binding antagonist is not avelumab.

E169. A method of treating cancer, comprising administering to a patient an amount of a PD-1 axis binding antagonist and an amount of an antibody or antigen-binding fragment thereof according to any of E1-E80, or an amount of a pharmaceutical composition according to E167-E168, where the amounts together are effective for treating cancer.

E170. A method of treating cancer comprising administering to a patient an amount of a PD-1 axis binding antagonist and an amount of an antibody or antigen-binding fragment thereof according to any of E1-E80, or an amount of a pharmaceutical composition according to E167-E169, wherein the amounts together are effective to treat cancer, and where avelumab is not a PD-1 binding antagonist.

E171. The method of E169-E170 wherein the amounts together provide a synergistic therapeutic effect in the treatment of cancer.

E172. The method of E169, wherein the PD-1 axis binding antagonist is an anti-PD-L1 antibody.

E173. The method of E169, wherein the PD-1 axis binding antagonist is an anti-PD-L1 antibody, provided that the anti-PD-L1 antibody is not avelumab.

E174. The method of E172, wherein the PD-L1 antibody is selected from the group consisting of avelumab, atezolizumab, or durvalumab.

E175. The method of E172, wherein the PD-L1 antibody is atezolizumab or durvalumab.

E176. The method of E172 wherein the PD-1 binding antagonist is avelumab and is administered intravenously at about 10 mg/kg Q2W or about 800 mg Q2W.

E177. The method of claim E168, wherein the PD-1 axis binding antagonist is an anti-PD-1 antibody.

E178. The method of claim E177, wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, spartalizumab, tiselizumab, pidilizumab, AMP-224, AMP-514, cemiplimab, and sasanlimab (PF-06801591, RN888, mAb7).

E179. The method of claim E178, wherein the PD-1 binding antagonist is sasanlimab and is administered subcutaneously in an amount of about 300 mg Q4W or about 600 mg Q6W.

E180. The method of any one of E170-E180, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, renal cell carcinoma, Merkel cell carcinoma, ovarian cancer, breast cancer, pancreatic cancer, urothelial cancer, and castration-resistant prostate cancer .

E181. Method according to E180, the cancer is renal cell carcinoma.

E182. Method according to E1181, the cancer is pancreatic cancer.

E183. A method for detecting GDF15 in a sample, tissue, or cell using an antibody or antigen-binding portion thereof according to any of E1-E80, comprising contacting the sample, tissue, or cell with the antibody and detecting the antibody.

E184. A method of treating cancer in a patient, comprising administering to the patient a combination therapy containing a synergistic therapeutically effective amount of a PD-1 axis binding antagonist and a synergistic therapeutically effective amount of a GDF15 inhibitor, where the amounts together provide a synergistic therapeutic effect, thereby curing the cancer.

E185. A method of treating cancer in a patient, comprising administering to the patient a combination therapy comprising a synergistic therapeutically effective amount of a PD-1 axis binding antagonist and a synergistic therapeutically effective amount of a GDF15 inhibitor, wherein the amounts together provide a synergistic therapeutic effect thereby curing the cancer, and wherein the PD-binding antagonist Axis 1 is not avelumab.

E186. The method of E184-E185, wherein the GDF15 inhibitor is an anti-GDF15 antibody or antigen-binding fragment thereof, of any one of E1-E80.

E187. The method of E186, wherein the anti-GDF15 antibody or antigen-binding fragment thereof comprises:

a) LCDR-1 containing the amino acid sequence of SEQ ID NO:95;

b) LCDR-2 containing the aa sequence of SEQ ID NO:28;

c) LCDR-3 containing the aa sequence of SEQ ID NO:9;

d) HCDR-1 containing the aa sequence of SEQ ID NO:32;

e) HCDR-2 containing the aa sequence of SEQ ID NO:165; And

f) HCDR-3 containing the aa sequence of SEQ ID NO:52.

E188. The method of E187, wherein the PD-1 axis binding antagonist is an antibody that specifically binds to PD-1 and contains:

a) LCDR-1 containing the amino acid sequence of SEQ ID NO:216;

b) LCDR-2 containing the aa sequence of SEQ ID NO:217;

c) LCDR-3 containing the aa sequence of SEQ ID NO:218;

d) HCDR-1 containing the aa sequence of SEQ ID NO:210;

e) HCDR-2 containing the aa sequence of SEQ ID NO:213; And

f) HCDR-3 containing the aa sequence of SEQ ID NO:215.

E189. The method of E186 wherein the PD-1 axis binding antagonist is an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, spartalizumab, pidilizumab, tislelizumab, AMP-224, AMP-514, cemiplimab, and sasanlimab (PF-06801591 ).

E190. The method according to item E188-E189, wherein the anti-PD-1 antibody is sasanlimab (PF-06801591).

E191. The method of claim 186, wherein the PD-1 axis binding antagonist is an anti-PD-L1 antibody.

E192. The method of claim 190 wherein sasanlimab is administered subcutaneously in an amount of about 300 mg Q4W or about 600 mg Q6W.

E193. The method of claim 191, wherein the PD-L1 antibody is selected from the group consisting of atezolizumab, durvalumab, BMS-936559, MEDI4736, and MPDL3280A.

E194. 187, wherein the anti-GDF15 antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:166 and a VL comprising the amino acid sequence of SEQ ID NO:163.

E195. The method according to E194, wherein the anti-GDF15 antibody comprises an HC containing the amino acid sequence of SEQ ID NO:164 and an LC containing the amino acid sequence of SEQ ID NO:162.

E196. The method according to E195, wherein the anti-PD-1 antibody comprises an HC containing the amino acid sequence of SEQ ID NO:197 and an LC containing the amino acid sequence of SEQ ID NO:199.

E197. The method of claim 31, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, renal cell carcinoma, Merkel cell carcinoma, ovarian cancer, breast cancer, pancreatic cancer, urothelial cancer, and castration-resistant prostate cancer.

E198. The method according to E184-E197, the cancer is renal cell carcinoma or pancreatic cancer.

E199. A cancer treatment kit containing a synergistic therapeutically effective amount of an anti-PD-1 antibody and a synergistic therapeutically effective amount of an anti-GDF15 antibody.

E200. A method for treating cancer, comprising administering to a patient a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any of E1-E80, or a pharmaceutical composition according to E104 or E105.

E201. The method according to E200, where the cancer is, for example, without limitation, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, esophageal cancer, gastric cancer, glioblastoma, glioma, brain tumor, head and neck cancer, kidney cancer , lung cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, liver cancer, uterine cancer, bone cancer, leukemia, lymphoma, sarcoma, blood cancer, thyroid cancer, thymus cancer, eye cancer, and skin cancer.

E202. The method of E200 or E201, wherein the method further comprises administering one or more additional therapeutic agents.

E203. The method of E202, wherein the additional therapeutic agent(s) is selected from chemotherapy, vaccine, CAR-T cell based therapy, radiotherapy, cytokine therapy, vaccine, bispecific antibody, inhibitor of other immunosuppressive pathways, angiogenesis inhibitors, T cell activator, metabolic pathway inhibitor , an mTOR inhibitor, an adenosine pathway inhibitor, a tyrosine kinase inhibitor including but not limited to INLYTA, ALK inhibitors and sunitinib, a BRAF inhibitor, an epigenetic modifier, inhibitors or depletors of Treg cells and/or myeloid suppressor cells, a JAK inhibitor, a STAT inhibitor, a cyclin-dependent kinase, biotherapeutic agent (including, but not limited to, antibodies to VEGF, VEGFR, EGFR, Her2/neu, other growth factor receptors, CD20, CD40, CD-40L, CTLA-4, OX-40, 4- 1BB and ICOS), an immunogenic agent (e.g., attenuated cancer cells, tumor antigens, antigen-presenting cells such as dendritic cells injected with tumor-derived antigen or nucleic acids, immunostimulatory cytokines (e.g., IL-2, IFNα2, GM-CSF) , cells transfected with genes encoding immunostimulatory cytokines such as, but not limited to, GM-CSF), a STING agonist, and a toll-like receptor agonist (eg, TLR3, TLR7, TLR8, TLR9).

E204. The method of E202 wherein the additional therapeutic agent is an anti-CD40 antibody.

E205. The method of E203, wherein the chemotherapy is thiotepa, cyclophosphamide (CYTOXAN), busulfan, improsulfan, piposulfan, benzodopa, carbokwone, meturedopa, uredopa, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamin; bullatacin, bullatacinone), delta-9-tetrahydrocannabinol (dronabinol, MARINOL), beta-lapachone, lapachol, colchicines, betulinic acid, topotecan (HYCAMTIN), CPT-11 (irinotecan, CAMPTOSAR), acetylcamptothecin, scopolelectin, 9-aminocamptothecin, bryostatin , pemetrexed, callistatin, CC-1065 (including its adoselesin, carcelesin and bicelesin synthetic analogs), podophyllotoxin, podophyllic acid, teniposide, cryptophycins, dolastatin, duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1), eleutherobin, pancratistatin, TLK-286, CDP323, oral alpha-4 integrin inhibitor, sarcodictin, spongistatin, chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembikhin, phenesterine, prednimustine, trofosfamide, uramustine, carmustine, chlorozotocin, fotemustine , lomustine, nimustine, ranimnustin, endediine antibiotics (including calicheamicin, calicheamicin gamma and calicheamicin omega), dynemycin, dynemycin A, esperamycin, neocarcistatin chromophore and related chromoprotein non-diine antibiotics chromophores, aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carsinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (incl. (DOXIL ) and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, kelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate, gemcitabine (GEMZAR ), tegafur (UFTORAL), capecitabine (XELODA), epothilone, 5-fluorouracil (5-FU), denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur , cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine, imatinib, aminoglutethimide, mitotane, trilostane, frolinic acid, aceglaton, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraksat, defofamine, de mecolcin, diaziquone, elfornithine, elliptin acetate , etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerin, pentostatin, fenamet, pyrarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxan, rizoxin, sisophy ran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2'-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, anguidin, urethane, vindesine (ELDISINE, FILDESIN), dacarbazine, mannomustine, mitobronite, mitolactol, pipobroman, hacytosine, arabinoside ("Ara-C"), thiotepa, paclitaxel (TAXOL), albumin-engineered nanoparticulate paclitaxel (ABRAXANE), doxetaxel (TAXOTERE), chlorambucil, 6-thioguanine, mercaptopurine, methotrexate, cisplatin, carboplatin, vinblastine (VELBAN), platinum , etoposide (VP-16), ifosfamide, mitoxantrone, vincristine (ONCOVIN), oxaliplatin, leucovovin, vinorelbine (NAVELBINE), novantrone, edatraxate, daunomycin, aminopterin, ibandronate, topoisomerase inhibitor RFS 2000, difluoromethylchylomitine (DMFO), antiestrogen and selective modulators estrogen receptor receptor (SERM) (including, for example, tamoxifen (including NOLVADEX tamoxifen), raloxifene (EVISTA), droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY 1 1 7018, onapristone and toremifene (FARESTON), antiprogesterones, estrogen receptor suppressors ( ERD), fulvestrant (FASLODEX), lulibirin (LHRF1) agonists (including leuprolide acetate (LUPRON and ELIGARD), goserelin acetate, buserelin acetate and tripterelin), antiandrogens (including fiutamide, nilutamide and bicalutamide); aromatase inhibitors (including 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE), exemestane (AROMASIN), formestani, fadrozole, vorozol (RJVISOR), letrozole (FEMARA) and anastrozole (ARIMIDEX), bisphosphonates (including clodronate (BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid/zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate (SKELID) and risedronate (ACTONEL), troxcitabine, antisense oligonucleotides (including PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R)), THERATOPE vaccine, gene therapy vaccine (including ALLOVECTIN vaccine, LEUVECTIN vaccine and VAXID® vaccine; topoisomerase 1 inhibitor (e.g. LURTOTECAN)), fulvestrant; imatinib, EXEL-0862, erlotinib, cetuximab, bevacizumab, arinotecan, rmRH (e.g. ABARELIX), lapatinib, lapatinib ditosylate (also known as GW572016), 17AAG, inotuzumab ozogamicin (BESPONSA), bosutinib (BOSULIF), palbociclib (IBRANCE), axitinib ( INLYTA), sunitinib malate (SUTENT), crizotinib (XALKORI), enzalutamide (XTANDI), and combinations of two or more pharmaceutically acceptable salts and/or acids or derivatives of any of the above.

E206. Method E203 wherein the chemotherapy is platinum-based chemotherapy.

E207. The method of any one of E200-E206, wherein said subject is a human.

E208. The method of any one of E200-E207, comprising subcutaneously administering said antibody, or antigen-binding fragment thereof, or pharmaceutical composition.

E209. The method according to any of E200-E206, which includes the introduction of the specified antibody or its antigennegative fragment or pharmaceutical composition intravenously.

E210. The method of any one of E200-E206, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered about twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once at five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months, once every three months or once every four months.

E211. The method of any of E200-E206, wherein said antibody or antigen-binding fragment or pharmaceutical composition is administered once a week at a dose of about 0.1 mg to about 60 mg.

E212. The method of any of E200-E206, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered once a week at a dose of about 2 mg to about 50 mg.

E213. The method of any one of E200-E206, wherein said antibody or antigen-binding fragment thereof or pharmaceutical composition is administered once a week at a dose selected from the group consisting of about 2 mg, about 5 mg, about 7 mg, about 10 mg, about 12 mg, about 15 mg, about 25 mg, about 40 mg and about 50 mg.

E214. The method of any one of E200-E206, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered biweekly at a dose of between about 0.1 mg and about 130 mg.

E215. The method of any one of E200-E206, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered biweekly at a dose of about 5 mg to about 125 mg.

E216. The method of any one of E200-E206, wherein said antibody or antigen-binding fragment thereof or pharmaceutical composition is administered biweekly at a dose selected from the group consisting of about 5 mg, about 12 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 60 mg, about 90 mg and about 125 mg.

E217. The method of any of E200-E206, wherein said antibody or antigen-binding fragment or pharmaceutical composition thereof is administered every four weeks at a dose of about 0.1 mg to about 400 mg.

E218. The method of any of E200-E206, wherein said antibody or antigen-binding fragment or pharmaceutical composition is administered every four weeks at a dose of about 15 mg to about 385 mg.

E219. The method of any one of E200-E206, wherein said antibody or antigen-binding fragment thereof or pharmaceutical composition is administered every four weeks at a dose selected from the group consisting of about 15 mg, about 40 mg, about 60 mg, about 75 mg, about 100 mg, about 115 mg, about 200 mg, about 300 mg and about 385 mg.

E220. A method for treating cancer in a patient, wherein the method comprises administering a therapeutically effective amount of an anti-GDF15 antibody or antigen-binding fragment thereof at E1-E80 or a pharmaceutical composition at E104 or E105.

E221. The method of E220, wherein the method further comprises administering to the subject an effective amount of one or more additional therapeutic agents.

E222. The method of E221 wherein the additional therapeutic agent is an anti-CD40 antibody or antigen-binding fragment thereof.

E223. The method of any one of E220-E222, wherein the cancer is selected from the group consisting of gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, head and neck squamous cell carcinoma, thymus cancer, epithelial cancer, saliva cancer, liver cancer , gastric cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, cancer colon, oral cancer, skin cancer and melanoma.

E224. The method of any one of E220-223, wherein the subject is a human.

E225. A method for improving the therapeutic effect of an immune modulator administered to a subject for the treatment of cancer, wherein the method comprises administering to the subject receiving the immune modulator an effective amount of an anti-GDF15 antibody or antigen-binding fragment thereof from E1-E80 or a pharmaceutical composition according to E104 or E105.

E226. The method of E225, wherein the cancer is selected from the group consisting of breast cancer, stomach cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, glioma, glioblastoma, kidney cancer, endometrial cancer, and rectal cancer.

E227. A method for treating or preventing cytokine release syndrome (CRS) in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of an anti-GDF15 antibody or an antigen-binding fragment thereof at E1-E80, or a pharmaceutical composition at E104 or E105, thereby curing or preventing subject's CRS.

E228. A method for reducing or inhibiting toxicity in a subject suffering from cytokine release syndrome (CRS) or cytokine storm, or susceptible to cytokine release syndrome or cytokine storm, comprising the step of administering a composition, comprising administering to the subject an effective amount of an anti-GDF15 antibody or an E1-antigen-binding fragment thereof. E80, or a pharmaceutical composition according to E104 or E105.

E229. The method of claim E227 or E228, wherein the production of at least one pro-inflammatory cytokine is reduced or inhibited in said subject compared to a subject suffering from cytokine release syndrome or cytokine storm or sensitive to cytokine release syndrome or cytokine storm and not receiving anti- GDF15 antibody or antigen-binding fragment thereof at E1-E80, or a pharmaceutical composition at E104 or E105.

E230. The method of any one of E227-E229, wherein the subject is undergoing cancer therapy and said method does not reduce the effectiveness of the cancer therapy.

E231. The method of E230 wherein the anti-cancer therapy comprises an immune modulator.

E232. The method according to E230 or E231, wherein the administration of the anti-GDF15 antibody or antigen-binding fragment thereof takes place before, simultaneously with, or after anti-cancer therapy.

E233. The method according to any of E231-E232, wherein the immune modulator is: an anti-CD40 antibody, an anti-CD47 antibody, an anti-CTLA4 antibody, an anti-4-1BB/CD137 antibody, interleukin 12 (IL-12) or IL-15.

E234. The method of any one of E228-E233, wherein the cause of the CRS or cytokine storm includes an infectious stimulus, condition, or syndrome, or wherein the cause of said cytokine release syndrome or cytokine storm includes a non-infectious stimulus, condition, or syndrome, or any combination thereof.

E235. The method of E234, wherein said infectious stimulus, condition, or syndrome includes influenza, avian influenza, severe acute respiratory syndrome (SARS), hemophagocytic lymphohistiocytosis (HLH) associated with Epstein-Barr virus, sepsis, gram-negative sepsis, malaria, Ebola virus, virus smallpox, systemic gram-negative bacterial infection, or Jarisch-Herxheimer syndrome, or where said non-infectious stimulus, condition, or syndrome includes hemophagocytic lymphohistiocytosis (HLH), sporadic HLH, macrophage activation syndrome (MAS), chronic arthritis, systemic juvenile idiopathic arthritis (sJIA) , Still's disease, cryopyrin-associated periodic syndrome (CAPS), familial cold autoinflammatory syndrome (FCAS), familial cold urticaria (FCU), Muckle-Wale syndrome (MWS), chronic childhood neurological skin and joint syndrome (CINCA), cryopyrinopathy containing inherited or de novo functional mutations in the NLRP3 gene, hereditary autoinflammatory disorder, acute pancreatitis, severe burns, trauma, acute respiratory distress syndrome, immunotherapy, monoclonal antibody therapy secondary to drug use, secondary to inhaled toxins, lipopolysaccharide ( LPS), gram-positive toxins, fungal toxins, glycosylphosphatidylinositol (GPI), or modulation of RIG-1 gene expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph depicting the transition temperatures (T _m 1 ) for an anti-GDF15 antibody of the invention as measured by differential scanning calorimetry (DSC). T _m 1 represents the temperature at which the C _H 2 antibody is 50% deployed.

FIG. 1B is a graph depicting the transition temperatures (T _m 2 ) for an anti-GDF15 antibody of the invention as measured by differential scanning calorimetry (DSC). T _m 2 represents the temperature at which the Fab of the antibody is 50% deployed.

FIG. 1C is a graph depicting the transition temperatures (T _m 3 ) for an anti-GDF15 antibody of the invention as measured by differential scanning calorimetry (DSC). T _m 3 represents the temperature at which the C _H 3 antibody is 50% deployed.

FIG. 2 shows the viscosity of GDF15_001 analyzed on an Anton Parr instrument. Acceptable limiting viscosity (20cP) is reached at about 140 mg/ml.

FIG. 3 shows the human plasma concentration of GDF15 following injection of human adeno-associated virus (AAV)-GDF15 into healthy male C57Bl6N mice. Plasma GDF15 is measured 13 and 14 days post AAV injection (corresponding to days -2 and -1 in Figure 3) via ELISA (R&D Systems DGD150). The horizontal lines represent the middle. n=9 per group.

FIG. 4 is a graph depicting the ability of GDF15_001 to reverse GDF15-induced weight loss in healthy mice. Arrows indicate the timing of AAV-GDF15 injection in humans (day -15) and the dose of the first monoclonal antibody (mAb) (day 0). Healthy male C57Bl6N mice are treated with GDF15_001 (30 mg/kg subcutaneously (SC) every third day (Q3D)) or control immunoglobulin G (IgG). Values are mean ± SOS. n=8 per group. Repeated measures ANOVA with an autoregressive (1) covariance design is used to compare percent change from baseline body weight between treatment groups during days 4-6 of the dosing period. Tukey-Kramer's multiple comparison test adjustment is used to control for the group error rate for comparing treatment groups in repeated measures analysis of variance (ANOVA). * p<0.0001 to control + IgG (days 4-6); †p<0.0001 to GDF15+GDF15_001 (days 4-6).

FIG. 5 is a graph depicting the ability of GDF15_001 to reverse GDF15-induced fat loss. Healthy male C57Bl6N mice were injected with human AAV-GDF15 on day -15 after the first dose of mAb, starting on day 0. Treatment with mAb GDF15_001 (30 mg/kg, PC, Q3D) or IgG control is continued for 6 days. Body composition is measured on day 6 using computed tomography (MRI). Values are mean ± SOS. n=8 per group. Statistical analysis is carried out using ANOVA. * p<0.0001 to control+IgG, † p<0.01 to GDF15+GDF15_001.

FIG. 6 is a graph depicting the ability of GDF15_001 to reverse GDF15-induced muscle mass loss. Healthy male C57Bl6N mice were injected with human AAV-GDF15 on day -15 after the first dose of mAb, starting on day 0. Treatment with GDF15_001 mAb (30 mg/kg, sc every third day) or IgG control is continued for 6 days. Body composition is measured on day 6 using MRI. Values are mean ± SOS. n=8 per group. Statistical analysis is carried out using ANOVA. * p<0.01 to control + IgG; †p<0.01 to GDF15+GDF15_001.

FIG. 7 is a graph depicting the plasma concentration of GDF15 in mice following injection of mouse AAV-GDF15 into healthy male C57Bl6N mice. Plasma GDF15 is measured 9 days post AAV injection (corresponding to day 9 in Figure 5) using ELISA (R&D Systems MGD150). The horizontal lines represent the middle. n=6 per group. Mean plasma GDF15 levels are compared between treatment groups using a t-test using Satterswaite's adjustment for non-uniform variance. * p<0.001 to control.

FIG. 8 is a graph depicting the ability of GDF15_001 to reverse GDF15-induced weight loss in healthy mice. Arrows indicate the time points of mouse AAV-GDF15 injection (day 0) and first mAb dose (day 11). Healthy male C57Bl6N mice are treated with GDF15_001 (30 mg/kg, PC, Q3D) or IgG control. Values are mean ± SOS. n=10 per group. Repeated ANOVA with unstructured covariance is used to compare percent of baseline body weights between treatment groups over a 12-16 day dosing period. In the presence of a statistically significant time-interaction treatment, comparisons between treatment groups are made at day 16 with percentage of baseline body weight determined using an ANOVA suitable for a fully randomized model. *p<0.0001 vs control + IgG on day 16, †p<0.0001 vs hGDF15+PF-06946860 on day 16.

FIG. 9 is a graph depicting the ability of GDF15_001 to increase food intake after GDF15 treatment in healthy mice. Healthy male C57Bl6N mice are treated with GDF15_001 (30 mg/kg, PC, Q3D) or IgG control. Food intake is measured daily and total food intake is calculated. Values are mean ± SOS. n=8-10 per group. ANOVA is used to compare cumulative food intake in treatment groups from days 11 to 16 using a Bonferroni correction for multiple comparisons. *p<0.001 to control + IgG, † p<0.001 to control+GDF15_001, p=0.0505 GDF15+GDF15_001 to GDF15+IgG.

FIG. 10 is a graph depicting the ability of GDF15_001 to reverse weight loss in mice bearing an HT-1080 tumor (human fibrosarcoma cell line). Arrows indicate the time points for subcutaneous implantation of HT-1080 cells (day -13) and the first mAb dosing (day 0). Female mice with severe combined immunodeficiency are treated with GDF15_001 (10 mg/kg, PC, Q3D) or IgG control. Values are mean ± SOS. n=9-10 per group. Repeated measures ANOVA with an autoregressive (1) covariance design is used to compare percent change from baseline body weight between treatment groups during days 9-11 of the dosing period (where disease is stabilized and animals do not begin to weed out due to body weight loss). * = p<0.0001 HT-1080+IgG to NTB+PBS and HT-1080+GDF15_001 (days 9-11).

FIG. 11 is a graph showing the ability of GDF15_001 to reverse fat loss in mice bearing an HT-1080 tumor (human fibrosarcoma cell line). Severe pooled immunodeficient female mice are implanted (subcutaneously) with HT-1080 cells on day -13 after the first mAb dosing, starting on day 0. Treatment with mAb GDF15_001 (10 mg/kg, PC, Q3D) or IgG control is continued for 18 days. Body composition is measured on day 18 using MRI echo. Values are mean ± SOS. n=9-10 per group. Statistical analysis is carried out using ANOVA. * = p<0.0001 HT-1080+IgG to NTB+PBS and HT-1080+GDF15_001.

FIG. 12 is a graph depicting the ability of GDF15_001 to reverse tumor-free muscle mass loss in mice bearing an HT-1080 tumor (human fibrosarcoma cell line). Severe pooled immunodeficient female mice are implanted (subcutaneously) with HT-1080 cells on day -13 after the first mAb dosing, starting on day 0. Treatment with mAb GDF15_001 (10 mg/kg, PC, Q3D) or IgG control is continued for 18 days. Body composition is measured on day 18 using MRI echo. Values are mean ± SOS. n=9-10 per group. Statistical analysis is carried out using ANOVA. * = p<0.001 HT-1080+IgG to NTB+PBS, † = p<0.0001 HT-1080+IgG to HT-1080+GDF15_001.

FIG. 13 is a graph depicting the ability of GDF15_001 to prolong survival in mice bearing an HT-1080 tumor (human fibrosarcoma cell line). Severe pooled immunodeficient female mice are implanted (subcutaneously) with HT-1080 cells on day -13 after the first mAb dosing, starting on day 0. Treatment with mAb with GDF15_001 (10 mg/kg, PC, Q3D) or IgG control is continued until death or euthanasia (defined by ill health and/or >30% weight loss according to Institutional Animal Care and Use Committee guidelines). n=9-10 per group. Kaplan-Meier survival curves are compared to failure time data using log-rank statistics to compare treatment groups. * p< 0.0001 to HT-1080+GDF15_001 group.

FIG. 14 is a graph depicting human plasma concentration of GDF15 in HT-1080 (human fibrosarcoma cell line) tumor-bearing mice housed under thermoneutral (86°F) conditions. Severe combined immunodeficient female mice are implanted (subcutaneously) with HT-1080 cells on day -13 after the first dose of mAb (IgG control, 10 mg/kg, SC, Q3D) starting on day 0. Plasma GDF15 is measured on day 18 using ELISA (R&D Systems DGD150). The horizontal line represents the middle. n=7.

FIG. 15 is a graph depicting the ability of GDF15_001 to reverse weight loss in HT-1080 (human fibrosarcoma cell line) tumor-bearing mice housed under thermoneutral (86°F) conditions. Arrows indicate the time points for subcutaneous implantation of HT-1080 cells (day -13) and the first mAb dosing (day 0). Female mice with severe combined immunodeficiency are treated with GDF15_001 (10 mg/kg, PC, Q3D) or IgG control. Values are mean ± SOS. n=10 per group. Repeated measures ANOVA with unstructured covariance is used to compare percent of baseline body weight between treatment groups over days 1-13 of the dosing period, followed by comparison between treatment groups on day 7 and day 13 using ANOVA. Comparisons of interest between treatment groups are then tested using t-tests using pooled variant scores from ANOVA. Bonferroni corrections for multiple comparisons are used to control for the group error rate for comparing treatment groups. * = p<0.0001 to NTB+IgG and HT-1080+GDF15_001 (days 7 and 13).

FIG. 16 is a graph depicting the human plasma concentration of GDF15 in mice with PA-1065 tumor (derived from liver metastases of a pancreatic tumor). Severe pooled immunodeficient female mice are implanted (subcutaneously) with PA-0165 tumor tissue on day -18 after the first dose of mAb (IgG control, 10 mg/kg, PC, Q3D) starting on day 0. Plasma GDF15 is measured on day 27 using ELISA (R&D Systems DGD150). The horizontal line represents the middle. n=14.

FIG. 17 is a graph depicting the ability of GDF15_001 to prevent weight loss in mice bearing a PA-0165 tumor (derived from liver metastases of a pancreatic tumor). Arrows point to the time points of subcutaneous implantation of PA-0165 tumor tissue (day -18) and the first mAb dosing (day 0). Female mice with severe combined immunodeficiency are treated with GDF15_001 (10 mg/kg, PC, Q3D) or IgG control. Values are mean ± SOS. n=10-17 per group. Analysis of variance with repeated measurements with an unstructured covariance structure is used to compare the percentage of initial body weight between treatment groups during the 10-16 days of the dosing period. If there is a statistically significant treatment by time interaction, comparisons between treatment groups are made at day 16 using t-tests using pooled estimates of variance based on repeated measures ANOVA. Bonferroni corrections for multiple comparisons are used to control for the group error rate for comparing treatment groups. * = p<0.0001 to NTB+PBS and PA-0165+GDF15_001.

FIG. 18 is a graph depicting the ability of GDF15_001 to prolong survival in mice bearing a PA-1065 tumor (derived from liver metastases of a pancreatic tumor). Severe pooled immunodeficient female mice are implanted (subcutaneously) with PA-0165 tumor tissue 18 days prior to the first mAb dosing starting at day 0. Treatment with mAb with GDF15_001 (10 mg/kg, PC, Q3D) or IgG control is continued until death or euthanasia (defined by ill health and/or >30% weight loss according to Institutional Animal Care and Use Committee guidelines). n=17 per group. Kaplan-Meier survival curves are compared to failure time data using log-rank statistics to compare survival rates among treatment groups. * p<0.01 to PA-0165+GDF15_001.

FIG. 19 is a graph depicting mouse plasma concentrations of GDF15 in RENCA tumor bearing mice (murine kidney adenocarcinoma cell line) under thermoneutral conditions (86°F). Balb/c female mice are implanted (subcutaneously) with RENCA cells and GDF15 in plasma measured at the end of the study (corresponding to Figure 17) using ELISA (R&D Systems MGD150). The values are average. n=10-12 per group. ANOVA with Kenward-Roger's adjustment for non-uniform variance is used to compare cumulative food intake and natural logarithmic transformation of GDF15 in plasma between treatment groups using Tukey-Kramer's multiple comparison adjustment to control for group error. Geometric means are obtained for natural log-transformed GDF15 means and 95% confidence interval estimates. All response variables are assessed for compliance with the normality assumption using the Shapiro-Wilks test and Q-Q plots. * p<0.0001 different from NTB + vehicle, ‡ p<0.0001 different from NTB + Sorafenib, † p<0.01 to RENCA + Sorafenib + IgG.

FIG. 20 is a graph depicting the ability of GDF15_001 to reverse weight loss in RENCA tumor mice (murine kidney adenocarcinoma cell line) with an anticancer agent and housed under thermoneutral conditions (86°F). Arrows indicate the times of subcutaneous implantation of RENCA cells (day -12), first dosing of Sorafenib (day -2) and first dosing of mAb (day 0). Balb/c female mice are treated with Sorafenib (15 mg/kg, PO, QD) and GDF15_001 (10 mg/kg, PO, Q3D) or IgG control. Values are mean ± SOS. n=10-12 per group. Repeated measures ANOVA with a heterogeneous autoregressive covariance design (1) is used to compare percent change from base masses between treatment groups during days 3-7 and 13-17 of the dosing period. The Tukey-Kramer multiple comparison test is used to control for the group error rate for comparing treatment groups in repeated measures ANOVA. ** p<0.0001 different from NTB+Vehicle (days 3-7), * p<0.0001 different from NTB+Sorafenib (days 13-17), †p<0.0001 to RENCA+Sorafenib+GDF15_001 ( days 13-17).

FIG. 21 is a graph depicting the ability of GDF15_001 to prolong survival in RENCA (murine kidney adenocarcinoma cell line) tumor bearing mice treated with an anticancer agent and housed under thermoneutral (86°F) conditions. Balb/c female mice are implanted (subcutaneously) with RENCA cells 10 days before the first dosing of Sorafenib, after the first mAb dosing 2 days later (corresponding to day 0). Sorafenib (15 mg/kg, PO, QD) and mAb treatment with GDF15_001 (10 mg/kg, PO, Q3D) or IgG control continues until death or euthanasia (defined by ill health and/or >30% weight loss according to Institutional Animal guidelines) Care and Use Committee). n=10-12 per group. Kaplan-Meier survival curves are compared to failure time data using log-rank statistics to compare survival rates between treatment groups. *p<0.01 for RENCA+Sorafenib+GDF15_001.

FIG. 22 is a graph depicting human plasma concentration of GDF15 in mice bearing NSX-26115 tumor (derived from human non-small cell lung carcinoma adenocarcinoma). Severe combined immunodeficient female mice are implanted (subcutaneously) with NSX-26115 tumor tissue and plasma GDF15 is measured at the end of the study (corresponding to Figure 20) using ELISA (R&D Systems DGD150). n=10-12 per group. Kenward-Roger ANOVA for heterogeneous variants is used to compare plasma levels of GDF15 between different treatment groups. Normality is assessed using histograms and the Shapiro-Wilk test, and probability homogeneity is assessed using Levene's test. Pairwise comparisons of treatment groups are adjusted for multiple comparisons using the Tukey-Kramer method. * p<0.01 to all other groups.

FIG. 23 is a graph depicting the ability of GDF15_001 to reverse weight loss in mice bearing NSX-26115 tumor (derived from human non-small cell lung carcinoma adenocarcinoma) with or without an anticancer agent. Arrows indicate the time points for subcutaneous implantation of NSX-26115 tumor tissue (day -30), first cisplatin dosing (day 0), and first mAb dosing (day 0). Female mice with severe combined immunodeficiency are treated with cisplatin (5 mg/kg, WB, Q7D) and/or GDF15_001 (10 mg/kg, PC, Q3D) or IgG control. Values are mean ± SOS. n=10-12 per group. Repeated ANOVA with a first order autoregressive covariance structure is used to compare percent change in body weight over time between treatment groups using data for days 17-19. At this time, the disease was in a stable state with no significant interaction between treatment and day. Multivariate normality is assessed using Mardia's tests for skewness and kurtosis. A one-way treatment ANOVA was performed on day 10 to evaluate the difference between treatment groups on the day of peak cisplatin exposure, as reported by the dosing regimen. All pairwise comparisons of treatment groups are adjusted for multiple comparisons using the Tukey-Kramer method. * p<0.0001 to NTB+Vehicle (days 17-19), ‡ p<0.0001 to NSX-26115+GDF15_001 (days 17-19), † p<0.001 to NTB+Cisplatin (days 17-19) , § p<0.0001 to NSX-26115+Cisplatin+GDF15_001 (days 17-19), ‡‡ p<0.01 to NTB+Vehicle (day 10), ** p<0.0001 to NSX-26115+ IgG (day 10).

FIG. 24 is a graph depicting the ability of GDF15_001 to prolong survival in mice bearing NSX-26115 tumor (derived from human non-small cell lung carcinoma adenocarcinoma) treated with an anticancer agent. Severe combined immunodeficient female mice are implanted (subcutaneously) with NSX-26115 tumor tissue 30 days prior to the first dose of Cisplatin and mAb (corresponding to day 0). Cisplatin (5 mg/kg, WB, Q7D) and mAb treatment with GDF15_001 (10 mg/kg, PC, Q3D) or IgG control until death or euthanasia (defined by ill health and/or >30% weight loss according to Institutional Animal guidelines) Care and Use Committee). n=10-12 per group. Kaplan-Meier survival curves using log-rank statistics are used to compare survival to premature euthanasia between groups. *p<0.0001 to NSX-26115+Cisplatin+GDF15_001.

FIG. 25 is a graph suggesting a weekly subcutaneous dose capable of reducing free GDF15 levels to less than 0.5 ng/mL in a subject, by dosing interval, for GDF15_001 as a function of the subject's baseline free GDF15 level.

FIG. 26 is a graph suggesting a weekly subcutaneous dose capable of reducing free GDF15 levels to less than 0.5 ng/mL in a subject, by dosing interval, for GDF15_001 as a function of the subject's baseline free GDF15 level.

FIG. 27 is a graph suggesting a weekly subcutaneous dose capable of reducing free GDF15 levels to less than 0.5 ng/mL in a subject, by dosing interval, for GDF15_001 as a function of the subject's baseline free GDF15 level.

FIG. 28 is a table showing SEQ ID NOs corresponding to GDF15 antibodies of the invention.

FIG. 29 is a graph summarizing the results of anti-GDF15 treatment (solid triangles), anti-CD40 antibody treatment (open circles), and combination treatment (inverted open triangles). MHCII expression levels are presented as the mean fluorescence intensity (MFI) of the population of macrophages infiltrating the tumor.

FIG. 30 is a graph summarizing the results of anti-GDF15 treatment (solid triangles), anti-CD40 antibody treatment (open circles), and combination treatment (open triangles).

FIG. 31 is a graph summarizing the results of anti-GDF15 treatment (inverted solid triangles), anti-CD40 antibody treatment (open triangles), and combination treatment (solid squares).

FIG. 32 is a graph summarizing the results of anti-GDF15_297 antibody treatment (open circles), anti-GD15_001 antibody treatment (open triangles).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to GDF15 and reduce or inhibit the activity of GDF15, including, but not limited to, the ability of GDF15 to interact with a GDNF family α receptor-like protein (GFRAL). The invention also provides methods for obtaining, preparing or producing GDF15 antibodies. Antibodies of the invention are useful in the diagnosis, prevention and/or treatment of disorders or conditions mediated or associated with GDF15 activity including, but not limited to, hyperproliferative diseases characterized by GDF15, muscle wasting, body weight loss, fat loss, reduced food intake and the like. The invention also encompasses the expression of antibodies and the preparation and manufacture of compositions containing the antibodies of the invention or antigen-binding fragments thereof, such as medicaments for the use of the antibodies.

Provided are polynucleotides encoding antibodies that bind GDF15 or antigen-binding portions thereof. Also provided are polynucleotides encoding the heavy chain or light chain of an antibody, or both. Presented are host cells that express anti-GDF15 antibodies. Methods of treatment using antibodies to GDF15 are presented. Such methods include, but are not limited to, methods for treating diseases associated with or mediated by GDF15 expression and/or GDF15 binding to GFRAL, including, but not limited to, inflammatory and immune diseases and hyperproliferative disorders.

The section headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter described.

All references cited herein, including patent applications, patent publications, and Genbank access numbers, are incorporated herein by reference as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety.

The methods and procedures described or referred to herein are generally well understood and commonly used by those skilled in the art using conventional methodology, such as the widely used methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3 ^rd edition ( 2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel, et al. eds., (2003)); in the METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (A. Doyle, JB Griffiths and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al, eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993); and their updated versions.

Antibodies

An “antibody” or “Ab” is an immunoglobulin molecule capable of recognizing and binding to a specific target or antigen (Ag), such as a carbohydrate, polynucleotide, fat, polypeptide, etc., through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. As used herein, the term "antibody" may encompass any type of antibody, including, but not limited to, monoclonal antibodies, polyclonal antibodies, antigen-binding fragments (or portions) of intact antibodies that retain the ability to specifically bind to a given antigen (e.g., GDF15).

The term "antigen" refers to a molecular entity used to immunize an immunocompetent vertebrate to produce an antibody that recognizes Ag, or to screen an expression library (eg, phage, yeast, or ribosome display libraries, among others). In this document, Ag is referred to more broadly and is generally intended to include target molecules that are specifically recognized by Ab, thus including fragments or molecular mimetics used in the immunization process to increase Ab or in library screening for Ab selection. Thus, for antibodies of the invention that bind to GDF15, full-length mammalian species GDF15 (e.g., human, monkey, mouse, and rat GDF15), including their monomers and multimers such as dimers, trimers, etc., as well as truncated and other variants of GDF15 are called an antigen.

An "antigen-binding fragment" of an antibody refers to a fragment of a full length antibody that retains the ability to specifically bind to an antigen (preferably with substantially the same binding affinity). Examples of an antigen-binding fragment include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of VL and VH domains of one arm of an antibody, (v) a dAb fragment (Ward et al., 1989 Nature 341:544-546) which consists of a VH domain; and (vi) isolated complementarity determining region (CDR), disulfide-linked Fvs (dsFv) and anti-idiotypic (anti-Id) antibodies and intrabodies. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be combined, using recombinant methods, with a synthetic linker that allows them to form a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single chain Fv (scFv)); see, for example, Bird et al. Science 242:423-426 (1988) and Huston et al., 1988, Proc. Natl. Acad. sci. USA 85:5879-5883. Other forms of single chain antibodies, such as diabodies, are also covered. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on the same polypeptide chain, but using a linker that is too short to allow pairing between two domains on the same chain, thereby causing the domains to pair with the complementary domains of the other. chains and create two antigen binding sites (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994, Structure 2:1121-1123).

The "variable domain" of an antibody refers to an antibody light chain variable region (VL) or an antibody heavy chain variable region (VH), either alone or in combination. As is known in the art, the heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs) and participate in the formation of the antigen-binding site of an antibody.

"Complementarity determining regions" (CDRs) may be identified according to the Cabot, Chothia, Cabot and Chothia co-definitions, AbM, contact, North, and/or conformational definitions, or any method of CDR determination well known in the art. See, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, ^5th ed. (hypervariable regions); Chothia et al., 1989, Nature 342:877-883 (structural loop structures). The identity of the amino acid residues in a particular antibody that make up the CDR can be determined using methods well known in the art. The AbM CDR definition is a compromise between Cabot and Chothia and uses Oxford Molecular's AbM antibody modeling software (Accelrys®). The definition of CDR "contact" is based on the observed antigen contacts described in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. The "conformational" definition of CDRs is based on residues that make an enthalpy contribution to antigen binding (see, for example, Makabe et al., 2008, J. Biol. Chem., 283:1156-1166). North has identified canonical CDR conformations using different preferred sets of CDR definitions (North et al., 2011, J. Mol. Biol. 406: 228-256). In another approach, referred to herein as "conformational definition" of CDRs, CDR positions can be identified as residues that contribute enthalpy to antigen binding (Makabe et al., 2008, J Biol. Chem. 283:1156-1166). Other delimiting definitions of CDRs may not strictly follow one of the above approaches, but will nonetheless overlap with the Cabot part of the CDR, although they may be shortened or lengthened in light of predictions or experimental results regarding particular residues or groups of residues, or even complete CDRs that do not significantly affect antigen binding. As used herein, CDRs may refer to CDRs determined by any approach known in the art, including combinations of approaches. The methods used herein may apply CDRs defined by any of these approaches. For any given embodiment involving more than one CDR, the CDRs (or other antibody residues) can be determined by any of Cabot, Chothia, North, extended, AbM, contact, and/or conformational definitions.

"Framework" (FR) residues are residues of the variable domain of an antibody other than CDR residues. The framework of the VH or VL domain contains four framework sub-regions, FR1, FR2, FR3 and FR4, arranged alternately with the CDR in the following structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.

As known in the art, the "constant region" of an antibody refers to an antibody light chain constant region or an antibody heavy chain constant region, alone or in combination.

The terms "Fc region", "Fc domain", and "Fc", used interchangeably herein, refer to the portion of an immunoglobulin (Ig) molecule that correlates with a crystallizable fragment produced by the papain digest of the Ig molecule. As used herein, refers to the constant region of an antibody, excluding the first domain of the immunoglobulin constant region, and also refers to portions of this region. Thus, Fc refers to the last two IgA constant region domains, IgD and IgG, and the last three IgE and IgM constant region domains and the flexible hinge N-terminal to these domains, or portions thereof. For IgA and IgM, Fc may include a J chain.

For IgG, Fc contains the immunoglobulin domains Cγ2 and Cγ3 (C gamma 2 and C gamma 3) and a hinge between Cγ1 (C gamma 1) and Cγ2 (C gamma 2). Although the boundaries of the Fc region may vary, the Fc region of the human IgG heavy chain is generally defined as containing C226 or P230 residues towards its carboxyl terminus, where numbering is according to the EU index from Edelman et al., 1969, Proc. Natl. Acad. sci. USA 63(1):78-85 as described in Kabat et al., 1991. Typically, the Fc domain contains amino acid residues from about 236 to about 447 of the human IgG1 constant domain. An exemplary amino acid sequence of the Fc domain of human wild-type IgG1 is shown in SEQ ID NO:31. An Fc polypeptide may refer to this region in isolation, or this region in the context of an antibody or antigen-binding portion thereof, or an Fc fusion protein.

The heavy chain constant domain contains an Fc region and further comprises a CH1 domain and a hinge, as well as CH2 and CH3 (and optionally CH4 IgA and IgE) IgG heavy chain domains.

In some embodiments, the antibody or antigen-binding fragment described herein contains an Fc domain. The Fc domain can be derived from IgA (eg, IgA ₁ or IgA ₂ ), IgD, IgE, IgM, or IgG (eg, IgG ₁ , IgG ₂ , IgG ₃ , or IgG ₄ ).

An "Fc fusion" protein may be a protein wherein one or more polypeptides are operably linked to an Fc polypeptide. An Fc fusion combines the Fc regions of an immunoglobulin with a fusion partner.

"Epitope" refers to the site or region of the antigen to which the antibody specifically binds, for example, the site or region containing residues that interact with the antibody. Epitopes may be linear or conformational.

At the most detailed level, an epitope for interaction between Ag and Ab can be defined by spatial coordinates defining the atomic contacts present in the Ag-Ab interaction, as well as information about their relative contribution to binding thermodynamics. At a less detailed level, an epitope can be characterized by spatial coordinates defining atomic contacts between Ag and Ab. At an even finer level, an epitope can be characterized by the amino acid residues it contains, as defined by specific criteria, such as the distance between atoms (eg, heavy, ie, non-hydrogen atoms) in Ab and Ag. At an even finer level, an epitope can be characterized in terms of function, such as competitive binding to other Abs. An epitope can also be defined more generally as containing amino acid residues whose replacement with a different amino acid will change the characteristics of the interaction between Ab and Ag (eg using alanine scanning).

From the fact that descriptions and definitions of epitopes, depending on the epitope mapping method used, are obtained at different levels of detail, it follows that comparison of epitopes for different Abs on the same Ag can similarly be carried out at different levels of detail.

Epitopes described at the amino acid level, such as those defined by x-ray structure, are considered identical if they contain the same set of amino acid residues. Epitopes are considered overlapping if at least one amino acid is shared between the epitopes. Epitopes are considered separate (unique) if the epitopes do not share common amino acid residues.

Epitopes with competitive binding are considered overlapping if the binding of the respective antibodies is mutually exclusive, i.e. binding of one antibody precludes simultaneous or sequential binding of another antibody. Epitopes are considered separate (unique) if the antigen is able to simultaneously bind both corresponding antibodies.

An antibody that "preferentially binds" or "specifically binds" (used interchangeably herein) to an epitope is a term well known in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is considered to exhibit "specific binding" or "preferential binding" if it interacts or binds more frequently, faster, for longer duration, and/or with greater affinity for a particular cell or substance than for alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more easily and/or longer than with other substances. For example, an antibody that specifically or preferentially binds to a GDF15, PD-1, or PD-L1 epitope is an antibody that binds that epitope with greater affinity, avidity, more readily, and/or for a longer duration than it binds to another GDF15 epitope. , PD-1 or PD-L1 or non-GDF15, PD-1, PD-L1 epitopes. Usually, but not necessarily, a binding reference means a preferred binding. "Specific binding" or "preferential binding" includes a compound, such as a protein, nucleic acid, antibody, and the like, that recognizes and binds to a particular molecule in a sample, but does not substantially recognize or bind other molecules in the sample. For example, an antibody or peptide receptor that recognizes and binds to a cognate ligand or binding partner (e.g., a human tumor antigen anti-human antibody that binds a tumor antigen, a PD-1 molecule that binds PD-L1 or PD-L2, etc.) d) in the sample, but does not substantially recognize or bind other molecules in the sample, specifically binds to that cognate ligand or binding partner. Thus, under the indicated assay conditions, said binding group (e.g., an antibody or antigen-binding portion thereof, or a receptor or ligand-binding portion thereof) preferentially binds to a particular target molecule and does not bind in significant amounts to other components present in the test sample.

A variety of assay formats can be used to select an antibody or peptide that specifically binds to a molecule of interest. For example, ELISA, immunoprecipitation, BIAcore™ (GE Healthcare, Piscataway, NJ), fluorescence-activated cell sorting (FACS), Octet™ (FortéBio, Inc., Menlo Park, CA) and Western blotting are among the many assays which can be used to identify an antibody that specifically interacts with an antigen or receptor, or a ligand-binding portion thereof, that specifically binds to a cognate ligand or binding partner. Typically, a specific or selective response will be at least twice the background signal or noise, and more often more than 10 times the background, even more specifically, an antibody is said to "specifically bind" an antigen if the equilibrium dissociation constant (K _D ) is ≤1 μM, preferably ≤100 nM, more preferably ≤10 nM, even more preferably ≤100 pM, even more preferably ≤10 pM, and even more preferably ≤1 pM.

The term "compete" as used herein with respect to an antibody means that binding of the first antibody or antigen-binding portion to an antigen reduces subsequent binding of the same antigen by a second antibody or antigen-binding portion thereof. In general, binding of the first antibody creates a steric hindrance, a conformational change, or binding to a common epitope (or portion thereof) such that the binding of the second antibody to the same antigen is reduced. Standard competition assays can be used to determine if two antibodies compete with each other. One suitable antibody competition assay involves the use of Biacore technology, which can measure the extent of interactions using surface plasmon resonance (SPR) technology, typically using a biosensor system (such as the BIACORE system). For example, SPR can be used in an in vitro competitive binding inhibition assay to determine the ability of one antibody to inhibit the binding of a second antibody. Another assay for measuring antibody competition uses an ELISA based approach.

In addition, a high throughput process for "sorting" antibodies based on their competition is described in International Patent Application No. WO2003/48731. Competition is present if one antibody (or fragment) reduces the binding of another antibody (or fragment) to GDF15. For example, a competitive sequential binding assay with sequential addition of different antibodies can be used. The first antibody may be added to achieve near saturation binding. Then a second antibody is added. If the binding of the second antibody to GDF15 is not detected or significantly reduced (e.g., reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%) compared to a parallel assay with no first antibody (the value of which can be set to 100%), the two antibodies are considered to compete with each other.

A variant antibody may contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and/or deletions and/or insertions from certain sequences and fragments discussed above. "Deletion" variants may include deletion of single amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of large amino acid regions such as deletion of certain amino acid domains or other features. "Insert" variants may include the insertion of single amino acids, the insertion of small groups of amino acids, such as 2, 3, 4, or 5 amino acids, or the insertion of large amino acid regions, such as the insertion of specific amino acid domains or other features. Variants of "substitution" preferably include replacing one or more amino acids with the same number of amino acids and making conservative substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. . Some properties of the 20 basic amino acids that can be used to select suitable substituents are as follows.

Substitutional variants have at least one amino acid residue removed from the antibody molecule and another residue inserted in its place. Sites of greatest interest for displacement mutagenesis include hypervariable regions, but framework changes are also considered. Conservative substitutions are shown in Table 1 under the heading "conservative substitutions". If such substitutions result in a change in biological activity, then more significant changes can be made, referred to as "type substitutions" shown below, or, as further described below with reference to amino acid classes, can be introduced and products screened.

TABLE 1
Amino acids and substitutions original balance Conservative substitutions Type substitutions alanine Ala(A) Val Val; Leu; ile arginine Arg(R) Lys Lys; gln; Asn asparagine Asn(N) Gln gln; His; Asp, Lys; Arg aspartic acid Asp(D) Glu Glu; Asn cysteine Cys(C) Ser Ser; Ala glutamine Gln (Q) Asn asn; Glu glutamic acid Glu(E) asp asp; Gln glycine Gly(G) Ala Ala histidine His(H) Arg asn; gln; Lys; Arg Isoleucine Ile(I) Leu Leu; Val; met; Ala; Ph; Norleucine leucine Leu(L) ile Norleucine; Ile; Val; met; Ala; Phe lysine Lys(K) Arg Arg; gln; Asn methionine Met(M) Leu Leu; Ph; ile phenylalanine Phe(F) Tyr Leu; Val; Ile; Ala; Tyr Proline Pro (P) Ala Ala serine Ser(S) Thr Thr threonine Thr(T) Ser Ser tryptophan Trp(W) Tyr Tyr; Phe tyrosine Tyr(Y) Phe trp; Ph; Thr; Ser valine Val(V) Leu Ile; Leu; met; Ph; Ala; Norleucine

Significant modifications of the biological properties of an antibody are achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of substitution, for example, in the form of a beta-sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the main part of the side chain. Naturally occurring residues are divided into groups based on common side chain properties:

Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;

Polar without charge: Cys, Ser, Thr, Asn, Gln;

Acidic (negatively charged): Asp, Glu;

Basic (positively charged): Lys, Arg;

Residues that affect chain orientation: Gly, Pro; And

Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by replacing a member of one of these classes with another class.

One type of substitution, for example, which can be carried out is to replace one or more cysteines in the antibody, which may be chemically reactive, with another residue such as, but not limited to, alanine or serine. For example, a substitution for a non-canonical cysteine is possible. The substitution may be in the CDR or framework region of a variable domain or in the constant region of an antibody. In some embodiments, the cysteine is canonical. Any cysteine residue not involved in maintaining the proper conformation of the antibody may also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to an antibody to improve its stability, especially if the antibody is an antibody fragment, such as an Fv fragment.

In a process known as "humanization", certain amino acids in the VH and VL sequences can be mutated to match those found in nature and the VH and VL germline sequences. In particular, the amino acid sequences of the framework regions in the VH and VL sequences can be mutated to match the germline sequences to reduce the risk of immunogenicity upon administration of the antibody. As used herein, the term "germline" refers to the nucleotide sequences and amino acid sequences of antibody genes and gene segments as they are passed from parent to offspring via germ cells. This germline sequence differs from the nucleotide sequence encoding antibodies in mature B cells, which have been altered by recombination and hypermutation events during maturation of B cells. An antibody that "uses" a particular germline has a nucleotide or amino acid sequence that most closely matches the nucleotide sequence or amino acid sequence of the germline it defines. Such antibodies often mutate from the germline sequence. The germline DNA sequences for the human VH and VL genes are known in the art (see, e.g., "Vbase" human germline sequence database; see also Kabat, E. A., et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Tomlinson et al., J. Mol. Biol. 24:827-836, 1994.)

Binding affinity

The binding affinity of an antibody can be expressed as a K _D value, which refers to the rate of dissociation of a particular antigen-antibody interaction. K _D is the ratio of the degree of dissociation, also called "rate of dissociation (k _off )" to the degree of association or "rate of association (k _on )". Thus, K _D is equal to k _off /k _on and is expressed as a molar concentration (M), and the smaller the K _D , the stronger the binding affinity. Antibody K _D values can be determined using methods well known in the art. One method for measuring Kd is surface plasmon resonance (SPR), typically using a biosensor system such as the BIACORE® system. BIAcore kinetic analysis includes analysis of antigen binding and dissociation from chips with immobilized molecules (eg, molecules containing epitope containing domains) on their surface. Another method for determining the Kd of an antibody involves the use of Biolayer Interferometry, typically using OCTET technology (Octet QKe system, ForteBio). Alternatively or in addition, the KinExA assay (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Id.) may also be used.

Anti-GDF15 antibodies

The invention provides anti-GDF15 antibodies. An anti-GDF15 antibody, preferably a high affinity antibody, can be effective in plasma and multiple tissue compartments where GDF15 is thought to act on its target cells. The antibodies of the invention have the potential to modify the pathway that controls the development and progression of cachexia associated with cancer, heart failure, or COPD, among others.

A neutralizing or "blocking" antibody refers to an antibody whose binding to GDF15 interferes, limits or inhibits the interaction between GDF15 or a GDF15 fragment and a GDF15 receptor such as GFRAL or a component of the GDF15 receptor; and/or (ii) results in inhibition of at least one biological function of GDF15. Assays for determining neutralization with an antibody of the invention are described elsewhere herein and are well known in the art.

As used herein, the term "GDF15" includes variants, isoforms, homologs, orthologs, and paralogs of human GDF15. In some aspects of the invention, the antibodies cross-react with non-human GDF15 species, such as mouse, rat, or non-human primate GDF15, as well as various forms of GDF15. In other aspects, the antibodies may be fully specific for human GDF15 and may not show species or other type of cross-reactivity. As used herein, the term GDF15 refers to naturally occurring human GDF15 unless otherwise specified by the context. Therefore, "GDF15 antibody", "anti-GDF15 antibody" or other similar designation means any antibody (as defined herein) that specifically associates, binds or interacts with a GDF15 type ligand or isoform, or a fragment or derivative thereof. The full-length mature form of human GDF15, provided under UniProtKB/Swiss-Prot accession number Q99988.1, is provided herein as SEQ ID NO: 1.

Without wishing to be bound by any particular theory, when interacting with GDF15, GFRAL interacts with the proto-oncogene receptor encoding tyrosine protein kinase Ret (RET) and induces cellular signaling through activation of the MAPK- and AKT- signaling pathways. RET signaling then induces or mediates phosphorylation of, for example, ERK, S6, among others. As used herein, the term "GFRAL" includes variants, isoforms, homologs, orthologs, and paralogs of human GFRAL. The full-length mature form of human GFRAL is represented by accession number UniProtKB/Swiss-Prot Q6UXV0. As used herein, the term "RET" includes variants, isoforms, homologs, orthologs, and paralogs of human RET. The full-length mature form of human RET is represented by accession number UniProtKB/Swiss-Prot P07949.

The "biological function" or "biological activity" of GDF15 includes the regulation of inflammatory and apoptotic pathways in tissues and the program of cellular response to stress after cellular injury. The "biological function" or "biological activity" of GDF15 includes indirect increases in: cachexia, reduced food intake, reduced appetite, decreased body weight, body weight loss, decreased fat mass, decreased muscle mass, GFRAL binding, RET activation, ERK phosphorylation, and S6 phosphorylation , among others currently known in the art or later identified. The biological function or biological activity of GDF15 may, but need not, be mediated by an interaction between GDF15 and its cognate receptor GFRAL.

The invention includes an antibody, or an antigen-binding portion thereof, that can modulate the biological activity of GDF15. That is, the invention includes an isolated antibody, or an antigen-binding portion thereof, that specifically binds to GDF15 and modulates at least one detectable GDF15 activity such that the antibody: (a) increases food intake; (b) increases appetite; (c) increases body weight; (d) reduces body weight loss; (e) increases fat mass; (f) increases muscle mass; (g) reduces fat loss, (h) reduces muscle loss, (i) reduces GDF15 binding to GFRAL; (j) reduces subsequent signaling mediated by RET; (k) reduces or inhibits ERK phosphorylation; (l) reduces or inhibits S6 phosphorylation; (m) reduces RET activation of the MAPK signaling pathway; (n) reduces RET activation of the AKT signaling pathway; and/or (o) reduces the activation of the PLC-γ1 signaling pathway.

Biological activity of GDF15 and GDF15-dependent signaling activity can be assessed in vitro using HEK293 or CHO cells co-expressing GFRAL and RET, among many assays known in the art. Activation of the MAPK pathway after GDF15 stimulation can be measured, among others, using a luciferase reporter gene system (eg, PathDetect, Agilent Technologies). Phosphoprotein assays based on time-resolved homogeneous fluorescence technology (Cisbio Inc.) can also be used as orthogonal approaches to measure activation of the MAPK and AKT pathways (eg, phospho-ERK1/2) in response to GDF15 binding to its receptor. The ability of neutralizing antibodies to prevent GDF15-dependent signaling can also be assessed by incubating cells with a fixed concentration of GDF15 in the absence or presence of increasing concentrations of anti-GDF15 antibody.

In one aspect of the invention, a GDF15 antibody of the invention comprises an antibody that competes for binding to human GDF15 c and/or binds the same epitope as the antibody or antigen-binding fragment thereof having the heavy chain variable region amino acid sequence indicated by SEQ ID NO : 166 and the amino acid sequence of the light chain variable region indicated as SEQ ID NO: 163.

In one aspect of the invention, the GDF15 antibody of the invention includes an antibody that inhibits or reduces the binding of GDF15 to GFRAL.

In one aspect, the invention encompasses an antibody that competes with an antibody, or an antigen-binding fragment thereof, having the heavy chain variable region amino acid sequence represented by SEQ ID NO:166 and the light chain variable region amino acid sequence represented by SEQ ID NO:163, in inhibition of GDF15 binding. with GFRAL.

In some aspects of the invention, the antibody, or antigen-binding fragment thereof, comprises an IgG1 heavy chain constant region, such as the GDF15 heavy chain shown in SEQ ID NO:164. In other aspects, the antibody, or antigen-binding fragment thereof, comprises a light chain kappa constant region, such as the GDF15 light chain represented by SEQ ID NO:162.

Table 2 lists the amino acid (protein) sequences and associated nucleic acid (DNA) sequences of an anti-GDF15 antibody of the present invention. CDR VH anti-GDF15 and VL anti-GDF15, as determined by Cabot and Chothia, are presented as separate sequences.

In some aspects, the CDRs comprise SEQ ID NOs: 171, 172, 173, 174, 175, and 176. These CDR sequences include consensus based on favorable sequence analysis and biophysical profile data presented in Examples 1-10 below. These CDR sequences offer advantages based on their sequence, binding, thermal stability, low pH stability and viscosity profiles.

Table 2 . Sequences of GDF15 peptides and anti-GDF15 antibodies SEQID NO Description Subsequence 1 human GDF15, mature form ARNGDHCPLG PGRCCRLHTV RASLEDLGWA DWVLSPREVQ VTMCIGACPS QFRAANMHAQ IKTSLHRLKP DTVPAPCCVP ASYNPMVLIQ KTDTGVSLQT YDDLLAKDCH CI 2 Dimeric mouse IgG1
Fc-human GDF-15 with FXa cleavage site GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLN VQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKIEGRMDGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 3 CH23 Human Fc-GDF15 with TEV cleavage site GGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVNLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLNSTLTVDKSRWQQGNV FSCSVLHEALHSHYTQKSLSLSPKGSENLYFQGARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 4 Dimeric mouse IgG1
Fc-cynomolgus monkey GDF-15 with FXa cleavage site GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLN VQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKIEGRMDGGGGSARNGDRCPLGPGRCCRLHTVHASLEDLGWADWVLSPREVQVTMCIGACPSQFREANMHAQIKMNLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCV 5 Dimeric mouse IgG1
GDF-15 mouse Fc with FXa cleavage site GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLN VQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKIEGRMDGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 6 GDF15_200LC EIVLTQSPAT LSLSPGERAT LSCRASQSVH SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSWPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 7 GDF15_200 LCDR-1LCDR-1 RASQSVHSYL A 8 GDF15_010 LCDR-2
GDF15_013 LCDR-2
GDF15_014 LCDR-2
GDF15_200 LCDR-2 DASNRAT 9 GDF15_001 LCDR-3
GDF15_002 LCDR-3
GDF15_005 LCDR-3
GDF15_007 LCDR-3
GDF15_009 LCDR-3
GDF15_200 LCDR-3 QQFWSWPWT 10 LC CL RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC eleven GDF15_200 VL EIVLTQSPAT LSLSPGERAT LSCRASQSVH SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSWPWTFGQ GTKVEIK 12 IgG1 JK light chain FGQGTKVEIK R 13 IgG1 Heavy chain Hinge epkscdktht cppcp 14 IgG1 CH2 heavy chain apeaagapsv flfppkpkdt lmisrtpevt cvvvdvshed pevkfnwyvd gvevhnaktk preeqynsty rvvsvltvlh qdwlngkeyk ckvsnkalpa piektiskak 15 Heavy chain CH3 gqprepqvyt lppsreemtk nqvsltclvk gfypsdiave wesngqpenn ykttppvlds dgsfflyskl tvdksrwqqg nvfscsvmhe alhnhytqks lslspg 16 GDF15_200HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNISWVRQA PGQGLEWMGG INPINGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 17 GDF15_200HCDR-1 GYTFSSYNIS 18 GDF15_200HCDR-2 GINPINGLAF YNQKFQG 19 GDF15_007 HCDR-3
GDF15_010 HCDR-3
GDF15_013 HCDR-3
GDF15_014 HCDR-3
GDF15_017 HCDR-3
GDF15_018 HCDR-3
GDF15_020 HCDR-3
GDF15_100 HCDR-3
GDF15_200HCDR-3 EAITTVGAMD Y 20 Heavy chain CH1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKK 21 GDF15_200 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNISWVRQA PGQGLEWMGG INPINGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS 22 Frame H1 QVQLVQSGAE VKKPGSSVKV SCKAS 23 Frame H2 WVRQAPGQGL EWMG 24 Frame H3 RVTITADEST STAYMELSSL RSEDTAVYYC AR 25 JH WGQGTLVTVS S 26 GDF15_100LC EIVLTQSPAT LSLSPGERAT LSCRTSQNVH SYLAWYQQKP GQAPRLLIYD ASTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSDPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 27 GDF15_008 LCDR-1LCDR-1
GDF15_009 LCDR-1
GDF15_100 LCDR-1 RTSQNVHSYL A 28 GDF15_001 LCDR-2
GDF15_004 LCDR-2
GDF15_012 LCDR-2
GDF15_018 LCDR-2
GDF15_020 LCDR-2
GDF15_100 LCDR-2 DASTRAD 29 GDF15_100 LCDR-3 QQFWSDPWT thirty GDF15_100 VL EIVLTQSPAT LSLSPGERAT LSCRTSQNVH SYLAWYQQKP GQAPRLLIYD ASTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSDPWTFGQ GTKVEIK 31 GDF15_100HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGQ INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 32 GDF15_001 HCDR-1
GDF15_002 HCDR-1
GDF15_004 HCDR-1
GDF15_021 HCDR-1
GDF15_100HCDR-1 GYTFSSYNID 33 GDF15_003 HCDR-2
GDF15_009 HCDR-2
GDF15_015 HCDR-2
GDF15_017HCDR-2
GDF15_100HCDR-2 QINPNNGLAF YNQKFQG 34 GDF15_100 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGQ INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS 35 GDF15_022LC EIVLTQSPAT LSLSPGERAT LSCRTSQSVH SYLAWYQQKP GQAPRLLIYD AKTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FSSDPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSST LT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 36 GDF15_022 LCDR-1 RTSQSVHSYL A 37 GDF15_005 LCDR-2
GDF15_022 LCDR-2 DAKTRAD 38 GDF15_003 LCDR-3
GDF15_012 LCDR-3
GDF15_017 LCDR-3
GDF15_022 LCDR-3 QQFSSDPYT 39 GDF15_022 VL EIVLTQSPAT LSLSPGERAT LSCRTSQSVH SYLAWYQQKP GQAPRLLIYD AKTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FSSDPYTFGQ GTKVEIK 40 GDF15_022HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNIDWVRQA PGQGLEWMGQ INPNNGLIFF NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREV ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSS VVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTK NQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 41 GDF15_010 HCDR-1
GDF15_022 HCDR-1 GYTFSDYNID 42 GDF15_022 HCDR-2 QINPNNGLIF FNQKFQG 43 GDF15_012 HCDR-3
GDF15_022 HCDR-3 EVITTVGAMD Y 44 GDF15_022 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNIDWVRQA PGQGLEWMGQ INPNNGLIFF NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREV ITTVGAMDYW GQGTLVTVSS 45 GDF15_021LC EIVLTQSPAT LSLSPGERAT LSCRTSENVH SYLAWYQQKP GQAPRLLIYD ASNLADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSDPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 46 GDF15_007 LCDR-1
GDF15_021 LCDR-1 RTSENVHSYL A 47 GDF15_021 LCDR-2 DASNLAD 48 GDF15_004 LCDR-3
GDF15_009 LCDR-3
GDF15_014 LCDR-3
GDF15_020 LCDR-3
GDF15_021 LCDR-3 QQFWSDPYT 49 GDF15_021 VL EIVLTQSPAT LSLSPGERAT LSCRTSENVH SYLAWYQQKP GQAPRLLIYD ASNLADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSDPYTFGQ GTKVEIK 50 GDF15_021HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGG INPINGLIFF NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDHW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSS VVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTK NQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 51 GDF15_021 HCDR-2 GINPINGLIF FNQKFQG 52 GDF15_001 HCDR-3
GDF15_021 HCDR-3 EAITTVGAMD H 53 GDF15_021 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGG INPINGLIFF NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDHW GQGTLVTVSS 54 GDF15_020LC EIVLTQSPAT LSLSPGERAT LSCRASQNLH SYLAWYQQKP GQAPRLLIYD ASTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSDPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 55 GDF15_020 LCDR-1 RASQNLHSYL A 56 GDF15_020 VL EIVLTQSPAT LSLSPGERAT LSCRASQNLH SYLAWYQQKP GQAPRLLIYD ASTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSDPYTFGQ GTKVEIK 57 GDF15_020HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNMDWVRQA PGQGLEWMGQ INPNNGLANY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSS VVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTK NQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 58 GDF15_005 HCDR-1
GDF15_012 HCDR-1
GDF15_013 HCDR-1
GDF15_015 HCDR-1
GDF15_020 HCDR-1 GYTFSDYNMD 59 GDF15_020 HCDR-2 QINPNNGLAN YNQKFQG 60 GDF15_020 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNMDWVRQA PGQGLEWMGQ INPNNGLANY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS 61 GDF15_018LC EIVLTQSPAT LSLSPGERAT LSCRASQNVH SYLAWYQQKP GQAPRLLIYD ASTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWNDPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSST LT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 62 GDF15_018 LCDR-1 RASQNVHSYL A 63 GDF15_008 LCDR-3
GDF15_018 LCDR-3 QQFWNDPYT 64 GDF15_018 VL EIVLTQSPAT LSLSPGERAT LSCRASQNVH SYLAWYQQKP GQAPRLLIYD ASTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWNDPYTFGQ GTKVEIK 65 GDF15_018HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYNIDWVRQA PGQGLEWMGQ INPNNGLIFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 66 GDF15_017 HCDR-1
GDF15_018 HCDR-1 GYTFTDYNID 67 GDF15_018 HCDR-2 QINPNNGLIF YNQKFQG 68 GDF15_018 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYNIDWVRQA PGQGLEWMGQ INPNNGLIFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS 69 GDF15_017LC EIVLTQSPAT LSLSPGERAT LSCRTSQSVH SYLAWYQQKP GQAPRLLIYD AKTRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FSSDPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSST LT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 70 GDF15_017 LCDR-2 DAKTRAT 71 GDF15_017 VL EIVLTQSPAT LSLSPGERAT LSCRTSQSVH SYLAWYQQKP GQAPRLLIYD AKTRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FSSDPYTFGQ GTKVEIK 72 GDF15_017HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYNIDWVRQA PGQGLEWMGQ INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED NAVYYCAREA ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 73 GDF15_017 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYNIDWVRQA PGQGLEWMGQ INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED NAVYYCAREA ITTVGAMDYW GQGTLVTVSS 74 GDF15_017 FW_H3 RVTITADEST STAYMELSSL RSEDNAVYYC AR 75 GDF15_015LC EIVLTQSPAT LSLSPGERAT LSCRTSQNVH SYLAWYQQKP GQAPRLLIYD ASNLADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FSNDPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSST LT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 76 GDF15_015 LCDR-3 QQFSNDPWT 77 GDF15_015 VL EIVLTQSPAT LSLSPGERAT LSCRTSQNVH SYLAWYQQKP GQAPRLLIYD ASNLADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FSNDPWTFGQ GTKVEIK 78 GDF15_015HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNMDWVRQA PGQGLEWMGQ INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGATDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 79 GDF15_015 HCDR-3 EAITTVGATD Y 80 GDF15_015 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNMDWVRQA PGQGLEWMGQ INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGATDYW GQGTLVTVSS 81 GDF15_014LC EIVLTQSPAT LSLSPGERAT LSCRTSQNVH NYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSDPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 82 GDF15_014 LCDR-1 RTSQNVHNYL A 83 GDF15_014 VL EIVLTQSPAT LSLSPGERAT LSCRTSQNVH NYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSDPYTFGQ GTKVEIK 84 GDF15_014HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGQ INPINGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 85 GDF15_014 HCDR-2 QINPINGLAF YNQKFQG 86 GDF15_014 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGQ INPINGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS 87 GDF15_013LC EIVLTQSPAT LSLSPGERAT LSCRTSESVH SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWNWPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 88 GDF15_004 LCDR-1
GDF15_013 LCDR-1 RTSESVHSYL A 89 GDF15_013 LCDR-3 QQFWNWPWT 90 GDF15_013 VL EIVLTQSPAT LSLSPGERAT LSCRTSESVH SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWNWPWTFGQ GTKVEIK 91 GDF15_013HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNMDWVRQA PGQGLEWMGG INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 92 GDF15_013 HCDR-2 GINPNNGLAF YNQKFQG 93 GDF15_013 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNMDWVRQA PGQGLEWMGG INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS 94 GDF15_012LC EIVLTQSPAT LSLSPGERAT LSCRTSQSVH NYLAWYQQKP GQAPRLLIYD ASTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FSSDPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 95 GDF15_001 LCDR-1
GDF15_012 LCDR-1 RTSQSVHNYL A 96 GDF15_012 VL EIVLTQSPAT LSLSPGERAT LSCRTSQSVH NYLAWYQQKP GQAPRLLIYD ASTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FSSDPYTFGQ GTKVEIK 97 GDF15_012HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNMDWVRQA PGQGLEWMGQ INPIFGLAFY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREV ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 98 GDF15_012 HCDR-2 QINPIFGLAF YAQKFQG 99 GDF15_012 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNMDWVRQA PGQGLEWMGQ INPIFGLAFY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREV ITTVGAMDYW GQGTLVTVSS 100 GDF15_010LC EIVLTQSPAT LSLSPGERAT LSCRTSQSLH SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWNDPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 101 GDF15_010 LCDR-1 RTSQSLHSYL A 102 GDF15_006 LCDR-3
GDF15_010 LCDR-3 QQFWNDPWT 103 GDF15_010 VL EIVLTQSPAT LSLSPGERAT LSCRTSQSLH SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWNDPWTFGQ GTKVEIK 104 GDF15_010HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNIDWVRQA PGQGLEWMGG INPNNGLAFF NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSS VVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTK NQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 105 GDF15_010 HCDR-2 GINPNNGLAF FNQKFQG 106 GDF15_010 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNIDWVRQA PGQGLEWMGG INPNNGLAFF NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS 107 GDF15_009LC EIVLTQSPAT LSLSPGERAT LSCRTSQNVH SYLAWYQQKP GQAPRLLIYD AKNRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSDPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSST LT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 108 GDF15_003 LCDR-2
GDF15_009 LCDR-2 DAKNRAD 109 GDF15_009 VL EIVLTQSPAT LSLSPGERAT LSCRTSQNVH SYLAWYQQKP GQAPRLLIYD AKNRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSDPYTFGQ GTKVEIK 110 GDF15_009HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNISWVRQA PGQGLEWMGQ INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMEYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 111 GDF15_009 HCDR-3 EAITTVGAME Y 112 GDF15_009 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNISWVRQA PGQGLEWMGQ INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMEYW GQGTLVTVSS 113 GDF15_008LC EIVLTQSPAT LSLSPGERAT LSCRTSQNVH SYLAWYQQKP GQAPRLLIYD ASNRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWNDPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSST LT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 114 GDF15_002 LCDR-2
GDF15_008 LCDR-2 DASNRAD 115 GDF15_008 VL EIVLTQSPAT LSLSPGERAT LSCRTSQNVH SYLAWYQQKP GQAPRLLIYD ASNRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWNDPYTFGQ GTKVEIK 116 GDF15_008HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFT SYNISWVRQA PGQGLEWMGQ INPNNGLIFF AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDQW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSS VVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTK NQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 117 GDF15_008 HCDR-1 GYTFTSYNIS 118 GDF15_008 HCDR-2 QINPNNGLIF FAQKFQG 119 GDF15_005 HCDR-3
GDF15_008 HCDR-3 EAITTVGAMD Q 120 GDF15_008 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFT SYNISWVRQA PGQGLEWMGQ INPNNGLIFF AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDQW GQGTLVTVSS 121 GDF15_007LC L SKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 122 GDF15_007 LCDR-2 DASTLAT 123 GDF15_007 VL EIVLTQSPAT LSLSPGERAT LSCRTSENVH SYLAWYQQKP GQAPRLLIYD ASTLATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSWPWTFGQ GTKVEIK 124 GDF15_007HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNISWVRQA PGQGLEWMGG INPIFGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 125 GDF15_007 HCDR-1 GYTFSDYNIS 126 GDF15_002 HCDR-2
GDF15_007 HCDR-2 GINPIFGLAF YNQKFQG 127 GDF15_007 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNISWVRQA PGQGLEWMGG INPIFGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDYW GQGTLVTVSS 128 GDF15_006LC EIVLTQSPAT LSLSPGERAT LSCRTSQSVS NYLAWYQQKP GQAPRLLIYD AKNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWNDPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSST LT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 129 GDF15_006 LCDR-1 RTSQSVSNYL A 130 GDF15_006 LCDR-2 DAKNRAT 131 GDF15_006 VL EIVLTQSPAT LSLSPGERAT LSCRTSQSVS NYLAWYQQKP GQAPRLLIYD AKNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWNDPWTFGQ GTKVEIK 132 GDF15_006HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYNISWVRQA PGQGLEWMGQ INPNNGLAFY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREF ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 133 GDF15_006 HCDR-1 GYTFTDYNIS 134 GDF15_006 HCDR-2 QINPNNGLAF YAQKFQG 135 GDF15_006 HCDR-3 EFITTVGAMD Y 136 GDF15_006 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DYNISWVRQA PGQGLEWMGQ INPNNGLAFY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREF ITTVGAMDYW GQGTLVTVSS 137 GDF15_005LC EIVLTQSPAT LSLSPGERAT LSCRTSESVS SYLAWYQQKP GQAPRLLIYD AKTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSWPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT L SKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 138 GDF15_005 LCDR-1 RTSESVSSYL A 139 GDF15_005 VL EIVLTQSPAT LSLSPGERAT LSCRTSESVS SYLAWYQQKP GQAPRLLIYD AKTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSWPWTFGQ GTKVEIK 140 GDF15_005HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNMDWVRQA PGQGLEWMGG INPNNGTAFY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDQW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 141 GDF15_005 HCDR-2 GINPNNGTAF YAQKFQG 142 GDF15_005 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DYNMDWVRQA PGQGLEWMGG INPNNGTAFY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDQW GQGTLVTVSS 143 GDF15_004LC L SKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 144 GDF15_004 VL EIVLTQSPAT LSLSPGERAT LSCRTSESVH SYLAWYQQKP GQAPRLLIYD ASTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSDPYTFGQ GTKVEIK 145 GDF15_004HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGQ INPNNGLANY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTIGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSS VVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTK NQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 146 GDF15_004 HCDR-2 QINPNNGLAN YAQKFQG 147 GDF15_004 HCDR-3 EAITTIGAMD Y 148 GDF15_004 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGQ INPNNGLANY AQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTIGAMDYW GQGTLVTVSS 149 GDF15_003LC EIVLTQSPAT LSLSPGERAT LSCRASQSLS SYLAWYQQKP GQAPRLLIYD AKNRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FSSDPYTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 150 GDF15_003 LCDR-1 RASQSLSSYL A 151 GDF15_003 VL EIVLTQSPAT LSLSPGERAT LSCRASQSLS SYLAWYQQKP GQAPRLLIYD AKNRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FSSDPYTFGQ GTKVEIK 152 GDF15_003HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFT SYNIDWVRQA PGQGLEWMGQ INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREQ ITTVGAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLY SLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 153 GDF15_003 HCDR-1 GYTFTSYNID 154 GDF15_003 HCDR-3 EQITTVGAMD Y 155 GDF15_003 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFT SYNIDWVRQA PGQGLEWMGQ INPNNGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREQ ITTVGAMDYW GQGTLVTVSS 156 GDF15_002LC EIVLTQSPAT LSLSPGERAT LSCRASQNVH NYLAWYQQKP GQAPRLLIYD ASNRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSWPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 157 GDF15_002 LCDR-1 RASQNVHNYL A 158 GDF15_002 VL EIVLTQSPAT LSLSPGERAT LSCRASQNVH NYLAWYQQKP GQAPRLLIYD ASNRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSWPWTFGQ GTKVEIK 159 GDF15_002HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGG INPIFGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDPW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSL SSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE M TKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 160 GDF15_002 HCDR-3 EAITTVGAMD P 161 GDF15_002 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGG INPIFGLAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDPW GQGTLVTVSS 162 GDF15_001LC EIVLTQSPAT LSLSPGERAT LSCRTSQSVH NYLAWYQQKP GQAPRLLIYD ASTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSWPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 163 GDF15_001VL EIVLTQSPAT LSLSPGERAT LSCRTSQSVH NYLAWYQQKP GQAPRLLIYD ASTRADGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ FWSWPWTFGQ GTKVEIK 95 GDF15_001 LCDR-1 RTSQSVHNYL A 28 GDF15_001 LCDR-2 DASTRAD 9 GDF15_001 LCDR-3 QQFWSWPWT 164 GDF15_001HC QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGG INPIFGTAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDHW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLY SLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG 32 GDF15_001 HCDR-1 GYTFSSYNID 165 GDF15_001 HCDR-2 GINPIFGTAF YNQKFQG 52 GDF15_001 HCDR-3 EAITTVGAMD H 166 GDF15_001 VH QVQLVQSGAE VKKPGSSVKV SCKASGYTFS SYNIDWVRQA PGQGLEWMGG INPIFGTAFY NQKFQGRVTI TADESTSTAY MELSSLRSED TAVYYCAREA ITTVGAMDHW GQGTLVTVSS 167 GDF15_001 VL DNA GAAATTGTGC TGACCCAGAG CCCGGCGACC CTGAGCCTGA GCCCGGGCGA ACGCGCGACC CTGAGCTGCC GCACCAGCCA GAGCGTTCAT AACTATCTGG CGTGGTATCA GCAGAAACCG GGCCAGGCGC CGCGCCTGCT GATTTATGAT GCGAGCACCC GTGCGGATGG CATTCCGGCA CGCTTTAGCG GCAGCGGCAG CGGC ACCGAT TTTACCCTGA CCATTAGCAG CCTGGAACCG GAAGATTTTG CGGTGTATTA TTGCCAGCAG TTTTGGAGCT GGCCGTGGAC CTTTGGCCAG GGCACCAAAG TGGAAATTAAA 168 GDF15_001 VH DNA CAGGTGCAGC TGGTGCAGAG CGGCGCGGAA GTGAAAAAAC CGGGCAGCAG CGTGAAAGTG AGCTGCAAAG CGAGCGGCTA TACCTTTAGC AGCTATAACA TTGATTGGGT GCGCCAGGCG CCGGGCCAGG GCCTGGAATG GATGGGCGGT ATTAACCCGA TTTTTGGCAC CGCATTTTAT AACCAGAAAT TTC AGGGCCG CGTGACCATT ACCGCGGATG AAAGCACCAG CACCGCGTAT ATGGAACTGA GCAGCCTGCG CAGCGAAGAT ACCGCGGTGT ATTATTGCGC ACGCGAAGCG ATTACCACCG TGGGCGCGAT GGATCATTGG GGCCAGGGCA CCCTGGTGAC CGTGAGCAGC 169 GDF15_001 LC DNA GAAATTGTGC TGACCCAGAG CCCGGCGACC CTGAGCCTGA GCCCGGGCGA ACGCGCGACC CTGAGCTGCC GCACCAGCCA GAGCGTTCAT AACTATCTGG CGTGGTATCA GCAGAAACCG GGCCAGGCGC CGCGCCTGCT GATTTATGAT GCGAGCACCC GTGCGGATGG CATTCCGGCA CGCTTTAGCG GCAGCGGCAG CGGC ACCGAT TTTACCCTGA CCATTAGCAG CCTGGAACCG GAAGATTTTG CGGTGTATTA TTGCCAGCAG TTTTGGAGCT GGCCGTGGAC CTTTGGCCAG GGCACCAAAG TGGAAATTAA ACGTACGGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTAT CCCAGAGAGG CCAAAGTACA GTGGAAGGTG GATAACGCCC TCCAATCGGG TAACTCCCAG GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG CTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC CTGAGCTCGC CCGTCACAAA GAGCTTCAAC AGGGGAGAGT GT 170 GDF15_001 HC DNA CAGGTGCAGC TGGTGCAGAG CGGCGCGGAA GTGAAAAAAC CGGGCAGCAG CGTGAAAGTG AGCTGCAAAG CGAGCGGCTA TACCTTTAGC AGCTATAACA TTGATTGGGT GCGCCAGGCG CCGGGCCAGG GCCTGGAATG GATGGGCGGT ATTAACCCGA TTTTTGGCAC CGCATTTTAT AACCAGAAAT TTC AGGGCCG CGTGACCATT ACCGCGGATG AAAGCACCAG CACCGCGTAT ATGGAACTGA GCAGCCTGCG CAGCGAAGAT ACCGCGGTGT ATTATTGCGC ACGCGAAGCG ATTACCACCG TGGGCGCGAT GGATCATTGG GGCCAGGGCA CCCTGGTGAC CGTGAGCAGC GCGTCGACCA AGGCCCATC GGTCTTCCCC CTGGCACC CT CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACCCAGACC TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG TGGACAAGAA AGTTGAGCCC AAATCTTGTG ACAAAACTCA CACATGCCCA CCGTGCCCAG CACCTG AAGC CGCTGGGGCA CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC CCGGACCCCT GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC CTGAGGTCAA GTTCAACTGG TACGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA GCAGTACAAC AGCACGTA CC GTGTGGTCAG CGTCCTCACC GTCCTGCACC AGGACTGGCT GAATGGCAAG GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCC CCATCGAGAA AACCATCTCC AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCC TGCCCCCATC CCGGGAGGAG ATGACCAAGA ACCAGGTCAG CCTGACCTGC CTGGTCAAAG GCTTCTATCC CAGCGACATC GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC GCCTCCCGTG CTGG ACTCCG ACGGCTCCTT CTTCCTCTAT AGCAAGCTCA CCGTGGACAA GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC CCCCGGA 171 GDF15 HCDR-1 Consensus sequence GYTFX ₁ X ₂ YNID
where X ₁ is S or T and X ₂ is S or D 172 GDF15 HCDR-2 Consensus sequence X ₃ INPX ₄ X ₅ GX ₆ AX ₇ X ₈ X ₉ QKFQG,
where X ₃ is G or Q; X ₄ is I or N; X ₅ is F or N; X ₆ is T or L; X ₇ is F or N; X ₈ is Y or F and X ₉ is N or A 173 GDF15 HCDR-3 Consensus sequence EX ₁₀ ITTX ₁₁ GAMDX ₁₂ ,
where X ₁₀ is A or Q; X ₁₁ is V or I; and X ₁₂ is H or Y 174 GDF15 LCDR-1 Consensus sequence RX ₁ SQX ₂ X ₃ X ₄ X ₅ YLA where X ₁ is T or A, X ₂ is S or N, X ₃ is V or L, X ₄ is H or S and X ₅ is N or S 175 GDF15 LCDR-2 Consensus sequence DAX ₆ X ₇ RAX ₈ where X ₆ is S or K; X ₇ is T or N; and X ₈ is D or T 176 GDF15 LCDR-3 Consensus sequence QQFX ₉ X ₁₀ X ₁₁ PX ₁₂ T, where X ₉ is W or S; X ₁₀ is S or N; X ₁₁ is W or D; and X12 is W or Y 177 hu01G06 VH QVQLVQSGAE VKKPGASVKV SCKASGYTFT DYNMDWVRQA PGQSLEWMGQ INPNNGLIFF NQKFQGRVTL TTDTSTSTAY MELRSLRSDD TAVYYCAREA ITTVGAMDYW GQGTLVTVSS 178 hu01G06 VL DIQMTQSPSS LSASVGDRVT ITCRTSENLH NYLAWYQQKP GKSPKLLIYD AKTLADGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQH FWSDPYTFGQ GTKLEIK 179 GDF15_0297 HCDR-1
GDF15_0301 HCDR-1
GDF15 0470 HCDR-1 GYPFEGWYIH 180 GDF15_0297 HCDR-2
GDF15_0301 HCDR-2
GDF15 0470 HCDR-2 WNNPRTGLTNHAQKFQG 181 GDF15_0297 HCDR-3
GDF15_0301 HCDR-3
GDF15 0470 HCDR-3 GVGADAAFDI 182 GDF15 0297 VH QVQLQQPGAE LVKPGASVKM SCKASGYPFE GWYIHWVKQR PGQGLEWMGW NNPRTGLTNH AQKFQGKVTM TRDTSSSTAY MQLSSLTSED SAVYYCARGV GADAAFDIWG QGTTLTVSS 183 GDF15 0297HC QVQLQQPGAE LVKPGASVKM SCKASGYPFE GWYIHWVKQR PGQGLEWMGW NNPRTGLTNH AQKFQGKVTM TRDTSSSTAY MQLSSLTSED SAVYYCARGV GADAAFDIWG QGTTLTVSSA KTTPPSVYPL APGSAAQTNS MVTLGCLVKG YFPEPVTVTW NSGSLSSGVH TFPAV KEQMA KDKVS LTCMITDFFP EDIVEWQWN GQPAENYKNT QPIMDTDGSY FIYSKLNVQK SNWEAGNTFT CSVLHEGLHN HHTEKSLSHS PGK 184 GDF15_0297 LCDR-1
GDF15_0301 LCDR-1
GDF15 0470 LCDR-1 RSSQSLLWKHGYNYLD 185 GDF15_0297 LCDR-2
GDF15_0301 LCDR-2
GDF15 0470 LCDR-2 LDRNRAH 186 GDF15_0297 LCDR-3
GDF15_0301 LCDR-3
GDF15 0470 LCDR-3 MQSFETPIT 187 GDF15_0297 VL DIVMTQSPSS LSVSAGEKVT MSCRSSQSLL WKHGYNYLDW YQQKPGQPPK LLIYLDRNRA HGVPDRFTGS GSGTDFTLTI SSVQAEDLAV YYCMQSFETP ITFGGGTKLE IK 188 GDF15_0297LC DIVMTQSPSS LSVSAGEKVT MSCRSSQSLL WKHGYNYLDW YQQKPGQPPK LLIYLDRNRA HGVPDRFTGS GSGTDFTLTI SSVQAEDLAV YYCMQSFETP ITFGGGTKLE IKRADAAPTV SIFPSSEQL TSGGASVVCF LNNFYPKDIN VKWKIDGSER QNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE ATHKTSTSPI VKSFNRNEC 189 GDF15 0301 VH QVQLQQPGAE LVKPGASVKM SCKASGYPFE GWYIHWVKQR PGQGLEWMGW NNPRTGLTNH AQKFQGKATL TVDTSSSTAY MQLSSLTSED SAVYYCARGV GADAAFDIWG QGTTLTVSS 190 GDF15 0301HC QVQLQQPGAE LVKPGASVKM SCKASGYPFE GWYIHWVKQR PGQGLEWMGW NNPRTGLTNH AQKFQGKATL TVDTSSSTAY MQLSSLTSED SAVYYCARGV GADAAFDIWG QGTTLTVSSA KTTPPSVYPL APGSAAQTNS MVTLGCLVKG YFPEPVTVTW NSGSLSSGVH TFPAVL QSDL YTLSSSVTVP SSTWPSETVT CNVAHPASST KVDKKIVPRD CGCKPCICTV PEVSSVFIFP PKPKDVLTIT LTPKVTCVVV AISKDDPEVQ FSWFVDDVEV HTAQTQPREE QFNSTFRSVS ELPIMHQDWL NGKEFKCRVN SAAFPAPIEK TISKTKGRPK APQVYTIPPP KEQMAK DKVS LTCMITDFFP EDIVEWQWN GQPAENYKNT QPIMDTDGSY FIYSKLNVQK SNWEAGNTFT CSVLHEGLHN HHTEKSLSHS PGK 191 GDF15_0301 VL DIVLTQSPSS LSVSAGEKVT MSCRSSQSLL WKHGYNYLDW YQQKPGQPPK LLIYLDRNRA HGVPDRFTGS GSGTDFTLTI SSVQAEDLAV YYCMQSFETP ITFGGGTKLE IK 192 GDF15_0301LC DIVLTQSPSS LSVSAGEKVT MSCRSSQSLL WKHGYNYLDW YQQKPGQPPK LLIYLDRNRA HGVPDRFTGS GSGTDFTLTI SSVQAEDLAV YYCMQSFETP ITFGGGTKLE IKRADAAPTV SIFPSSEQL TSGGASVVCF LNNFYPKDIN VKWKIDGSER QNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE ATHKTSTSPI VKSFNRNEC 193 GDF15 0470 VH QVQLVQSGAE VKKPGASVKV SCKASGYPFE GWYIHWVRQA PGQGLEWMGW NNPRTGLTNH AQKFQGRVTM TRDTSISTAY MELSRLRSDD TAVYYCARGV GADAAFDIWG QGTMVTVSS 194 GDF15 0470HC QVQLVQSGAE VKKPGASVKV SCKASGYPFE GWYIHWVRQA PGQGLEWMGW NNPRTGLTNH AQKFQGRVTM TRDTSISTAY MELSRLRSDD TAVYYCARGV GADAAFDIWG QGTMVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG Y TLPPSREEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPG 195 GDF15_0470 VL EIVLTQSPA TLSLSPGER ATLSCRSSQ SLLWKHGYN YLDWYQQKP GQAPRLLIY LDRNRAHGI PARFSGSGS GTDFTLTIS SLEPEDFAV YYCMQSFET PITFGQGTK VEIK 196 GDF15_0470LC EIVLTQSPA TLSLSPGER ATLSCRSSQ SLLWKHGYN YLDWYQQKP GQAPRLLIY LDRNRAHGI PARFSGSGS GTDFTLTIS SLEPEDFAV YYCMQSFET PITFGQGTK VEIKRTVAA PSVFIFPPS DEQLKSGTA SVVCLLNNF YPREAKVQW KVDNALQSG NSQESVTEQ DSKDSTY SL SSTLTLSKA DYEKHKVYA CEVTHQGLS SPVTKSFNR GEC

In some embodiments, the substitution is a human germline substitution in which a CDR residue (donor) is replaced with a corresponding human germline residue (acceptor) to increase human amino acid content and potentially reduce the immunogenicity of the antibody, as described, for example, in Application Publication U.S. Patent No. 2017/0073395 and Townsend et al., 2015, Proc. Nat. Acad. sci. USA 112(50):15354-15359). For example, if a human germline IGHV1-69*01 framework is used and a generic antibody, GDF15_001 VH (SEQ ID NO:166) is compared, the alignment of the HCDR-1 GDF15_001 antibody (SEQ ID NO:32) and IGHV1-69*01 germline person is as follows:

Position 26 27 28 29 thirty 31 32 33 34 35 IGHV1-69*01 human germline G G T F S S Y A I S GDF15_001VH (SEQ ID NO:166) G Y T F S S Y N I D

For amino acid positions 26, 28, 29, 30, 31, 32, and 34 ( italic ), the human germline residue (acceptor) and the corresponding GDF15_001 residues (donor) match and germline replacement is not possible. For positions 27, 33 and 35 (bold and underlined), the human germline residue (acceptor) and the corresponding GDF15_001 residue (donor) are different. The GDF15_001 residues at these positions can be replaced with the corresponding human germline IGHV1-69*01 residue to further increase the content of human residues. The same process can be performed for each CDR heavy and light chain to increase the content of human amino acid residues while maintaining binding characteristics, e.g., epitope binding, affinity, and the like, while minimizing the content of mouse residues, thereby reducing any potential immunogenicity, e.g., human immune response. anti-mouse antibody (HAMA) to human antibody.

Methods and libraries for introducing human germline residues into antibody CDRs are described in detail in US Patent Application Publication No. 2017/0073395 and Townsend et al., 2015, Proc. Natl. Acad. sci. USA. 112(50):15354-15359, both incorporated herein by reference in their entirety.

Anti-GDF15 antibodies, or antigen-binding fragments thereof, may comprise a VH framework containing a human germline VH framework sequence. In some aspects, VH scaffolds from the following germlines may be used: IGHV1-2*02, IGHV1-3*01, IGHV1-46*01, IGHV1-69*01, IGHV1-69*02, IGHV1-8*01, IGHV3 -13*01, IGHV3-23*01, IGHV3-23*04, IGHV3-30*01, IGHV3-30*18, IGHV5-10-1*01, IGHV5-10-1*04 or IGHV5-51*01 (germline names are based on the IMGT germline definition). In some aspects, VL scaffolds from the following germlines may be used: IGKV1-12*01, IGKV1-13*02, IGKV1-33*01, IGKV1-39*01, IGKV1-5*01, IGKV3-11*01, IGKV3 -15*01, IGKV3-20*01, IGKV3D-20*02 and IGKV4-1*01 (germline names based on IMGT germline definition). Human germline scaffold sequences are available from various public databases such as V-base, IMGT, NCBI or Abysis.

Anti-GDF15 antibodies, or antigen-binding fragments thereof, may comprise a VL framework containing the human germline VL framework sequence. The VL framework may contain one or more amino acid substitutions, additions or deletions while still retaining functional and structural similarity to the germline from which it is derived. In some aspects, the VL frame is at least 53%, 58%, 60%, 63%, 71%, 72%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, or 99% identical to the human germline sequence from which it is derived. In some aspects, the antibody or antigen-binding fragment thereof comprises a VL framework containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions, additions, or deletions to the human germline VL framework sequence. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, or deletions are only in the framework regions. In some aspects, the share (%) identity is based on the similarity with VL except for those parts that are defined herein as CDRs.

Anti-GDF15 antibodies, or antigen-binding fragments thereof, may comprise a VH framework containing a human germline VH framework sequence. The VH framework may contain one or more amino acid substitutions, additions or deletions while still retaining functional and structural similarity to the germline from which it is derived. In some aspects, the VH frame is at least 72%, 74%, 75%, 77%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the human germline sequence from which it is derived. In some aspects, the antibody or antigen-binding fragment thereof comprises a VH framework containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions, additions, or deletions to the human germline VH framework sequence. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, or deletions are only in the framework regions. In some aspects, % identity is based on similarity to VH except for those parts defined herein as CDRs.

Anti-GDF15 antibodies or antigen-binding fragments thereof may comprise a VH containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:6. The VH may contain an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs: 21, 34, 44, 53, 60, 68, 73, 80, 86, 93, 99, 106, 112, 120, 127, 136, 142, 148, 155, 161 and 166. VH may contain the amino acid sequence of SEQ ID NOs: 21, 34, 44 , 53, 60, 68, 73, 80, 86, 93, 99, 106, 112, 120, 127, 136, 142, 148, 155, 161 and 166.

The antibody or antigen binding fragment may comprise a VL containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1. VL may contain an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NOs: 11, 30, 39, 49, 56, 64, 71, 77, 83, 90, 96, 103, 109, 115, 123, 131, 139, 144, 151, 158 and 163. VL may contain the amino acid sequence of SEQ ID NOs: 11, 30, 39 , 49, 56, 64, 71, 77, 83, 90, 96, 103, 109, 115, 123, 131, 139, 144, 151, 158 and 163.

In some aspects, the antibody, or antigen-binding portion thereof, comprises LCDR-1, LCDR-2, and LCDR-3 represented in the amino acid sequence of at least one of SEQ ID NOs: 11, 30, 39, 49, 56, 64, 71 , 77, 83, 90, 96, 103, 109, 115, 123, 131, 139, 144, 151, 158 and 163.

In some aspects, the antibody or antigen-binding portion thereof further comprises HCDR-1, HCDR-2 and HCDR-3 presented in the amino acid sequence of at least one of SEQ ID NOs: 21, 34, 44, 53, 60, 68, 73 , 80, 86, 93, 99, 106, 112, 120, 127, 136, 142, 148, 155, 161 and 166.

In some aspects, the antibody or antigen-binding portion thereof comprises LCDR-1, LCDR-2, LCDR-3 represented by the amino acid sequence of SEQ ID NO:163, and HCDR-1, HCDR-2 and HCDR-3 represented by the amino acid sequence of SEQ ID NO: :166.

The antibody, or antigen-binding portion thereof, may comprise a VL containing an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:163. VL may contain an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:163. VL may contain the amino acid sequence of SEQ ID NO:163.

The antibody, or antigen-binding portion thereof, may comprise a VH containing an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:166. The VH may contain an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:166. VH may contain the amino acid sequence of SEQ ID NO:166.

The antibody or antigen binding fragment may comprise a HC containing an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ. ID NO:164. HC may contain the amino acid sequence of SEQ ID NO:164.

The antibody or antigen binding fragment may comprise an LC containing an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO :162. The LC may contain the amino acid sequence of SEQ ID NO:162.

PD-1 axis binding antagonists

The term "PD-1 axis binding antagonist" as used herein refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more binding partners to eliminate T cell dysfunction resulting from signaling along the signaling axis. PD-1 pathway (also called the "PD-1/PD-L pathway" or "PD-1/PD-L signaling pathway"), resulting in the restoration or enhancement of T cell function. As used herein, a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. In some embodiments, the PD-1 axis binding antagonist is an anti-PD-1 antibody. In some embodiments, the PD-1 axis binding antagonist is an anti-PD-L1 antibody. In some embodiments, the PD-1 axis binding antagonist is an anti-PD-L2 antibody.

In some aspects, the PD-1 axis antagonist, PD-1 axis binding antagonist, PD-1 binding antagonist, and anti-PD-L1 antibody do not include avelumab. That is, optionally, avelumab is excluded from agents that inhibit the signaling axis of the PD-1 axis.

Exemplary PD-1 axis binding antagonists for use in the treatment, medicaments, and uses of the present invention include, but are not limited to, nivolumab, pembrolizumab, AMP-224 with or without a signal sequence as described in International Patent Application Publication No. WO2010/027827 and WO2011/066342, mAb7 and mAb15 as described in International Patent Application Publication No. WO2016/092419, and avelumab as described in WO2013/079174. Descriptions WO2010/027827, WO2011/066342, WO2016/092419 and WO2013/079174 are incorporated by reference in their entirety. Table 3 lists the different sequences of some of the presented PD-1 axis binding antagonists.

Table 3 SEQID NO Description Subsequence 197 mAb7 (RN888) or mAb15 full length heavy chain (HC) with underlined CDRs including terminal lysine (K). QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWINWVRQA PGQGLEWMGN IYPGSSLTNY NEKFKNRVTM TRDTSTSTVY MELSSLRSED TAVYYCARLS TGTFAYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SL SSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKN QV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK (SEQ ID NO: 1 in US 62/750579) (SEQ ID NO:29 in WO 16/092419) 198 mAb7 (RN888) or mAb15 full-length heavy chain without C-terminal lysine, with underlined CDRs. QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWINWVRQA PGQGLEWMGN IYPGSSLTNY NEKFKNRVTM TRDTSTSTVY MELSSLRSED TAVYYCARLS TGTFAYWGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SL SSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKN QV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLG (SEQ ID NO: 2 in US 62/750579) (SEQ ID NO:38 in WO 16/092419) 199 mAb7 (RN888) full-length light chain, with CDRs underlined. DIVMTQSPDS LAVSLGERAT INCKSSQSLW DSGNQKNFLT WYQQKPGQPP KLLIYWTSYR ESGVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYCQNDYFY PHTFGGGTKV EIKRGTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C (SEQ ID NO:3 in US 62/750579) (SEQ ID NO:39 in WO 16/092419) 200 mAb7 (RN888) light chain variable region, with underlined CDRs. (SEQ ID NO:4 in US 62/750579) (SEQ ID NO:8 in WO 16/092419) 201 mAb7 (RN888) and mAb15 heavy chain variable region, with CDRs underlined. (SEQ ID NO:5 in US 62/750579) (SEQ ID NO:4 in WO 16/092419) 202 mAb15 light chain variable region, with CDRs underlined. DIVMTQSPDS LAVSLGERAT INCKSSQSLWD SGNQKNFLT WYQQKPGQPP KLLIYWTSYR ESGVPDRFSG SGSGTDFTLTI SSLQAEDVA VYYCQNDYFY PHTFGGGTKV EIK (SEQ ID NO:6 in US 62/750579) 203 nivolumab, MDX1106, full length heavy chain from WO2006/121168 QVQLVESGGG WQPGRSLRLD CKASGITFSN SGMHWVRQAP GKGLEWVAVR WYDGSKRYYA DSVKGRFTIS RDNSKNTLFL QMNSLRAEDT AVYYCATNDD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSS SLGTT YTCNVDHKPS NTKVDRVESY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLM ISRTPEVTCW VDVSQEDPEV QFNWYYDGVE VHNATKPREE QFNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKGLPSSIEK TISKAGQPRE PQVYTLPPSQ EEMTKNQVSL TCLVKGFYPS DIAVE WESNG QPEKNYKTTP PVLDSDGSFF LYSRLTVDKS RWQEGNVFSC SVMHEALHNH YTQKSLSLSL GK 204 nivolumab, MDX1106, full length light chain from WO2006/121168 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQPG QAPRLLIYDA SNRATGIPAR FSGSGSGTDF TLTISSLEPE DFAVYYCQQS SNWPRTFGQG TKVEIRTVAA PSVFIFPPSD EQLSGTASVV CLLNNFYPRE AVQWKVDNAL QSGNSQESVT EQDSDSTYSL SSTLTLSKAD YEK HKVYACE VTHQGLSSPV TSFNRGEC 205 pembrolizumab, MK3475, full length heavy chain from WO2009/114335 QVQLVQSGVE VKKPGASVK VSCKASGYTF TNYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVT LTTDSSTTTA YMELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVS SASTKGPSVF PLAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSG VHTFPAVLQS SGLYSL SSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVE SKYGPPCPPC PAPEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQE DPEVQFNWYV DGVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEY KCKVSNKGLP SSIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLV KGFYPSDIAV EWESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQE GNVFSCSVMH EALHNHYTQKS LSLSLGK 206 pembrolizumab, MK3475, full length light chain from WO2009/114335 EIVLTQSPAT LSLSPGERA TLSCRASKGV STSGYSYLHWY QQKPGQAPRL LIYLASYLES GVPARFSGS GSGTDFTLTI SSLEPEDFAVY YCQHSRDLPL TFGGGTKVEI KRTVAAPSV FIFPPSDEQL KSGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTE QDSKDSTYSL SS TLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 207 AMP-224, without signal sequence from WO2010/027827 and WO2011/066342 LFVTVPKEL YIIEHGSNVT LECNFDTGSH VNLGAITASL QKVENDTSPH RERATLLEEQ LPLGKASFHI PQVQVRDEGQ YQCIIIYGVA WDYKYLTLKV KASYRKINTH ILKVPETDEV ELTCQATGYP LAEVSWPNVS VPANTSHSRT PEGLYQVTSV LRLKPPPGRN FSCVFWNTHV RE LTLASIDL QSQMEPRTHP TWEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCWV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFY PS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 208 YW243.55.S70, MPDL3280A) heavy chain variable region from WO2010/077634 EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGKGLEWVAW ISPYGGSTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSA 209 YW243.55.S70, MPDL3280A, light chain variable region from WO2010/077634 DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKR

The term "PD-1 binding antagonist" as used herein refers to a molecule that specifically binds to PD-1 and reduces, blocks, inhibits, abrogates, or interferes with signal transduction by the interaction of PD-1 with one or more of its binding partners, such as like PD-L1, PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partners. In a specific aspect, the PD-1 binding antagonist specifically binds to PD-1 and thereby inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, a PD-1 binding antagonist includes anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abolish, or interfere with signaling resulting from the interaction of PD-1 with PD- L1 and/or PD-L2. In one embodiment, the PD-1 binding antagonist specifically binds to PD-1 and thereby reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated through signal transduction through PD-1, thus making dysfunctional T cell is less non-dysfunctional. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody, including but not limited to nivolumab, pembrolizumab, spartalizumab, tiselizumab, pidilizumab, AMP-224, AMP-554, cemiplimab, and PF-06801591.

PF-06801951 also called sasanlimab (CAS Registry No. 2206792-50-7), RN888 and described in International Patent Application Publication No. WO 2016/092419, which is hereby incorporated by reference in its entirety. Sasanlimab is a humanized, hinge-stabilized IgG4-kappa (κ) monoclonal antibody. The amino acid sequences of sasanlimab (PF-06801951; RN888) are shown in Table 4 below.

In a specific aspect, the PD-1 binding antagonist is nivolumab. In another specific aspect, the PD-1 binding antagonist is pembrolizumab. In another specific aspect, the PD-1 binding antagonist is pidilizumab.

The term "PD-L1 binding antagonist" as used herein refers to a molecule that specifically binds to PD-L1 and reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-L1 with any or more of its binding partners. such as PD-1, B7-1. In some embodiments, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In some aspects, the PD-L1 binding antagonist does not include avelumab. In a specific aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonist includes anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abolish, or interfere with signal transduction arising from PD- L1 with one or more of its binding partners such as PD-1, B7-1. In one embodiment, the PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated through signaling through PD-L1, thereby making a dysfunctional T cell less non-dysfunctional. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific aspect, the anti-PD-L1 antibody is avelumab (described as A09-246-2, in International Patent Application Publication No. WO2013/079174). In some aspects, avelumab is not included as a PD-1 axis antagonist.

In another specific aspect, the anti-PD-L1 antibody is atezolizumab. In another specific aspect, the anti-PD-L1 antibody is durvalumab. In another specific aspect, the anti-PD-L1 antibody is BMS-936559 (MDX-1105).

As used herein, an anti-human PD-L1 antibody refers to an antibody that specifically binds to mature human PD-L1 or a portion thereof, wherein the mature human PD-L1 molecule consists of amino acids 19-290 of the following sequence: KDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTH LEET (SEQ ID NO: 221).

Table 4. ANTI-HUMAN PD-L1 MONOCLONAL ANTIBODY SASANLIMAB (PF-06801951, RN888, mAb7) SEQUENCES SEQID NO Description Amino acid sequence 210 HCDR-1 (Chothia) (SEQ ID NO:14 in WO 2016/092419) GYTFTSY 211 HCDR-1 (extended) (SEQ ID NO:13 in WO 2016/092419) GYTFTSYWIN 212 HCDR-1 (Cabot) (SEQ ID NO:15 in WO 2016/092419) SYWIN 213 HCDR-2 (Chothia) (SEQ ID NO:16 in WO 2016/092419) NIYPGSSL 214 HCDR-2 (extended) (SEQ ID NO:17 in WO 2016/092419) NIYPGSSLTNYNEKFK 215 HCDR-3 (SEQ ID NO:23 in WO 2016/092419) LSTGTFAY 216 LCDR-1 (SEQ ID NO:10 in WO 2016/092419) KSSQSLWDSGNQKNFLT 217 LCDR-2 (SEQ ID NO:20 in WO 2016/092419) WTSYRES 218 LCDR-3 (SEQ ID NO:21 in WO 2016/092419) QNDYFYPHT 219 VH (SEQ ID NO:4 in WO 2016/092419)
Chothia (bold), Cabot (underlined), and extended (both) CDRs are indicated; QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSLTNYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTLVTVSS 220 VL (SEQ ID NO:8 in WO 2016/092419) CDRs are in bold and underlined DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKPGQPPKLLIYWTSYRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPHTFGGGTKVEIK 197 Heavy chain (HC) (SEQ ID NO:29 in WO 16/092419)
Terminal lysine (K) optional;
Chothia CDRs are in bold;
Cabot CDRs are underlined; extended CDRs (both) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSLTNYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(K) 199 Light chain (LC) (SEQ ID NO:39 in WO 2016/092419)
CDRs are in bold and underlined DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKPGQPPKLLIYWTSYRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPHTFGGGTKVEIKRGTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 221 human PD-L1 protein; mature human PD-L1 consists of amino acid residues 29-290; residues not included in the mature protein are underlined MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTH LVILGAILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET

The term "PD-L2 binding antagonists" as used herein refers to a molecule that specifically binds to PD-L2 and reduces, blocks, inhibits, abrogates, or interferes with signal transduction by the interaction of PD-L2 with one or more of its binding partners, such as like PD-1. In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partners. In a specific aspect, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that specifically bind PD-L2 and reduce, block, inhibit, abolish, or interfere with signaling in the result of the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, the PD-L2 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated by PD-L2 signaling to make dysfunctional T cells less non-dysfunctional. In some embodiments, the PD-L2 binding antagonist is a PD-L2 immunoadhesin.

Nucleic acids

The invention also relates to polynucleotides encoding any of the antibodies of the invention, including the antibody portions and modified antibodies described herein. The invention also provides a method for producing any of the polynucleotides described herein. Polynucleotides can be prepared and expressed using methods known in the art.

The sequence of the desired antibody, its antigen-binding fragment, and the nucleic acid encoding that antibody, or antigen-binding fragment thereof, can be determined using standard sequencing techniques. The nucleic acid sequence encoding the desired antibody, or antigen-binding fragment thereof, can be inserted into various vectors (such as cloning and expression vectors) for recombinant production and characterization. A nucleic acid encoding a heavy chain or a heavy chain antigen-binding fragment and a nucleic acid encoding a light chain or a light chain antigen-binding fragment may be cloned into the same vector or into different vectors.

In one aspect, the invention provides polynucleotides encoding the amino acid sequences of any of the following GDF15 antibodies and antigen-binding portions thereof: GDF15_001, GDF15_002, GDF15_003, GDF15_004, GDF15_005, GDF15_006, GDF15_007, GDF15_008, GDF15_009, GDF1 5_010, GDF15_011, GDF15_012, GDF15_013, GDF15_014, GDF15_015, GDF15_017, GDF15_018, GDF15_020, GDF15_021, GDF15_022, GDF15_100, GDF15_200, GDF15_297, GDF15_301, GDF15-470.

The invention provides polynucleotides encoding one or more proteins containing an amino acid sequence selected from the group consisting of: (i) SEQ ID NOs: 21, 34, 44, 53, 60, 68, 73, 80, 86, 93, 99 , 106, 112, 120, 127, 136, 142, 148, 155, 161, 166, 11, 30, 39, 49.56, 64, 71, 77, 83, 90, 96, 103, 109, 115, 123 , 131, 139, 144, 151, 158,163, 166, 183, 187, 189, 191, 193 and 195.

The invention provides polynucleotides containing the nucleic acid sequence indicated as one or more SEQ ID NOs: 167, 168, 169 and 170. The invention provides a polynucleotide containing the nucleic acid sequence indicated as SEQ ID NO: 167. The invention provides a polynucleotide, containing the nucleic acid sequence indicated as SEQ ID NO:168. The invention provides a polynucleotide containing the nucleic acid sequence indicated as SEQ ID NO:169. The invention provides a polynucleotide containing the nucleic acid sequence indicated as SEQ ID NO:170. Due to the degeneracy of the genetic code, the invention additionally provides a nucleic acid sequence, where the nucleotide at position number 1344 of SEQ ID NO: 170 may be A, C, G, T, and/or the nucleotide at position 1347 may be A, C, G , T. The last two codons shown in SEQ ID NO:170 additionally code for proline and glycine, respectively.

The invention provides a polynucleotide containing a nucleic acid sequence of interest from a plasmid deposited with ATCC and having Accession No. PTA-125038 encoding the VH domain of GDF15_001. The invention also provides a polynucleotide containing a nucleic acid sequence of interest from a plasmid deposited with ATCC and having Accession No. PTA-125039 encoding the VL domain of GDF15_001. In addition, the invention provides a polypeptide containing an amino acid sequence encoded by a DNA insert of a plasmid deposited with ATCC and having Accession No. PTA-125038 encoding the VH domain of GDF15_001. The invention also provides a polypeptide containing an amino acid sequence encoded by an insert of a plasmid deposited with ATCC and having Accession No. PTA-125039 encoding the VL domain of GDF15_001.

The invention also provides a polynucleotide containing a nucleic acid sequence of interest from a plasmid deposited with ATCC and having accession number PTA-125038, encoding the VH domain of GDF15_001, and a nucleic acid sequence of interest from a plasmid deposited by ATCC and having accession number PTA-125039 encoding the VL domain of GDF15_001.

In another aspect, the invention provides polynucleotides and variants thereof encoding an anti-GDF15 antibody, wherein such polynucleotide variants share at least 70%, at least 75%, at least 80%, at least 85 %, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleic acid sequence identity in any of the nucleic acid sequences described or indicated by reference in this document. These amounts are not limiting, and the steps between the indicated percentages are expressly provided as part of the description.

The invention provides polypeptides encoded by the nucleic acid molecules described herein.

In one embodiment, the VH and VL domains or their antigen-binding portion or full-length HC or LC are encoded by separate polynucleotides. Alternatively, both VH and VL, or an antigen-binding portion thereof, or HC and LC, are encoded by a separate polynucleotide.

Polynucleotides complementary to any such sequences are also covered by the present description. Polynucleotides can be single stranded (coding or antisense) or double stranded and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules that contain introns and one-to-one correspond to a DNA molecule, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may optionally be present in a polynucleotide of the present disclosure, and the polynucleotide may optionally be linked to other molecules and/or support materials.

Polynucleotides may contain a nucleic acid sequence that encodes an antibody or portion thereof, or may contain a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions, and/or insertions, such that the binding characteristics of the encoded polypeptide are not impaired compared to the native antibody molecule. The effect on binding characteristics of a polypeptide encoded by a nucleic acid sequence variant can generally be assessed as described herein. In some embodiments, polynucleotide variants exhibit at least about 70% identity, in some embodiments at least about 80% identity, in some embodiments at least about 90% identity, and in some embodiments implementation, at least about 95% identity of the polynucleotide sequence encoding the original (parent) antibody, not containing any substitutions, additions, deletions and/or insertions or parts thereof. This share of identity is not intended to be limiting, and the steps between these shares are expressly provided as part of the description.

Two polynucleotide or polypeptide sequences are considered "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum match, as described below. Comparison between two sequences is typically performed by comparing sequences in a comparison window to identify and compare local areas of sequence similarity. "Comparison window" as used herein refers to a segment of at least about 20 contiguous positions, typically 30 to about 75 or 40 to about 50, in which a sequence can be compared to a reference sequence with the same number of consecutive positions after the optimal alignment of two sequences.

Optimal sequence alignment for comparison can be performed using the MegAlign® program in the Lasergene® Bioinformatics Suite (DNASTAR®, Inc., Madison, WI), using default parameters. This program includes several alignment schemes described in the following references: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5:151-153; Myers, E.W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E.D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 1983, Proc. Natl. Acad. sci. USA 80:726-730.

In some embodiments, "percent sequence identity" is determined by comparing two optimally aligned sequences in a comparison window of at least 20 positions, where a portion of the polynucleotide or polypeptide sequences in the comparison window may contain additions or deletions (i.e., gaps) at 20 percent or less, typically 5 to 15 percent or 10 to 12 percent, compared to reference sequences (which contain no addition or deletion) to optimally align the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e. window size) and multiplying the results by 100 to obtain percent sequence identity.

Variant polynucleotides may also, or alternatively, be substantially homologous to a gene, or portion thereof, or complement. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding an antibody (or a complementary sequence).

Suitable "moderately stringent conditions" include a pre-wash in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at about 50°C to 65°C, 5X SSC (0.75 M NaCl, 0.075 M sodium citrate), overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

As used herein, "significantly stringent conditions" or "high stringency conditions" include those where: (1) low ionic strength and high washing temperature are used, such as 0.015 M sodium chloride/0.0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50°C; (2) a denaturing agent such as formamide is applied during hybridization, e.g. 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM buffer based on sodium phosphate at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) 50% formamide, 5X SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50 µg/ml), 0.1% SDS and 10% dextran sulfate at 42°C, with a 42°C wash in 0.2X SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high hardness wash consisting of 0.1X SSC containing EDTA at 55°C. One skilled in the art will understand how to adjust temperature, ionic strength, etc. if necessary, taking into account factors such as sample length and the like.

Those skilled in the art will appreciate that, as a result of the degeneracy of the genetic code, there are multiple nucleotide sequences that encode for a polypeptide as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequences of any native gene. However, polynucleotides that differ due to differences in codon usage are specifically addressed in the present specification. In addition, the alleles of the genes containing the polynucleotide sequences presented herein are included in the scope of the present description. Alleles are endogenous genes that change as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles can be identified using standard methods (such as hybridization, amplification, and/or comparison of sequence databases).

Polynucleotides of this description can be obtained using chemical synthesis, recombinant methods or PCR. Methods for the chemical synthesis of polynucleotides are well known in the art and need not be described in detail herein. One skilled in the art can use the sequences provided herein and a commercial DNA synthesizer to obtain the desired DNA sequence.

To obtain polynucleotides using recombinant techniques, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and this vector can in turn be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct capture, endocytosis, transfection, F-pairing, or electroporation. Once introduced, the exogenous polynucleotide may be maintained in the cell as a non-integrated vector (such as a plasmid) or integrated into the genome of the host cell. The thus amplified polynucleotide can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al. , 1989.

Alternatively, PCR allows the reproduction of DNA sequences. PCR technology is well known in the art and is described in US Patent Nos. 4,683,195; 4,800,159 ; eds., Birkauswer Press, Boston, 1994.

RNA can be prepared by using the isolated DNA in a suitable vector and inserting it into a suitable host cell. Once the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using techniques well known to those skilled in the art, as outlined in Sambrook et al. , 1989, for example.

As used herein, "vector" means a construct that is capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors coupled with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

Suitable cloning and expression vectors may include a variety of components such as a promoter, enhancer, and other transcriptional regulatory sequences. The vector can also be designed to allow subsequent cloning of the antibody variable domain into various vectors. Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. Although the cloning vector chosen may vary depending on the host cell to be used, useful cloning vectors will typically be self-replicating, may have a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used. when selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g. pUC18, pUC19, Bluescript (e.g. pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors such as pSA3 and pAT28 . These and many more cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen. Expression vectors are also provided. Expression vectors are typically replicable polynucleotide constructs that contain a polynucleotide as described. The expression vector is intended to replicate in host cells either as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO 87/04462. Vector components may typically include, but are not limited to, one or more of the following: a signal sequence; replication source; one or more marker genes; suitable transcriptional control elements (such as promoters, enhancers and terminator). Expression (ie, translation) also typically requires one or more translational control elements, such as ribosome binding sites, translation initiation sites, and stop codons.

Vectors containing the polynucleotides of interest and/or the polynucleotides themselves can be introduced into the host cell by any of a variety of suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microparticle bombardment; lipofection; and infection (for example, if the vector is an infectious agent such as vaccinia virus). The choice of administration vectors or polynucleotides often depends on the characteristics of the host cell.

Thus, a "host cell" includes an individual cell or cell culture that can be or has been a recipient of polynucleotides and/or vector(s) containing polynucleotides to include polynucleotides and/or vectors. Host cells include the progeny of a single host cell, and these progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or intentional mutation. A host cell includes cells transfected and/or transformed in vivo with a polynucleotide of the present invention.

The antibody, or antigen-binding fragment thereof, can be produced recombinantly using a suitable host cell. The nucleic acid encoding the antibody or antigen-binding fragment thereof can be cloned into an expression vector, which can then be introduced into a host cell such as an E. coli cell, a yeast cell, an insect cell, a monkey COS cell, a Chinese hamster ovary (CHO ) or a myeloma cell, where the cell does not otherwise produce an immunoglobulin protein, to obtain antibody synthesis in a recombinant host cell. Preferred host cells include a CHO cell, a human embryonic kidney (HEK) 293 cell, an NS0 cell, or an Sp2.0 cell, among many cells well known in the art. An antibody fragment can be produced by proteolytic or other degradation of a full-length antibody, by recombinant methods, or by chemical synthesis. An antibody polypeptide fragment, especially shorter polypeptides of up to 50 amino acids, can be conveniently prepared by chemical synthesis. Methods for the chemical synthesis of proteins and peptides are known in the art and are commercially available.

An antibody or antigen-binding fragment thereof of the invention may be affinity matured. For example, an affinity matured antibody can be prepared by methods known in the art (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91 :3809-3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7):3310-9; Hawkins et al., 1992, J. Mol. Biol., 226:889-896; and WO2004/058184).

Immunogenicity

Immunogenicity is a major barrier to the development and use of protein therapeutics, including antibodies and Fc fusion proteins. Several factors can contribute to the immunogenicity of a protein, including, but not limited to, protein sequence, route and frequency of administration, and patient population. Although immune responses are usually most severe for non-human proteins, such as mouse antibodies, even therapeutics containing mostly or entirely human sequences can be immunogenic. Immunogenicity is a complex series of responses to a substance that is perceived as foreign and may include the production of neutralizing and non-neutralizing antibodies, immune complex formation, complement activation, mast cell activation, inflammation, and anaphylaxis. Unwanted immune responses can reduce the effectiveness of antibody and Fc fusion protein therapeutics by directly interfering with antigen recognition, altering interactions with effector molecules, or altering the serum half-life or tissue distribution of the therapeutic.

Protein therapeutics can be analyzed to predict the presence of potential immunogenic epitopes using commercially available services such as those provided by Epivax, Inc. from Providence, R.I. In some embodiments, electronic algorithms can predict epitopes that bind to class II MHC molecules. Analysis of a polypeptide dataset with such algorithms provides predictive epitopes. Predicted epitopes are used to generate peptides produced by standard automated peptide synthesis or recombinant DNA techniques. The estimated information provided by Epivax can serve as an indicator of how widespread the predicted epitope is as recognized by the population.

As described in Example 10 below, antibodies of the present invention are screened for the presence of epitopes recognized by T cells, also referred to herein as T cell epitopes, "T-regitopes" or "tReg", using the EpiMatrix algorithm developed by EpiVax. The antibody sequences are disassembled into overlapping 9-mer frames, where each frame overlaps the last one by 8 amino acids. Each of the resulting frames is evaluated for predicted binding affinity for a panel of eight common MHC class II HLA alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301 and DRB1 *1501). The raw scores are normalized to the results of a large sample of randomly generated peptides and a final "Z" score is produced.

The overall sequence score, the corrected tReg score, can be calculated using the EpiMatrix Z-score to predict antibody immunogenicity. As described in Example 10, the adjusted tReg score is calculated by summing the EpiMatrix Z-scores for the 9-mer frames (subtotal) and taking into account the number of HLA-type observations. All individual combinations of 9-mer and HLA type ("observations") are considered, regardless of whether the 9-mer is an epitope. If a particular observation indicates that a peptide is in the top 5% binding agents for that HLA type, the EpiMatrix Z-score for that observation is added to the running sum associated with the entire protein sequence. The total number of observations studied is also recorded. The only exception is that all 9-mer observations identified by the ISPRI software package developed by EpiVax as "T-regiotopes" are assumed to have EpiMatrix scores of zero. As used herein, "T-regitopes" are sequential sequences within the framework region of a monoclonal antibody that can potentially activate natural regulatory T cells and reduce unwanted immune responses. The adjusted tReg score is calculated as follows: Adjusted tReg score = (Subtotal) * 1000 / (Number of observations). As a subtotal, a baseline score of 0.05 * 2.2248 is subtracted from each observation (including T regitopes). A lower adjusted tReg predicts a lower risk potential for immunogenicity.

Application

Ways to treat cachexia

In some aspects, the invention provides therapeutic methods for reducing or inhibiting GDF15 activity using an anti-GDF15 antibody or antigen-binding fragment thereof, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof. A treated disorder is any disease or condition that is ameliorated, alleviated, inhibited, or prevented by removing, inhibiting, or reducing GDF15 activity or signaling.

The invention includes a method for reducing the level of free GDF15 in a patient. The method includes determining the level of free GDF15 in a subject, administering a therapeutic amount of an antibody of the invention or an antigen-binding fragment thereof, and comparing the pre-administration GDF15 level with the free GDF15 level after administration of the antibody or antigen-binding fragment thereof, thereby reducing the free GDF15 level in the subject.

In one embodiment, reducing the level of free GDF15 reduces unwanted, harmful or unnecessary biological activity of GDF15. Such GDF15 activity includes, but is not limited to, (a) reduced food intake; (b) decreased appetite; (c) weight loss; (d) increased body weight loss; (e) reduction of fat mass; (e) decrease in muscle mass; (g) increased fat loss, (h) increased muscle loss, (i) binding to GFRAL; (j) amplification of downstream signaling mediated by RET; (k) increasing ERK phosphorylation; (l) increasing S6 phosphorylation; (m) enhancing RET-mediated activation of the MAPK signaling pathway; (n) enhancing activation of the RET AKT signaling pathway; and (o) enhancing activation of the PLC-γ1 signaling pathway.

In one embodiment, the invention includes a method for reducing the biological activity of GDF15 in a patient. The method includes administering a therapeutic amount of an antibody of the invention, or an antigen-binding fragment thereof, thereby reducing the biological activity of GDF15.

In one aspect, the biological activity of GDF15 includes, but is not limited to, (a) reduced food intake; (b) decreased appetite; (c) weight loss; (d) increased body weight loss; (e) reduction of fat mass; (e) decrease in muscle mass; (g) increased fat loss, (h) increased muscle loss, (i) binding to GFRAL; (j) amplification of downstream signaling mediated by RET; (k) increasing ERK phosphorylation; (l) increasing S6 phosphorylation; (m) enhancing RET-mediated activation of the MAPK signaling pathway; (n) enhancing activation of the RET AKT signaling pathway; and (o) enhancing activation of the PLC-γ1 signaling pathway.

The terms "treatment" or "treated" include prophylactic and/or therapeutic treatment. If it is given before the clinical manifestation of the condition, the treatment is considered prophylactic. Therapeutic treatment includes, for example, alleviating or lessening the severity of the disease or shortening the duration of the disease.

As used herein, the term "cachexia" includes a metabolic disorder and comorbidities that occur with several chronic diseases, including cancer, chemotherapy, chemotherapy in combination with immuno-oncological therapy, chronic heart failure, congestive heart failure, sarcopenia, chronic obstructive pulmonary disease (COPD) , sarcopenia and chronic kidney disease (CKD).

The invention encompasses methods of treating a disease, disorder, or condition mediated or associated with GDF-15. In one aspect, the disorder is cachexia. In other aspects, the disorder is cachexia associated with cancer, chemotherapy, chemotherapy in combination with immuno-oncological therapy, chronic obstructive pulmonary disease, chronic kidney disease, chronic heart failure, congestive heart failure, or sarcopenia. In some aspects, the cancer is a solid tumor, pancreatic cancer, lung cancer, non-small cell lung cancer, colorectal cancer, prostate cancer, ovarian cancer, cervical cancer, or testicular cancer. In some aspects, the chemotherapy is platinum-based chemotherapy. The disease or disorder or symptom may be alleviated or reduced in severity, duration, or frequency of occurrence.

The invention also includes an antibody, or an antigen-binding fragment thereof, or a pharmaceutical composition as defined herein, for use in certain treatments. In embodiments that relate to a method of treatment described herein, such embodiments are also further embodiments relating to an antibody or antigen-binding fragment thereof, a pharmaceutical composition for use in that treatment, or, alternatively, for the manufacture of a drug for use in this treatment.

Antibodies and antibody fragments may be administered in combination with one or more additional therapeutically active compounds. Additional therapeutically active compounds include agents used to treat chronic disorders associated with cachexia, anti-cancer agents (eg, immune therapy and chemotherapy), anti-cancer agents that cause cellular stress (eg, platinum-based chemotherapeutic agents such as cisplatin), muscle anabolics (eg, selective androgen receptor modulators (SARMs), myostatin inhibitors, and an Activin A receptor inhibitor), anti-inflammatory agents (eg, JAK inhibitors, IL-6 inhibitors, IL-8 inhibitors), appetite stimulants (eg, ghrelin mimetics, melanocortin 4) and agents that improve metabolism (eg metformin). Combination therapy prophylactic or therapeutic agents, including antibodies or antigen-binding fragments thereof, may be administered to a subject in a single pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapy may be administered to the subject simultaneously in separate pharmaceutical compositions. Prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.

A "chemotherapeutic agent" is a chemical compound used in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbochone, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamin; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analog of topotecan (HYCAMTIN), CPT-11 (irinotecan, CAMPTOSAR), acetylcamptothecin, scopolectin and 9-aminocamptothecin); bryostatin; pemetrexed; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues of KW-2189 and CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral alpha-4 integrin inhibitor; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterin, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as endiine antibiotics (eg calicheamicin, especially calicheamicin gamma I and calicheamicin omega l (see, for example, Nicolaou et al, Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemycin , including dynemycin A; esperamycin; as well as neocarzinostatin chromophore and related chromoprotein enediine antibiotic, chromophores), aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo- 5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolin-doxorubicin, doxorubicin HCl liposome injection (DOXIL) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such how mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, kelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR), tegafur (UFTORAL), capecitabine (XELODA), epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, floxuridine, and imatinib (a 2-phenylaminopyrimidine derivative), as well as other c-it inhibitors; adrenal depressants such as aminoglutethimide, mitotane, trilostane; a folic acid supplement such as frolinic acid; aceglatone; aldofosfamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elfornitine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenamet; pyrarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide complex PSK (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidin); urethane; vindesine (ELDISINE, FILDESIN); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, for example, paclitaxel (TAXOL), a composition of paclitaxel (ABRAXANE) and doxetaxel (TAXOTERE) in the form of nanoparticles created on the basis of albumin; chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine (VELBAN); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN); oxaliplatin; leucovins; vinorelbine (NAVELBINE); novantron; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylchylomitin (DMFO); retinoids such as retinoic acid; inotuzumab ozogamicin (BESPONSA), bosutinib (BOSULIF), palbociclib (IBRANCE), axitinib (INLYTA), sunitinib malate (SUTENT), crizotinib (XALKORI), enzalutamide (XTANDI); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above, such as CHOP, an abbreviation for combination therapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for an oxaliplatin (ELOXATIN) regimen in combination with 5-FU and leucovin.

Additional examples of chemotherapeutic agents include antihormonal agents that act to regulate, decrease, block or inhibit the action of hormones that can promote cancer growth, and are often administered in the form of systemic or whole body treatments. It could be the hormones themselves. Examples include antiestrogen and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX tamoxifen), raloxifene (EVISTA), droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY 11 7018, onapristone, and toremifene (FARESTON® )); anti-progesterones; estrogen receptor suppressors (ERD); estrogen receptor antagonists such as fulvestrant (FASLODEX); agents that suppress or close the ovaries, for example lulibirin (LHRF1) agonists such as leuprolide acetate (LUPRON and ELIGARD), goserelin acetate, buserelin acetate and tripterelin; antiandrogens such as futamide, nilutamide and bicalutamide; and aromatase inhibitors, which inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, eg 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE), exemestane (AROMASIN), formestane, fadrozole, vorozol (RJVISOR), letrozole (FEMARA) ) and anastrozole (ARIMIDEX). In addition, this definition of chemotherapeutic agents includes bisphosphonates such as clodronate (e.g., BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid/zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate ( SKELID) or risedronate (ACTONEL); as well as troxocitabine (an analogue of 1,3-dioxolannucleosidecytosine); antisense oligonucleotides, especially those that inhibit the expression of genes in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE vaccine and gene therapy vaccines such as ALLOVECTIN vaccine, LEUVECTIN vaccine and VAXID vaccine; a topoisomerase 1 inhibitor (eg, LURTOTECAN); an antiestrogen such as fulvestrant; a Kit inhibitor such as imatinib or EXEL-0862 (a tyrosine kinase inhibitor); an EGFR inhibitor such as erlotinib or cetuximab; an anti-VEGF inhibitor such as bevacizumab; arinotecan; rmRH (for example, ABARELIX); lapatinib and lapatinib ditosylate (a small molecule ErbB-2 dual tyrosine kinase and EGFR inhibitor, also known as GW572016); 17AAG (a derivative of geldanamycin which is a heat shock protein (Hsp) 90 poison) and pharmaceutically acceptable salts, acids or derivatives of any of the above.

"Chemotherapy" as used herein refers to a chemotherapeutic agent as defined above, or a combination of two, three or four chemotherapeutic agents for the treatment of cancer. When the chemotherapy consists of more than one chemotherapeutic agent, the chemotherapeutic agents may be administered to the patient on the same day or on different days of the same treatment cycle.

"Platinum-based chemotherapy" as used herein refers to chemotherapy wherein at least one chemotherapeutic agent is a platinum coordination complex. Typical platinum-based chemotherapy includes, without limitation, cisplatin, carboplatin, oxaliplatin, nedaplatin, gemcitabine in combination with cisplatin, carboplatin in combination with pemetremed.

"Platinum-based doublet" as used herein refers to chemotherapy containing two and no more than two chemotherapeutic agents and where at least one chemotherapeutic agent is a platinum coordination complex. An exemplary platinum-based doublet includes, but is not limited to, gemcitabine in combination with cisplatin, carboplatin in combination with pemetrexed.

As used herein, the term "systemic cancer therapy" refers to the systemic administration of pharmaceutical agents approved by the regulatory authorities of any country in the world, or in human clinical trials conducted by the regulatory authorities of any country in the world with the general intention of changing the outcome of cancer. Systemic cancer therapy includes, but is not limited to, chemotherapy, hormonal therapy, targeted cancer therapy, cancer vaccines, oncolytic vaccines, and adoptive T cell therapy.

cancer treatment method

The invention includes a method of treating cancer comprising administering to a patient an effective amount of a GDF15 antibody. In some embodiments, the cancer is selected from the group consisting of gastric cancer, sarcoma, lymphoma, leukemia, head and neck cancer, thymus cancer, epithelial cancer, saliva cancer, liver cancer, gastric cancer, thyroid cancer, lung cancer, ovarian cancer , breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer, and melanoma. In some embodiments, the subject is a previously treated adult patient with locally advanced or metastatic melanoma, head and neck squamous cell carcinoma (SCHNC), ovarian carcinoma, sarcoma, or relapsed or refractory classical Hodgkin's lymphoma (cHL). In some embodiments, the cancer may be platinum-resistant and/or platinum-refractory cancer, such as, for example, platinum-resistant and/or refractory ovarian cancer, platinum-resistant and/or refractory breast cancer, or platinum-resistant and/or refractory to platinum lung cancer.

In another aspect, the invention provides a method of inhibiting tumor growth or progression in a subject having a tumor, comprising administering to the subject an effective amount of a pharmaceutical composition as described herein.

In another aspect, the invention provides a method for inhibiting or preventing cancer cell metastasis in a subject, comprising administering to a patient an effective amount of a pharmaceutical composition as described herein.

In another aspect, the invention provides a method of inducing tumor regression in a subject having a tumor, comprising administering to the subject an effective amount of a pharmaceutical composition as described herein.

In some embodiments, the method may further comprise administering an effective amount of the second therapeutic agent. In some embodiments, the second therapeutic agent is, for example, an immunomodulator. The term "immunomodulator" refers to a substance capable of altering (e.g., inhibiting, decreasing, increasing, enhancing, or stimulating) the immune response (as defined herein) or the operation of any component of the innate, humoral, or cellular immune system of a mammalian host. Thus, the term "immunomodulator" encompasses "immune effector cell enhancer" as defined herein and "immunosuppressive cell inhibitor" as defined herein, as well as a substance that affects other components of the mammalian immune system. In some embodiments, the immunomodulator may be an anti-CD40 agonist antibody.

The invention also includes an antibody or antigen-binding fragment or pharmaceutical composition as defined herein for use in certain treatments. In embodiments that relate to a method of treatment as described herein, such embodiments are also further embodiments relating to an antibody or antigen-binding fragment thereof, a pharmaceutical composition for use in that treatment, or alternatively for the manufacture of a drug for use in this treatment.

Thus, an anti-GDF15 antibody, or an antigen-binding fragment thereof, or a pharmaceutical composition provided herein for use in treating cancer or for inhibiting tumor growth or progression in a patient is also provided.

Also contemplated is the use of any of the anti-GDF15 antibodies provided herein in the manufacture of a medicament for the treatment of cancer or for inhibiting the growth or progression of a tumor in a patient.

Antibodies and antibody fragments may be administered in combination with one or more additional therapeutically active compounds. Additional therapeutically active compounds include agents used to treat chronic disorders associated with cachexia, anti-cancer agents (eg, immunotherapy and chemotherapy), anti-cancer agents that cause cellular stress (eg, platinum-based chemotherapeutic agents such as cisplatin), muscle anabolics (eg, selective androgen receptor modulators (SARMs), myostatin inhibitors, and Activin A receptor inhibitor), anti-inflammatory agents (eg, JAK inhibitors, IL-6 inhibitors, IL-8 inhibitors), appetite stimulants (eg, ghrelin mimetics, melanocortin receptor inhibitors 4) and agents that improve metabolism (eg metformin).

Combination therapy prophylactic or therapeutic agents, including antibodies or antigen-binding fragments thereof, may be administered to a subject in a single pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapy may be administered to the subject simultaneously in separate pharmaceutical compositions. Prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.

The term "immune response" refers to any detectable response of the immune system of a mammalian host to a specific substance (e.g., antigen or immunogen), e.g., innate immune response (e.g., activation of the toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen-specific T cells and non-specific cells of the immune system) and humoral immune responses (eg, responses mediated by B cells, such as the formation and secretion of antibodies into plasma, lymph and/or tissue fluids).

The term "immunogenic" refers to the ability of a substance to cause, elicit, stimulate, or induce an immune response, or to ameliorate, enhance, increase, or prolong a pre-existing immune response against a particular antigen, whether alone or associated with a carrier, in the presence or absence of an adjuvant. .

The compositions and methods for treating cancer provided herein may additionally contain one or more other immunomodulators.

In some embodiments, the immunomodulator may be an anti-CD40 agonist antibody. The antibody may be, for example, a human, humanized or partially human chimeric anti-CD40 antibody. Examples of specific monoclonal anti-CD40 antibodies include monoclonal antibodies G28-5, mAb89, EA-5 or S2C6, CP870893 or APX005M. In a specific embodiment, the anti-CD40 agonist antibody is CP870893 or dacetuzumab (SGN-40).

CP-870,893 is a fully human anti-CD40 monoclonal agonist that has been clinically investigated as an anticancer therapy. The structure and preparation of CP870.893 is described in WO2003040170, in which the antibody CP870.893 is identified as antibody "21.4.1". The amino acid sequences of the heavy chain and light chain of CP-870,893 are shown in SEQ ID NO: 46 and SEQ ID NO: 48, respectively, and also in Table 7 of WO2003040170. In clinical trials, CP870,893 is administered by intravenous infusion at doses typically in the range of 0.05-0.25 mg/kg per infusion. In the cancer treatment methods provided herein, CP-870,893 can be administered intradermally, subcutaneously, or topically.

Dacetuzumab (also known as SGN-40 or huS2C6; CAS number 88-486-59-9) is another generic anti-CD40 agonist antibody that has been investigated in clinical trials for indolent lymphomas, diffuse large B-cell lymphomas, and multiple myeloma. . In the cancer treatment methods provided herein, dacetuzumab can be administered intradermally, subcutaneously, or topically.

Cytokine release syndrome (CRS)

The invention relates to a method for treating CRS comprising administering to a patient an effective amount of a GDF15 antibody. CRS is a systemic inflammatory response sometimes seen after administration of monoclonal antibodies and T cell immunotherapeutic agents. (Shimabukuro-Vornhagen et al., Journal for ImmunoTherapy of Cancer 6, 56(2018). Little is known about the pathophysiology of CRS and the initiating events that trigger the massive release of various cytokines that perpetuate the systemic inflammatory response of CRS.

In some embodiments? the invention provides a method of treating CRS in a patient, comprising administering to the subject an effective amount of a pharmaceutical composition as described herein.

In other embodiments, the method may further comprise administering an effective amount of the second therapeutic agent. Second therapeutic agents include anti-inflammatory agents such as IL-6 inhibitors, tumor necrosis factor alpha (TNF-α) inhibitors, interferon gamma (IFN-γ) inhibitors, corticosteroids, antihistamines), antipyretics, and/or antibiotics. In some embodiments, the second therapeutic agent is an antibody, such as tocilizumab and/or siltuximab.

The invention also provides a GDF15 antibody, an antigen-binding fragment thereof, or a pharmaceutical composition as defined herein, for use in a method of treating CRS. The invention also provides the use of a GDF15 antibody, or an antigen-binding fragment thereof, or a pharmaceutical composition as defined herein, in the manufacture of a medicament for the treatment of CRS. In embodiments that relate to a method of treatment described herein, such embodiments are also further embodiments relating to an antibody or antigen-binding fragment thereof, a pharmaceutical composition for use in such treatment, or alternatively for the manufacture of a medicament for use in such treatment.

Combination Therapy of GDF15 Antibody and PD-1 Axis Binding Antagonist

The invention encompasses a method of treating cancer by administering to a patient a combination of a GDF15 antibody with a PD-1 binding antagonist of the axis, wherein the combination is effective in treating cancer. That is, the data disclosed herein demonstrate that the combination of an anti-GDF15 antibody and a PD-1 axis antagonist provides a therapeutic effect in the treatment of cancer. The data further demonstrate that the combination of anti-GDF-15 and a PD-1 axis antagonist provides a synergistic therapeutic effect, which is a therapeutic effect that is greater than the predicted additive effect of each therapy administered alone.

The invention encompasses a method of treating cancer comprising administering to a patient an amount of a GDF15 inhibitor in combination with an amount of a PD-1 binding antagonist that is effective in treating cancer. The invention also encompasses a method of treating cancer comprising administering to a patient an amount of a GDF15 inhibitor and an amount of PD-1 axis antagonist binding, where these amounts together are effective in treating the cancer. In another embodiment, the invention relates to a method of treating cancer comprising administering to a patient an amount of a GDF15 inhibitor and an amount of a PD-1 axis binding antagonist, where these amounts together provide a synergistic effect in the treatment of cancer, i.e. the combination is "synergistic". In one aspect, the GDF15 inhibitor is the anti-GDF15 antibody GDF15_001 and the PD-1 axis binding antagonist is selected from the group consisting of avelumab, PF-06801591 (sasanlimab, RN-888), nivolumab, pembrolizumab, atezolizumab, and durvalumab. The invention encompasses a pharmaceutical composition containing a GDF15 inhibitor and a PD-1 axis binding antagonist and a pharmaceutically acceptable carrier for use in the treatment of cancer. The invention encompasses a pharmaceutical composition containing a synergistically effective amount of a GDF15 inhibitor, a synergistically effective amount of a PD-1 axis binding antagonist, and a pharmaceutically acceptable carrier, for use in the treatment of cancer. The composition may further contain an additional therapeutic agent, such as, but not limited to, at least one chemotherapeutic agent.

One skilled in the art will appreciate, based on the description provided herein, that a method of treating cancer of the invention comprises administering a synergistic therapeutically effective amount of an anti-GDF15 antibody and a synergistic therapeutically effective amount of a PD-1 binding antagonist (e.g., anti-PD- 1-antibody, anti-PD-L1 antibody, etc.) to a patient who has either previously received or is currently receiving at least one additional therapeutic agent for the treatment of cancer. Such additional therapeutic agent includes an agent that is standard in the treatment of cancer. That is, the combination therapy of the invention may be added to a therapeutic regimen for a cancer patient already receiving other therapy, including, but not limited to, surgery, radiation therapy, chemotherapy, and any other therapy known in the art.

Those skilled in the art will be able to determine, in accordance with known methods, the appropriate amount, dose, or dosage of each compound that is used in the combination of the present invention for administration to a patient, taking into account factors such as age, body weight, general health, administered the compound, the route of administration, the nature and stage of the cancer to be treated, and the presence of other drugs.

In an embodiment, the GDF15 inhibitor is an anti-GDF15 antibody, or an antigen-binding portion thereof, and is administered intravenously (IV) or subcutaneously (SC) at an initial dose of about 0.025 mg/kg to about 20 mg/kg. The initial dose may be followed by one or more subsequent doses. In some embodiments, one or more subsequent doses may be administered, at least to a lesser extent, once a week, every other week, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, or every twelve weeks.

In some embodiments, the GDF15 inhibitor is an anti-GDF15 antibody, wherein the anti-GDF15 antibody is administered intravenously (IV) or subcutaneously (SC) at a fixed dose of about 0.1 to about 60 mg every week. In some embodiments, the anti-GDF15 antibody is administered in a fixed dose of about 2 mg, about 5 mg, about 7 mg, about 10 mg, about 12 mg, about 15 mg, about 25 mg, about 40 mg, and about 50 mg weekly.

In some embodiments, the GDF15 inhibitor is an anti-GDF15 antibody, wherein the anti-GDF15 antibody is administered intravenously (IV) or subcutaneously (SC) at a fixed dose of about 0.1 to about 130 mg every other week. In some embodiments, the anti-GDF15 antibody is administered in a fixed dose of about 5 mg, about 10 mg, about 12 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg and about 125 mg every other week.

In some embodiments, the GDF15 inhibitor is an anti-GDF15 antibody, wherein the anti-GDF15 antibody is administered intravenously (IV) or subcutaneously (SC) at a fixed dose of about 0.1 to about 400 mg every 21 days (±2 days). In some embodiments, the anti-GDF15 antibody is administered in a fixed dose of about 15 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 75 mg, about 100 mg, about 115 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, and about 385 mg every 21 days ( ±2 days).

In some embodiments, the GDF15 inhibitor is an anti-GDF15 antibody, wherein the anti-GDF15 antibody is administered intravenously (IV) or subcutaneously (SC) at a fixed dose of about 0.1 to about 400 mg every 28 days (±2 days). In some embodiments, the anti-GDF15 antibody is administered in a fixed dose of about 15 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 75 mg, about 100 mg, about 115 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, and about 385 mg every 28 days ( ±2 days).

The practice of the method of the present invention can be carried out using various schemes of administration or dosing. The combination compounds of the present invention may be administered sequentially, simultaneously or sequentially. In an embodiment, the compounds of the combination of the present invention may be administered in a co-dosing regimen.

Repetition of administration or dosing regimens may be carried out as needed to achieve the desired reduction or contraction of cancer cells. A "continuous dosing regimen" as used herein is an administration or dosing regimen without interruption of the dose, eg, no days without treatment. The repetition of 21- or 28-day courses of treatment without interrupting the dose between courses of treatment is an example of a continuous dosing regimen. In an embodiment, the compounds of the combination of the present invention may be administered in a continuous dosing regimen. In an embodiment, the compounds of the combination of the present invention may be administered simultaneously in a continuous dosing regimen.

In an embodiment, the GDF15 inhibitor is an anti-GDF15 antibody. In some embodiments, the anti-GDF15 antibody is GDF-001.

In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody such as, but not limited to, MEDI4736, MPDL3280A (YW243.55.s70), BMS-936559 (MDX-1105), avelumab, atezolizumab, and durvalumab. In some embodiments, the anti-PD-L1 antibody is avelumab and may be administered intravenously at a dose of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/kg at intervals of about 14 days (±2 days) or about 21 days (±2 days) or about 30 days (±2 days) during the course of treatment. In some embodiment, avelumab is administered at a base dose of about 80, 150, 160, 200, 240, 250, 300, 320, 350, 400, 450, 480, 500, 550, 560, 600, 640, 650, 700, 720 , 750, 800, 850, 880, 900, 950, 960, 1000, 1040, 1050, 1100, 1120, 1150, 1200, 1250, 1280, 1300, 1350, 1360, 1400, 1440, 1500, 1520, 1550 or 1600 mg, preferably 800 mg, 1200 mg or 1600 mg at intervals of about 14 days (±2 days) or about 21 days (±2 days) or about 30 days (±2 days) during the course of treatment. In some embodiments, the subject is administered an intravenous (IV) infusion of a drug containing any of the PD-1 axis binding antagonists described herein. In a specific embodiment, the subject is administered a subcutaneous (SC) infusion of a drug containing any of the PD-1 axis binding antagonists described herein.

In some embodiments, the PD-1 axis antagonist is a PD-1 axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist is an anti-PD-1 antibody, e.g. -GDF15 antibody is administered intravenously or subcutaneously, but preferably subcutaneously, at a dose of about 1, 2, 3, 4, 5, 6, 7, or 8 mg/kg at intervals of about 14 days (±2 days) or about 21 days ( ±2 days) or approximately 30 days (±2 days) during the course of treatment. In some embodiments, PF-06801591 (sasanlimab, RN-888) described in US 2016/159905 is administered at a base dose of about 80, 150, 160, 200, 240, 250, 300, 320, 350, 400, 450, 500, 550 or 600 mg, preferably 300 mg, at intervals of about 14 days (±2 days) or about 21 days (±2 days) or about 30 days (±2 days) or about 35 days (±2 days), or approximately 42 days (±2 days). In some embodiments, PF-06801591 (sasanlimab, RN888) is administered subcutaneously in an amount of 300 mg Q4W. In some embodiments, PF-06801591 (sasanlimab, RN888, mAb7) is administered subcutaneously in an amount of 600 mg Q6W.

In addition, the invention relates to a method of treating cancer, comprising administering to a patient an amount of a GDF15 antibody and an amount of a PD-1 binding antagonist, where these amounts together provide a synergistic effect in the treatment of cancer, which is greater than the therapeutic effect of the anti-GDF15 antibody and the therapeutic effect PD-1 axis binding inhibitor, where the individual effects are summarized. The method or use of the invention relates to a synergistic combination of targeted therapeutic agents, in particular a GDF15 antibody and a PD-1 axis binding antagonist. In one aspect of the embodiment, the GDF15 inhibitor is an anti-GDF15 antibody and the PD-1 axis binding antagonist is selected from the group consisting of nivolumab, pembrolizumab, tislizumab, pidilizumab, AMP-224, AMP-514, cemiplimab, PF-06801591 (sasanlimab, RN888, mAb7), avelumab, atezolizumab, and durvalumab. In one aspect of the embodiments, the GDF15 inhibitor is an anti-GDF15 antibody and the PD-1 axis binding antagonist is selected from the group consisting of nivolumab, pembrolizumab, tislelizumab, pidilizumab, AMP-224, AMP-514, cemiplimab, PF-06801591 (sasanlimab , RN888, mAb7), atezolizumab, durvalumab, and is not avelumab.

In accordance with the present invention, an amount of a first compound or component, such as a GDF15 inhibitor, is administered with an amount of a second compound or component, such as a PD-1 axis binding antagonist (which does not include avelumab), and these amounts together are effective in treating cancer. Amounts which together are effective relieve to some extent one or more symptoms of the disease being treated. With regard to the treatment of cancer, an effective amount refers to an amount that has the effect of (1) reducing the size of a tumor, (2) inhibiting (i.e. slowing to some extent, preferably stopping) the occurrence of tumor metastases, (3) suppressing to some extent (then there is slowing to some extent, preferably stopping) tumor growth or tumor invasiveness and/or (4) attenuating to some extent (or preferably eliminating) one or more signs or symptoms associated with cancer. The therapeutic or pharmacological efficacy of dosages and administration regimens can also be characterized as the ability to induce, enhance, maintain or prolong disease control and/or overall survival in patients with these particular tumors, which can be measured as the prolongation of time to disease progression.

The invention provides methods for administering an anti-GDF15 antibody, or antigen-binding portion thereof, as described, alone or in combination with other therapies, to a subject in need thereof. Combination therapies (eg, anti-GDF15 and PD-1 axis antagonists) as described herein may be administered to a subject simultaneously or sequentially. Combination therapy with anti-GDF15 and PD-1 axis antagonists of the present disclosure can also be administered cyclically. Cyclic therapy involves administering a first therapy (eg, a first prophylactic or therapeutic agent) for a specified period of time, followed by administering a second therapy (eg, a second prophylactic or therapeutic agent) for a specified period of time, and repeating this sequential administration, i.e., a course of to reduce the development of resistance to one of the therapies (eg, agents), to avoid or reduce the side effects of one of the therapies (eg, agents), and/or to improve the effectiveness of the therapies.

The invention also encompasses an anti-GDF15 antibody or antigen-binding fragment or pharmaceutical composition as defined herein for use in certain treatments in which the antibody or antigen-binding fragment or pharmaceutical composition is administered in combination with a PD-1 binding antagonist. , as defined in this document. In embodiments that relate to a method of treatment described herein, such embodiments are also further embodiments relating to an antibody or antigen-binding fragment thereof, or a pharmaceutical composition for use in such treatment, or alternatively for the manufacture of a medicament for use in such treatment.

The combination therapy therapies (eg, prophylactic or therapeutic agents) of the present disclosure may be administered to a subject simultaneously. The term "simultaneously" is not limited to administering therapies (e.g., prophylactic or therapeutic agents) at the same time, but rather means that a pharmaceutical composition comprising a GDF15 antibody, or an antigen-binding portion thereof, is described as being administered to a subject sequentially and within such time frame. interval that the antibody can act together with other therapy(s) (e.g., a PD-1 axis antagonist, which may not include avelumab) to provide increased benefit greater than if otherwise administered, more preferably, combination therapy provides " "synergistic therapeutic effect" in the sense that the therapeutic effect is greater than the additive effect of the two therapies administered separately. For example, each therapy may be administered to a subject simultaneously or sequentially in any order at different points in time; however, if they are not administered simultaneously, they should be administered close enough in time to provide the desired therapeutic or prophylactic effect. Each therapy may be administered to a subject separately, in any suitable form, and by any suitable route. The anti-GDF15 antibody or antigen-binding fragment thereof may be any antibody of the invention, preferably GDF-15_001. A PD-1 axis antagonist includes, but is not limited to, a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. In other aspects, the PD-1 binding antagonist is an anti-PD-1 antibody or antigen-binding fragment thereof. In other aspects, the anti-PD-1 antibody includes, but is not limited to, nivolumab, pembrolizumab, spartalizumab, tiselizumab, pidilizumab, AMP-224, AMP-514, cemiplimab, and PF-06801591 (sasanlimab, RN888). In other aspects, the PD-L1 binding antagonist is an anti-PD-L1 antibody or antigen-binding fragment thereof. In other aspects, the anti-PD-L1 antibody is BMS-936559 (MDX-1105), avelumab, atezolizumab, durvalumab. In other aspects, the PD-L1 binding antagonist is an anti-PD-L1 antibody or antigen-binding fragment thereof, but is not avelumab.

In various embodiments, the anti-GDF15 antibody or antigen-binding fragment thereof is administered to the subject at less than 15 minutes intervals, less than 30 minutes intervals, less than 1 hour intervals, about 1 hour intervals, from about 1 hour to about 2 hours, from about 2 hours to about 3 hours, from about 3 hours to about 4 hours, from about 4 hours to about 5 hours, from about 5 hours to about 6 hours, from about 6 hours to about 7 hours, from about 7 hours to about 8 hours, from about 8 hours to about 9 hours, from about 9 hours to about 10 hours, from about 10 hours to about 11 hours hours, at intervals of about 11 hours to about 12 hours, 24 hours, 48 hours, 72 hours, or 1 week from administration of a PD-1 axis antagonist (e.g., anti-PD-1 antibody, anti-PD-L1 antibody, which may do not include avelumab, or an anti-PD-L2 antibody, among others). In other embodiments, two or more therapies (eg, an anti-GDF15 antibody, an anti-PD-L1 antibody, which may not include avelumab, and a chemotherapeutic agent) are administered to a patient during the same visit.

The prophylactic or therapeutic agents of combination therapy may be administered to a subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies may be administered to the subject simultaneously in separate pharmaceutical compositions. Prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.

Diagnostic methods

Anti-GDF15 antibodies, antibody compositions and methods of the present invention can be used in vitro and in vivo , including immunoassays, and used to diagnose and evaluate the treatment of disorders mediated by GDF15. The methods are particularly suitable for diagnosing, evaluating, and treating human patients suffering from a disorder associated with the existence of GDF15, and more preferably with elevated GDF15 levels, where the elevated GDF15 level includes elevated levels in excess of plasma concentrations of free GDF15 in healthy volunteers. This disorder associated with the existence of GDF15 includes, but is not limited to, cachexia associated with cancer, chemotherapy, chemotherapy in combination with immuno-oncological therapy, chronic obstructive pulmonary disease, chronic kidney disease, chronic heart failure, congestive heart failure, or sarcopenia.

The invention provides a method for detecting the presence of GDF15 in a sample, wherein the method comprises contacting a sample suggestive of GDF15 with an antibody specific for GDF15 and detecting the presence of GDF15 bound to the antibody, thereby detecting GDF15 in the sample. Methods for detecting GDF15 bound to an antibody are well known in the art, including, but not limited to, an assay in which GDF15 is bound to a solid support and a sample is added to it, allowing the antibody to bind GDF15 in the sample. A second GDF15 antibody is added that is either the same as or different from the antibody bound to the solid support and can be detected either by direct labeling (i.e. the second antibody is conjugated to a detectable label) or by adding a third antibody, e.g. , from another species that interacts with the constant domain of the second antibody, and which contains a detectable label. Thus, the assay can be used to detect the presence or absence of GDF15 in a sample.

In another embodiment, the invention includes a kit for detecting the presence of GDF15 in a sample, where the kit contains an antibody specific for GDF15, an applicator and instructions for its use.

The invention also provides a method for determining the concentration of GDF15 in a sample, wherein said method comprises providing a labeled competitor containing GDF15 associated with the label to be determined; providing an antibody, or antigen-binding fragment thereof, that specifically binds GDF15; combining the sample, antibody and labeled competitor, where GDF15 in the sample competes with the labeled competitor for binding to the antibody; and determining the concentration of GDF15 in said sample by measuring the amount of labeled competitor not bound to the antibody by detecting the label. The amount of labeled competitor bound to the antibody in the absence of the sample is compared to the amount of labeled competitor bound to the antibody when the sample is added. The degree of reduction of bound labeled competitor in the presence of a sample is indicative of the amount of unlabeled GDF15 present in a sample, so the assay can be used to assess the presence and level of GDF15 in a sample.

In one embodiment, the invention provides a method for evaluating the efficacy of treating a disease or disorder associated with elevated GDF15 levels in a subject, wherein the method comprises administering treatment to the subject and comparing the GDF15 level in a sample obtained from the subject prior to treatment with the GDF15 level in an identical sample obtained from a subject after treatment, where the level of GDF15 in the sample is estimated using a specific antibody to GDF15 and, further, where the lower level of GDF15 in the sample taken from the subject after treatment, compared with the level of GDF15 in the sample taken from the subject before treatment, is indicator of the effectiveness of the course of treatment.

The term "labeled" in relation to a GDF15-specific antibody or labeled competitor includes direct labeling by binding (i.e., physical binding) of the analyte to the antibody or labeled competitor, as well as indirect labeling of the antibody or labeled competitor by combining it with another reagent that is directly marked. An example of indirect labeling includes detection of a primary antibody using a fluorescently labeled secondary antibody. In vitro methods for detecting polypeptides of the invention include enzyme-linked immunosorbent assays (ELISA), Western blotting, immunoprecipitation, and immunofluorescence.

The term "biological sample" includes tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present in a subject.

The antibodies, labeled competitors, and potential therapeutic compounds described herein are also suitable for use with any of a variety of other homogeneous and heterogeneous immunoassays with multiple detection systems.

Compositions

The GDF15 antibodies of the invention may be formulated into a pharmaceutical composition. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient and/or stabilizer (Remington: The Science and practice of Pharmacy 21st Ed., 2005, Lippincott Williams and Wilkins, Ed. K. E. Hoover) as a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to recipients in dosages and concentrations, and may contain buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexane; 3-pentane; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

The pharmaceutical composition of the present disclosure may further comprise a PD-1 axis antagonist as described herein and a GDF15 inhibitor as described herein. In one embodiment, the GDF15 inhibitor is the anti-GDF15 antibody GDF15_001 or GDF15_297, and the PD-1 axis antagonist is selected from the group consisting, optionally, of avelumab, PF-06801591 (also called "sasanlimab" and "RN-888" and mAb7 , all of which are described in WO 2016/092419), nivolumab, pembrolizumab, atezolizumab, and durvalumab. In one embodiment, the PD-1 axis antagonist does not include avelumab.

The pharmaceutical compounds of the present disclosure may include one or more pharmaceutically acceptable salts. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphoric and the like, as well as from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids. ., hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium and the like, as well as from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

The pharmaceutical composition as described may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and complex organic injectable esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization methods and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be provided by the inclusion of absorption delaying agents such as aluminum monostearate and gelatin.

Pharmaceutical compositions generally must be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. In many cases it is advantageous to include isotonic agents, for example sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by the inclusion in the composition of an agent that slows down the absorption, for example, salts of monostearates and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of the ingredients listed above, if required, followed by microfiltration sterilization.

Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the other necessary ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredient from its previously sterile filtered solution.

The pharmaceutical composition of the present invention may be formulated, packaged or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops, including, for example, a 0.1%-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may additionally contain buffering agents, salts, or one or more of the other additional ingredients described herein. Other formulations suitable for ophthalmic administration include those containing the active ingredient in microcrystalline form or in a liposomal formulation.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and loosening agents; binding agents; lubricating agents; sweeteners; flavors; dyes; preservatives; physiologically degradable compositions such as gelatin; aqueous carriers and solvents; oil carriers and solvents; suspending agents; dispersing or wetting agents; emulsifiers, softeners; buffers; salt; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in pharmaceutical compositions of the present disclosure are known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Genaro, ed., Mack Publishing Co., Easton, PA (1985), which are included in this document as a reference.

In one embodiment, the GDF15 antibody, or antigen-binding portion thereof, is administered as an intravenous formulation as a sterile aqueous solution containing 5 mg/mL, or in some embodiments, about 10 mg/mL, or, in some embodiments, about 15 mg/ml, or, in some embodiments, about 20 mg/ml of antibody, or, in some embodiments, about 25 mg/ml, or, in some embodiments, about 50 mg/ml, of sodium acetate, polysorbate 80, and sodium chloride at a pH of about 5 to 6. In some embodiments, the intravenous formulation is a sterile aqueous solution containing 5 or 10 mg/ml of antibody with 20 mM sodium acetate, 0.2 mg/ml polysorbate 80, and 140 mM sodium chloride at pH 5.5. In addition, the solution containing the antibody or antigen-binding portion thereof may contain, among many other compounds, histidine, mannitol, sucrose, trehalose, glycine, poly(ethylene)glycol, EDTA, methionine, and any combination thereof, and many other compounds known in this field of technology.

In one embodiment, the pharmaceutical composition of the present description contains the following components: 50 mg/ml GDF15 antibody or antigen-binding portion of the present description, 20 mm histidine, 8.5% sucrose and 0.02% polysorbate 80, 0.005% EDTA at pH 5.8; in another embodiment, the pharmaceutical composition of the present invention contains the following components: 100 mg/ml GDF15 antibody or antigen-binding portion of the present disclosure, 10 mM histidine, 5% sucrose, and 0.01% polysorbate 80 at pH 5.8. This composition may be presented as a liquid formulation or a lyophilized powder. When the powder is reconstituted to full volume, the composition retains the same composition. Alternatively, the powder may be reconstituted at half volume, in which case the composition comprises 100 mg of the GDF15 antibody or antigen-binding portion thereof as described herein, 20 mM histidine, 10% sucrose, and 0.02% polysorbate 80 at pH 5.8.

In one embodiment, a portion of the dose is administered as an intravenous bolus and the remainder as an infusion of the antibody formulation. For example, a 0.01 mg/IV injection of the GDF15 antibody, or antigen-binding portion thereof, may be given as a bolus, and the remainder of the antibody may be given by intravenous injection. A predetermined dose of the GDF15 antibody, or antigen-binding portion thereof, may be administered over, for example, one and a half to two hours to five hours.

With regard to a therapeutic agent, where the agent is, for example, a small molecule, it may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, for example, in combination with a physiologically acceptable cation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or further developed in the field of pharmacology. Typically, such preparations include the step of bringing the active ingredient into association with the carrier or one or more other accessory ingredients and then, if necessary or desired, forming or packaging the product into the desired single or multi-dose unit.

In one embodiment, the compositions described are pyrogen-free compositions that are substantially free of endotoxins and/or related pyrogens. Endotoxins include toxins that are contained within a microorganism and are released when the microorganism is destroyed or dies. Pyrogens also include fever-inducing thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension, and shock when administered to humans. Due to potential harmful effects, it is useful to remove even small amounts of endotoxins from intravenously administered pharmaceutical solutions. The Food and Drug Administration ("FDA") has set an upper limit of 5 units of endotoxin (EU) per dose per kilogram of body weight per hour for intravenous drug administration (The United States Pharmacopeia Convention, Pharmacopeia Forum 26(1):223 (2000) ). When therapeutic proteins are administered in amounts of several hundred or thousand milligrams per kilogram of body weight, it is beneficial to remove even trace amounts of endotoxin. In one embodiment, the levels of endotoxin and pyrogen in the composition are less than 10 EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0. 01 EU/mg, or less than 0.001 EU/mg. In another embodiment, the levels of engdotoxin and pyrogen in the solution are less than about 10 EU/mg, or less than about 5 EU/mg, or less than about 1 EU/mg, or less than about 0.1 EU/mg, or less than about 0.01 EU/mg, or less than about 0.001 EU/mg.

In one embodiment, the invention includes administering a composition wherein said administration is oral, parenteral, intramuscular, intranasal, vaginal, rectal, lingual, sublingual, buccal, buccal, intravenous, dermal, subcutaneous, or transdermal.

In another embodiment, the description further includes administering the composition in combination with other treatments such as surgery, chemotherapy, hormonal therapy, biological therapy, immunotherapy, or radiation therapy.

Dosage

For the preparation of pharmaceutical or sterile compositions comprising a GDF15 antibody, or an antigen-binding portion thereof, as described, the antibody is mixed with a pharmaceutically acceptable carrier or excipient. Therapeutic and diagnostic agent formulations can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, suspensions, aqueous solutions, lotions, or suspensions (see, for example, Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, New York, N.Y.; Avis, et al. (eds.) ( 1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY Lieberman, et al (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY Lieberman, et al (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

The choice of regimen for administering a therapeutic agent depends on several factors, including the turnover rate of the substance in serum or tissue, the level of symptoms, the immunogenicity of the substance, and the availability of target cells in the biological matrix. In some embodiments, the implementation schedule maximizes the amount of therapeutic drug delivered to the patient, in accordance with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular substance and the severity of the condition being treated. Guidelines for selecting appropriate doses of antibodies, cytokines and small molecules are available (see e.g. Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.), 1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N. Y.; Baert, et al., 2003, New Engl. J. Med. 348: 601-608 Milgrom, et al., 1999, New Engl. J. Med. 341:1966-1973; Slamon, et al., 2001, New Engl. J. Med. 344:783-792; Beniaminovitz, et al ., 2000, New Engl. J. Med. 342:613-619; Ghosh, et al., 2003, New Engl. J. Med. 348:24-32; Lipsky, et al., 2000, New Engl. J Med. 343:1594-1602).

Determination of an appropriate dose is made by the physician, for example, using parameters or factors known or suspected in the art to affect treatment or expected to affect treatment. Typically, the dose will be started at a slightly less than optimal dose and then increased in small increments until the desired or optimal effect is achieved with respect to any negative side effects.

The terms "decrease in GDF15" or "decrease in GDF15" as used herein means the reduction in free GDF15 levels compared to free GDF15 levels prior to any therapeutic intervention. As used herein, "free GDF15" means GDF15 that is not bound or otherwise complexed with another molecule (eg, an antibody or binding molecules present, for example, in plasma).

The level of GDF15 includes the level of free GDF15 in the subject, where the level is assessed using the methods disclosed herein, or any other method for assessing the level of free GDF15 known in the art.

In one embodiment, the level of free GDF15 is reduced compared to the level of GDF15 in the subject prior to administration of the antibody of the invention. In one embodiment, the free GDF15 level is reduced compared to a standard free GDF15 level that is associated with or indicates that the subject is not suffering from a disease, disorder, or condition associated with or mediated by elevated free GDF15 levels. In one embodiment, the standard or reference level of free GDF15 is from about 0.05 ng/ml to about 3 ng/ml in plasma. In another embodiment, the standard or reference level of free GDF15 is in the range, the lower value of which is selected from the group consisting of 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0, 2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2, 7, 2.8, 2.9 ng/ml, and the upper value of which is selected from the group consisting of 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3 , 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1 .6, 1.7,1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 , 2.9 and 3.0 ng/ml. In another embodiment, the standard or reference level of free GDF15 is less than 1 ng/mL, preferably less than 0.9 ng/mL, even more preferably less than 0.8 ng/mL, even more preferably less than more than 0.7 ng/ml, even more preferably less than 0.6 ng/ml, even more preferably less than 0.5 ng/ml and even more preferably less than 0.4 ng/ml . In one embodiment, the free GDF15 level is the plasma level.

The invention is not limited to free GDF15 levels of less than 0.5 ng/ml; instead, one of skill in the art will appreciate that the therapeutic level may be below or above 0.5 ng/mL for a particular subject. Therefore, the invention includes reducing the level of free GDF15 to a level at which there is a reduction or no detectable harmful effect(s) mediated by or associated with an increased level of free GDF15. Such effects include, but are not limited to, cachexia, decreased food intake, decreased appetite, decreased body weight, weight loss, decreased fat mass, decreased muscle mass, and the like.

As used herein, an "effective dosage", "effective dose", "effective amount", or "therapeutically effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more useful or desired results. For prophylactic use, beneficial or desirable outcomes include eliminating or reducing the risk, reducing the severity or delaying the onset of the disease, including the biochemical, histological and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the course of the disease. For therapeutic use, useful or desirable results include observable clinical results such as reduction or reduction in the rate of weight loss or reduction in one or more symptoms resulting from high expression of active GDF15 (e.g., reduced food intake, reduced appetite, decreased body weight, weight loss, reduction in fat mass and reduction in muscle mass), reducing the dose of other drugs needed to treat the disease, enhancing the effect of another drug and / or slowing down the progression of the disease in patients. An effective dose may be administered in one or more administrations. For the purposes of this invention, an effective dosage of a drug, compound or pharmaceutical composition is an amount sufficient to effect a prophylactic or therapeutic treatment directly or indirectly. As will be apparent from the clinical context, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective dose" may be considered in the context of the administration of one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if, in combination with one or more other agents, the desired result can be or is achieved.

In some embodiments, an effective dosage of an antibody or antigen-binding fragment thereof of the invention is based on the plasma concentration of free GDF-15 in healthy volunteers and in affected patients. The total effective dose depends on the initial plasma concentration of free GDF-15 in the affected patient. In one embodiment, an effective dosage may be a dose capable of reducing or decreasing free GDF15 levels to the same or lower mean levels as measured in healthy volunteers over the entire steady state dosing period. In another embodiment, an effective dosage may be a dosage capable of reducing or decreasing free GDF15 levels in a patient to less than 0.5 ng/ml over the entire steady state dosing period. In yet another embodiment, an effective dosage may be a dosage administered to a 70 kg subject that can reduce or decrease the subject's free GDF15 level to less than 0.5 ng/mL over the entire steady state dosing period.

A "human", "patient", or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats. In some embodiments, the individual is considered to be at risk for a disease, disorder, or condition mediated by or associated with GDF15 binding to its receptor and signaling mediated by it. In some embodiments, the subject has cachexia associated with cancer, chemotherapy, chemotherapy in combination with immuno-oncological therapy, chronic heart failure, congestive heart failure, sarcopenia, chronic obstructive pulmonary disease (COPD), sarcopenia, and chronic kidney disease (CKD).

In some embodiments, the method or use comprises administering an initial dose of about 0.025 mg/kg to about 20 mg/kg of an antibody or antigen-binding fragment thereof, or a pharmaceutical composition of the invention. The initial dose may be followed by one or more subsequent doses. In some embodiments, one or more subsequent doses may be administered at least weekly, every second week, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, or every twelve weeks.

In some embodiments, the method or use comprises administering a fixed dose of about 0.25 mg to about 2000 mg of an antibody or antigen-binding fragment thereof of the invention. In some embodiments, the antibody or antigen-binding fragment thereof is administered weekly, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks. every eleven weeks or every twelve weeks.

In other embodiments, the method or use comprises administering a fixed dose of about 0.1 to about 60 mg of an antibody or antigen-binding fragment of the invention each week. In some embodiments, the fixed dose of an antibody or antigen-binding fragment thereof of the invention is about 2 mg, about 5 mg, about 7 mg, about 10 mg, about 12 mg, about 15 mg, about 25 mg, about 40 mg, and about 50 mg. weekly.

In some embodiments, the method or use comprises administering a fixed dose of about 0.1 to about 130 mg of an antibody or antigen-binding fragment of the invention every week. In some embodiments, the fixed dose of an antibody or antigen-binding fragment thereof of the invention is about 5 mg, about 12 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 60 mg, about 90 mg, and about 125 mg. every two weeks.

In some embodiments, the method or use comprises administering a fixed dose of about 0.1 to about 400 mg of an antibody or antigen-binding fragment of the invention every four weeks. In some embodiments, the fixed dose of an antibody or antigen-binding fragment thereof of the invention is about 15 mg, about 40 mg, about 60 mg, about 75 mg, about 100 mg, about 115 mg, about 200 mg, about 300 mg, and about 385 mg. every four weeks.

Sets

The invention also provides kits or a finished product containing an antibody or antigen-binding fragment of the invention, and instructions for use. Accordingly, in some embodiments, a kit or article of manufacture is provided comprising a container, a composition within the container containing an anti-GDF15 antibody, and a package insert containing instructions for administering a therapeutically effective amount of an anti-GDF15 antibody to treat a patient in need thereof.

In some embodiments, the kit may comprise a first container containing dried protein and a second container containing an aqueous formulation. In some embodiments, kits containing single and multi-chamber pre-filled syringes (eg, liquid syringes and lyophilisate syringes) are included.

In one embodiment, the invention provides a kit for determining the concentration of GDF15 in a sample, the kit contains a labeled competitor containing GDF15 linked to a detectable label; an antibody or antigen-binding fragment thereof that specifically binds to GDF15; applicator; and instructions for their use.

The invention also provides a competitive immunoassay kit for determining the amount of GDF15 in a test sample, wherein the competitive immunoassay comprises an antibody or antigen-binding fragment thereof that specifically binds to GDF15; a labeled competitor containing GDF15 conjugated to a detectable label; where the labeled competitor competes with GDF15 in the test sample for binding to the antibody and further, where the label provides a signal indicating the amount of GDF15 in the test sample. In an exemplary embodiment, the reduction in label associated with an antibody in a test sample compared to the label associated with an antibody in an otherwise identical sample that does not contain GDF15 is indicative of the amount of GDF15 in the test sample.

In one embodiment, the invention provides a kit for determining the concentration of GDF15 in a sample, where the kit contains a labeled competitor containing GDF15 associated with a detectable label; an antibody or antigen-binding fragment thereof that specifically binds to GDF15; applicator; and instructions for their use.

In an alternative embodiment, the invention provides a kit for identifying a human patient at risk of cachexia, containing a specific antibody to GDF15 or an antigen-binding fragment, an applicator and instructions for their use.

In some embodiments, a kit or product is provided comprising a first container, a composition within the container containing an anti-GDF15 antibody, a second container, a composition within the second container containing a PD-1 binding antagonist, and a package insert containing instructions for administration. a therapeutically effective amount of an anti-GDF15 antibody and a PD-1 axis binding antagonist to treat a patient in need thereof.

The invention includes a kit or finished product comprising a first container, a composition within the container containing a synergistic therapeutically effective amount of an anti-GDF15 antibody, a second container, a composition within a second container containing a therapeutically effective therapeutic amount of a PD-1 axis binding antagonist, and a package insert, containing instructions for administering a synergistic therapeutically effective amount of an anti-GDF15 antibody and a PD-1 axis binding antagonist for combined treatment of a patient in need thereof.

In some aspects, the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 antibody or antigen-binding fragment thereof, a PD-L1 antibody or antigen-binding fragment thereof, and a PD-L2 antibody or antigen-binding fragment thereof. In some aspects, the PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, spartalizumab, tiselizumab, pidilizumab, AMP-224, AMP-514, cemiplimab, and PF-06801591 (sasanlimab, RN888). In other aspects, the PD-L1 antibody is selected from the group consisting of, optionally, avelumab, atezolizumab, durvalumab. In other aspects, the PD-L1 antibody is not avelumab.

In other embodiments, a kit or article of manufacture is provided comprising a first container, a composition within the container containing an anti-GDF15 antibody, a second container, a composition within a second container containing an anti-cancer therapeutic agent, and a package insert containing instructions for administering a therapeutically effective amount an anti-GDF15 antibody; and an anti-cancer therapeutic agent for treating a patient in need thereof. In some aspects, the anti-cancer therapeutic agent is an anti-CD40 antibody.

The invention includes a kit or finished product containing a first container, a composition within the container containing a synergistic therapeutically effective amount of an anti-GDF15 antibody, a second container, a composition within a second container containing a therapeutically effective therapeutic amount of an anti-cancer therapeutic agent, and a package insert containing instructions for administering a synergistic therapeutically effective amount of an anti-GDF15 antibody and an anti-cancer therapeutic agent for combined treatment of a patient in need thereof. In some embodiments, the anti-cancer therapeutic agent is an anti-CD40 antibody.

Instructions relating to the use of an antibody or antigen-binding fragment of the invention typically include information on dosage, dosing regimen, and route of administration for the intended treatment. The containers may be unit doses, bulk packs (eg, multi-dose packs), or sub-unit doses. The instructions supplied in the kits of the invention are usually written instructions on a label or package insert (eg, a sheet of paper included in the kit), but machine-readable instructions (eg, instructions stored on a magnetic or optical disk) are also acceptable.

The kits of the present invention are in suitable packaging. Suitable packaging includes but is not limited to vials, bottles, jars, flexible packaging (eg sealed mylar or plastic bags) and the like. Packages are also provided for use in conjunction with a particular device such as an inhaler, a nasal device (eg a nebulizer) or an infusion device such as a minipump. The kit may have a sterile access port (for example, the container may be a bag of intravenous solution or a vial with a stopper that can be pierced with a hypodermic needle). The container may also have a port for sterile access (for example, the container may be an intravenous solution bag or a vial having a stopper that can be pierced with a hypodermic needle). The container may further contain a second pharmaceutically active agent.

Sets may further represent additional components such as buffers and interpretive information. Typically, the kit includes a container and a label or insert(s) on or associated with the container.

Definitions

"About" or "approximately", unless otherwise defined herein, when used in connection with a measurable numeric variable, refers to the specified value of the variable and to all values of the variable that are within the experimental error of the specified value (e.g., within 95 % confidence interval for the mean) or within 10 percent of the specified value, whichever is greater. Numeric ranges include numbers that define the range.

The term "identity" as known in the art refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by sequence comparison. In the art, "identity" also means the degree of sequence relatedness between polypeptides or nucleic acid molecules of the sequences, as the case may be, as determined by the correspondence between strands of nucleotides or amino acid sequences. "Identity" measures the proportion of identical matches between two or more gap-aligned sequences that are accounted for by a particular mathematical model of computer programs (ie, "algorithms").

The term "similarity" is a related concept, but unlike "identity", refers to a measure of similarity that includes both identical matches and matches with conservative substitutions. Since conservative substitutions apply to polypeptides and not to nucleic acid molecules, similarity only concerns comparisons of polypeptide sequences. If two polypeptide sequences have, for example, 10 of 20 identical amino acids, and all the rest are non-conservative substitutions, then the proportion of identity and the proportion of similarity will both be 50%. If in the same example there are 5 more positions where there are conservative substitutions, then the share of identity will remain 50%, but the share of similarity will be 75% (15 out of 20). Therefore, in cases where there are conservative substitutions, the degree of similarity between two polypeptide sequences will be greater than the proportion of identity between the two sequences.

The use of the term "or" in the claims is used to mean "and/or" unless it is explicitly stated that it refers only to alternatives or alternatives are mutually exclusive, although the description supports a definition that refers only to alternatives and "and/or". In this document, specifications "a" or "an" may mean one or more, unless explicitly stated otherwise. In the context of this document, in claims, when used together with the word "comprising", the words "a" or "an" can mean one or more than one. As used herein, "other" may mean at least the second or more. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings commonly understood by those skilled in the art. In addition, unless otherwise required by the context, singular terms must include the plural, and plural terms must include the singular. The words "comprises/comprising" and the words "having/comprising", when used herein with reference to the present invention, are used to indicate the presence of the claimed functions, integers, steps or components, but do not exclude the presence or addition of one or more others. functions, integers, stages, components or their groups.

In this document, to describe the amount of carboplatin administered to a patient, the terms "CAP 3 Estimated Dose", "AUC 4 Estimated Dose", "CAP 5 Estimated Dose", "CAP 6 Estimated Dose", etc. refer to the amount of carboplatin, calculated according to the Calvert equation based on a target area under the curve (AUC) of 3, 4, 5, and 6 mg⋅min/mL, respectively, and the patient's glomerular filtration rate (GFR, ml/min): carboplatin dose (mg) = Targeted AUC (mg min/mL) x (GFR +25) as described in the National Comprehensive Cancer Network® (NCCN) Chemotherapy Order Templates Appendix B, updated February 2018.

As used herein, the term "cytokine" generally refers to proteins released by one cell population that act on another cell as intercellular mediators or have an autocrine effect on cells producing these proteins. Examples of such cytokines include lymphokines, monokines; interleukins ("IL") such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL -10, IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18 - IL-29 (such as IL-23), IL-31, including PROLEUKIN® rIL-2 ; tumor necrosis factor such as TNF-a or TNF-β, TGF-I-3; and other polypeptide factors including leukemia inhibitory factor ("LIF"), ciliary neurotrophic factor ("CNTF"), CNTF-like cytokine ("CLC"), cardiotrophin ("CT"), and kit ligand ("L").

As used herein, the term "chemokine" refers to soluble factors (eg, cytokines) that have the ability to selectively induce chemotaxis and leukocyte activation. They also trigger the processes of angiogenesis, inflammation, wound healing and tumorigenesis. Examples of chemokines include IL-8, the human homologue of mouse keratinocyte (KC) chemoattractant.

The terms "cancer", "cancerous" or "malignant" refer to or describe the physiological condition of mammals, which is usually characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), multiple myeloma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, gastric cancer, urinary cancer bladder, hepatoma, breast cancer, colon cancer, and head and neck cancer. Another specific example of cancer includes renal cell carcinoma.

The term "treatment" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the development of, or preventing the disorder or condition to which such term applies, or one or more of the symptoms of such disorder or condition.

A "patient" treated in accordance with the present invention includes any warm-blooded animal such as, but not limited to, a human, ape or other lower order primate, a horse, a dog, a rabbit, a guinea pig, or a mouse. For example, the patient is a human. Those skilled in the art of medicine can readily identify individual patients with non-small cell lung cancer in need of treatment.

The terms "treatment regimen", "dosing protocol", and dosing regimen are used interchangeably to refer to the dose and timing of administration of each therapeutic agent in the combination of the invention.

"Relief" means the reduction or improvement of one or more symptoms compared to no treatment. "Relief" also includes a reduction or reduction in the duration of a symptom.

"Tumor", in relation to a subject diagnosed or suspected of having cancer, refers to a malignant or potentially malignant neoplasm or tissue mass of any size, and includes primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or fluid. The different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas and lymphomas. Leukemias (blood cancers) usually do not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

"Tumor mass", also referred to as "tumor burden", refers to the total amount of tumor material distributed throughout the body. Tumor mass refers to the total number of cancer cells or the total size of the tumor(s) throughout the body, including lymph nodes and bone marrow. Tumor mass can be determined by various methods known in the art, for example, by measuring the size of the tumor(s) when removed from the subject, for example, using calipers, or while in the body using imaging techniques, for example, ultrasound, bone scanning, computed tomography (CT) or magnetic resonance imaging (MRI).

The term "tumor size" refers to the overall size of the tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by various methods known in the art, such as, for example, measuring the size of the tumor(s) when removed from the subject, such as with a caliper, or while in the body using imaging techniques, such as bone scanning. , ultrasound, CT or MRI.

An "individual response" or "response" may be assessed using any endpoint indicating a benefit to an individual, including but not limited to (1) inhibition to some extent of disease progression (e.g., cancer progression), including slowing or stopping ; (2) reduction in tumor size; (3) inhibition (ie, reduction, slowdown or complete cessation) of cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibition (ie, reduction, slowdown or complete cessation) of metastasis; (5) alleviate, to some extent, one or more symptoms associated with the disease or disorder (eg, cancer); (6) increasing or extending the duration of survival, including overall survival and progression-free survival; and/or (7) reduction in mortality at a certain point in time after treatment.

"Effective response" of a patient or "responsiveness" of a patient to treatment with a drug and like language refers to a clinical or therapeutic benefit conferred on a patient at risk for or suffering from a disease or disorder such as cancer. In one embodiment, such benefit includes any one or more of: increased survival (including overall survival and/or progression-free survival); obtaining an objective answer (including a full or partial answer); or improvement in signs or symptoms of cancer.

"Objective response" refers to a measurable response, including complete response (CR) or partial response (PR). In some embodiments, the "objective response rate (ORR)" refers to the sum of the complete response rate (CR) and the partial response rate (PR).

"Complete response" or "CR" in this document means the disappearance of all signs of cancer (eg, the disappearance of all target lesions) in response to treatment. This does not always mean that the cancer is cured.

As used herein, "partial response" or "PR" refers to a decrease in the size of one or more tumors or lesions or the extent of cancer in the body in response to treatment. For example, in some embodiments, PR refers to at least a 30% reduction in the sum of the largest diameters (SLD) of target lesions, taking baseline SLD as a reference.

"Sustained response" refers to the sustained effect of reducing tumor growth after cessation of treatment. For example, the size of the tumor may be the same size or smaller compared to the size at the beginning of the drug administration phase. In some embodiments, the sustained response has a duration at least the same as the duration of treatment, at least 1.5x, 2x, 2.5x, or 3x the duration of treatment, or more.

As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment during which a treatable disease (eg, cancer) does not worsen. Progression-free survival may include the amount of time patients experienced a complete or partial response, as well as the amount of time patients experienced stable disease.

In some embodiments, the anti-cancer effect of a cancer treatment method, including "objective response", "complete response", "partial response", "progressive disease", "stable disease", "progression-free survival", "duration of response", as used herein, defined and evaluated by investigators using RECIST v1.1 (Eisenhauer et al, Eur J of Cancer 2009; 45(2):228-47) in patients with locally advanced or metastatic solid tumors other than metastatic CRPC, and RECIST v1.1 and PCWG3 (Scher et al, J Clin Oncol 2016 Apr 20; 34(12):1402-18) in patients with metastatic CRPC. Descriptions by Eisenhauer et al, Eur J of Cancer 2009; 45(2):228-47 and Scher et al, J Clin Oncol 2016 Apr 20; 34(12):1402-18 are incorporated herein by reference in their entirety.

In some embodiments, the anti-cancer effect of the treatment, including "immunity-related objective response" (irOR), "immunity-related complete response" (irCR), "immune partial response" (irCR), "immune-related progressive disease" (irPD ), "immune-related stable disease" (irSD), "immune-related progression-free survival" (irPFS), "immune response duration" (irDR), as used herein, are as defined and evaluated by the criteria for immune-related response (irRECIST, Nishino et. al. J Immunother Cancer 2014; 2:17) for patients with locally advanced or metastatic solid tumors other than metastatic CRPC. Description of Nishino et. al. J Immunother Cancer 2014; 2:17 is incorporated herein by reference in its entirety.

In this document, "overall survival" (OS) refers to the proportion of people in a group who are likely to be alive after a given period of time.

By "prolongation of survival" is meant the prolongation of overall survival or progression-free survival in a treated patient compared to an untreated patient (ie, compared to a patient not treated with a drug).

In this document, the terms "drug-related toxicity", "infusion-related reactions", and "immune-associated adverse events" ("irAE"), and their severity or extent, are exemplary and defined by the National Cancer Institute's Common Terminology Criteria for Adverse Events v 4.0 (NCI CTCAE v 4.0).

As used herein, "in combination with" or "in combination with" refers to the use of one treatment in addition to another treatment. Thus, "in combination with" or "in combination with" refers to the administration of one therapeutic agent to an individual before, during, or after administration of another therapeutic agent.

"Low dose amount" as used herein refers to an amount or dose of a substance, agent, compound, or composition that is lower than the amount or dose normally used in a clinical setting.

The term "common" as used herein in relation to solid tumors includes locally advanced (non-metastatic) disease and metastatic disease. Locally advanced solid tumors that may or may not be treated with radical cure and metastatic disease that cannot be treated with radical cure are included within the scope of "advanced solid tumors" as used in the present invention. One of skill in the art will be able to recognize and diagnose advanced solid tumors in a patient.

"Duration of response" for the purposes of the present invention means the time from documenting the growth inhibition of a tumor model due to treatment with a drug until a restored growth rate is achieved, similar to the growth rate before treatment.

The term "additive" is used to mean that the result of the combination of two compounds, components or target agents does not exceed the sum of each compound, component or target agent taken separately. The term "additive" means that there is no improvement in the disease state or disorder being treated when each compound, component, or target agent is used alone.

The terms "synergy" or "synergistic" are used to indicate that the effect of the combination of two compounds, components, or targeted agents is greater than the sum of the effect of each agent alone. The terms "synergy" or "synergistic" means that there is an improvement in the disease state or disorder being treated as compared to the individual use of each compound, component, or target agent alone. This improvement in the disease state or disorder being treated is a "synergistic effect" or "synergistic therapeutic effect". A "synergistic amount", "synergistically effective amount", or "synergistic therapeutically effective amount" is an amount of a compound, component, or target agent, when administered in combination, that results in a synergistic effect, as "synergistically" is defined herein. By determining the synergistic interaction between two or more components, the optimal range for effect and the ranges of absolute doses of each component for effect can be finally measured by introducing components in different ranges of the ratio wt./mass. (weight by weight) and doses to patients in need of treatment. However, the observation of synergy in in vitro models or in vivo models can be predictive of effect in humans and other species, and there are in vitro models or in vivo models, as described herein, to measure synergistic effect, and the results of such studies can also be used to predict effective dose ranges and ratios of plasma concentrations and absolute doses and plasma concentrations required in humans and other species by applying pharmacokinetic/pharmacodynamic methods.

Equivalents

The above description and the following examples describe in detail certain specific embodiments of the description and describe the best mode envisioned by the inventors. However, it should be appreciated that no matter how detailed the foregoing may appear in the text, the disclosure may be practiced in many ways and the disclosure is to be construed in accordance with the appended claims and any equivalents thereof.

Although the disclosed ideas have been described with reference to various uses, methods, kits, and compositions, it should be understood that various changes and modifications can be made without departing from the ideas herein and as claimed in the claims. The following examples are provided to better illustrate the ideas disclosed and are not intended to limit the scope of the ideas presented herein. While the present ideas have been described in terms of these exemplary embodiments, one of ordinary skill in the art will readily appreciate that numerous variations and modifications to these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present teachings.

All references cited herein, including patents, patent applications, articles, textbooks, and the like, as well as references cited herein, to the extent not already indicated, are incorporated herein by reference in their entirety. In the event that one or more of the included literature and the like differ from or contradict this application, including, but not limited to, certain terms, use of terms, methods described, and the like, this application shall govern.

General Methods

It should be understood that this invention is not limited to specific synthetic methods of obtaining, which, of course, may vary. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings commonly understood by those skilled in the art. In addition, unless otherwise required by the context, singular terms must include the plural, and plural terms must include the singular. Generally, the nomenclatures used in connection with the methods, cell and tissue cultures, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art.

The practice of the present invention will use, unless otherwise indicated, conventional methods of molecular biology (including recombinant methods), microbiology, cell biology, biochemistry and immunology, which are within the skill of those skilled in the art. Such methods are fully described in the literature, for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Enzymatic reactions and purification methods are performed according to the manufacturer's specifications, as is commonly done in the art, or as described herein. The nomenclatures used in connection with the laboratory procedures and methods of analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, medicinal and pharmaceutical chemistry described herein are well known and widely used in the art. Standard methods are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment.

biological deposits

The type materials of the present invention are deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, April 4, 2018. The GDF15_001-VH vector, ATCC accession number PTA-125038, contains a plasmid containing a DNA insert encoding the heavy chain variable region of the GDF15_001 antibody, and the GDF15_001-VL vector, accession number PTA-125039 , contains a plasmid containing a DNA insert encoding the variable region of the light chain of the antibody GDF15_001. The deposits were made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations (Budapest Treaty). This ensures that a viable deposit culture is maintained for 30 years from the date of deposit. The deposit will be made available to ATCC in accordance with the terms of the Budapest Treaty and subject to agreement between Pfizer Inc. and ATCC, which guarantees the continued and unrestricted availability of deposit culture progeny to the public upon the grant of the relevant U.S. patent or upon the publication of any U.S. or foreign patent application, whichever comes first, and ensures progeny is available for one U.S. Commissioner of Patents and Trademarks entitled to it under 35 USC Section 122 and the Commissioner's rules under it (including Section 1.14 of 37 C.F.R. with specific reference to 886 OG 638).

The patentee of the present application has agreed that if the culture of materials on deposit dies, is lost or destroyed when cultivated under suitable conditions, the materials will be immediately replaced upon notification with others of the same kind. The availability of deposited materials should not be construed as a license to practice the invention in violation of the rights granted by any government under its patent laws.

Examples

Example 1: Anti-GDF15 antibodies

An antibody panel (see Tables 2 and 5 and Figure 29) is generated and compared across a range of binding assays and biophysical assays.

Anti-GDF15 antibodies of the present invention are analyzed based on their amino acid sequences and the presence of "hot spots" in CDR regions (eg, potential glycosylation, oxidation, and chemical degradation sites). Sequence analysis of anti-GDF15 antibody hotspots are presented in Table 5 below. GDF15_005, GDF15_006, GDF15_007, GDF15_008, GDF15_009 and GDF15_200 show the presence of N-linked glycosylation sites in the CDR region and are not selected for further study.

Table 5. Sequence analysis of anti-GDF15 antibodies Antibody HCDR1 HCDR2 HDDR3 LCDR1 LCDR2 LCDR3 GDF15_001 GYTFSSYNID
(SEQ ID NO:32) GINPIFGTAFYNQKFQG
(SEQ ID NO:165) EAITTVGAMDH
(SEQ ID NO:52) RTSQSVHNYLA
(SEQ ID NO:95) DASTRAD
(SEQ ID NO:28) QQFWSWPWT
(SEQ ID NO:9) GDF15-002 GYTFSSYNID
(SEQ ID NO:32) GINPIFGLAFYNQKFQG
(SEQ ID NO:126) EAITTVGAMDP
(SEQ ID NO:160) RASQNVHNYLA
(SEQ ID NO:157) DASNRAD
(SEQ ID NO:114) QQFWSWPWT
(SEQ ID NO:9) GDF15-003 GYTFTSYNID
(SEQ ID NO:153) QINPNNGLAFYNQKFQG
(SEQ ID NO:33) EQITTVGAMDY
(SEQ ID NO:154) RASQSLSSYLA
(SEQ ID NO:150) DAKNRAD
(SEQ ID NO:108) QQFSSDPYT
(SEQ ID NO:38) GDF15-004 GYTFSSYNID
(SEQ ID NO:32) QINPNNGLANYAQKFQG
(SEQ ID NO:146) EAITTIGAMDY
(SEQ ID NO:147) RTSESVHSYLA
(SEQ ID NO:88) DASTRAD
(SEQ ID NO:28) QQFWSDPYT
(SEQ ID NO:48) GDF15-005 GYTFSDYNMD
(SEQ ID NO:58) GINPNNGTAFYAQKFQG
(SEQ ID NO:141) EAITTVGAMDQ
(SEQ ID NO:119) RTSESVSSYLA
(SEQ ID NO: 138) DAKTRAD
(SEQ ID NO:37) QQFWSWPWT
(SEQ ID NO:9) GDF15-006 GYTFTDYNIS
(SEQ ID NO:133) QINPNNGLAFYAQKFQG
(SEQ ID NO:134) EFITTVGAMDY
(SEQ ID NO:135) RTSQSVSNYLA
(SEQ ID NO:129) DAKNRAT
(SEQ ID NO:130) QQFWNDPWT
(SEQ ID NO:102) GDF15-007 GYTFSDYNIS
(SEQ ID NO:125) GINPIFGLAFYNQKFQG
(SEQ ID NO:126) EAITTVGAMDY
(SEQ ID NO:19) RTSENVHSYLA
(SEQ ID NO: 46) DASTLAT
(SEQ ID NO:122) QQFWSWPWT
(SEQ ID NO:9) GDF15-008 GYTFTSYNIS
(SEQ ID NO:117) QINPNNGLIFFAQKFQG
(SEQ ID NO:118) EAITTVGAMDQ
(SEQ ID NO:119) RTSQNVHSYLA
(SEQ ID NO:27) DASNRAD
(SEQ ID NO:114) QQFWNDPYT
(SEQ ID NO:63) GDF15-009 GYTFSSYNIS
(SEQ ID NO:17) QINPNNGLAFYNQKFQG
(SEQ ID NO:33) EAITTVGAMEY
(SEQ ID NO:111) RTSQNVHSYLA
(SEQ ID NO:27) DAKNRAD
(SEQ ID NO:108) QQFWSDPYT
(SEQ ID NO:48) GDF15-010 GYTFSDYNID
(SEQ ID NO:41) GINPNNGLAFFNQKFQG
(SEQ ID NO:105) EAITTVGAMDY
(SEQ ID NO:19) RTSQSLHSYLA
(SEQ ID NO:101) DASNRAT
(SEQ ID NO:8) QQFWNDPWT
(SEQ ID NO:102) GDF15-012 GYTFSDYNMD
(SEQ ID NO:58) QINPIFGLAFYAQKFQG
(SEQ ID NO:98) EVITTVGAMDY
(SEQ ID NO:43) RTSQSVHNYLA
(SEQ ID NO:95) DASTRAD
(SEQ ID NO:28) QQFSSDPYT
(SEQ ID NO:38) GDF15-013 GYTFSDYNMD
(SEQ ID NO:58) GINPNNGLAFYNQKFQG
(SEQ ID NO:92) EAITTVGAMDY
(SEQ ID NO:19) RTSESVHSYLA
(SEQ ID NO:88) DASNRAT
(SEQ ID NO:8) QQFWNWPWT
(SEQ ID NO:89) GDF15-014 GYTFSSYNID
(SEQ ID NO:32) QINPINGLAFYNQKFQG
(SEQ ID NO:85) EAITTVGAMDY
(SEQ ID NO:19) RTSQNVHNYLA
(SEQ ID NO:82) DASNRAT
(SEQ ID NO:8) QQFWSDPYT
(SEQ ID NO:48) GDF15-015 GYTFSDYNMD
(SEQ ID NO:58) QINPNNGLAFYNQKFQG
(SEQ ID NO:33) EAITTVGATDY
(SEQ ID NO:79) RTSQNVHSYLA
(SEQ ID NO: 27) DASNLAD
(SEQ ID NO:47) QQFSNDPWT
(SEQ ID NO:76) GDF15-017 GYTFTDYNID
(SEQ ID NO:66) QINPNNGLAFYNQKFQG
(SEQ ID NO:33) EAITTVGAMDY
(SEQ ID NO:19) RTSQSVHSYLA
(SEQ ID NO:36) DAKTRAT
(SEQ ID NO:70) QQFSSDPYT
(SEQ ID NO:38) GDF15-018 GYTFTDYNID
(SEQ ID NO:66) QINPNNGLIFYNQKFQG
(SEQ ID NO:67) EAITTVGAMDY
(SEQ ID NO: 19) RASQNVHSYLA
(SEQ ID NO: 62) DASTRAD
(SEQ ID NO:28) QQFWNDPYT
(SEQ ID NO:63) GDF15-020 GYTFSDYNMD
(SEQ ID NO:58) QINPNNGLANYNQKFQG
(SEQ ID NO:59) EAITTVGAMDY
(SEQ ID NO: 19) RASQNLHSYLA
(SEQ ID NO: 55) DASTRAD
(SEQ ID NO:28) QQFWSDPYT
(SEQ ID NO:48) GDF15-021 GYTFSSYNID
(SEQ ID NO:32) GINPINGLIFFNQKFQG
(SEQ ID NO:51) EAITTVGAMDH
(SEQ ID NO: 52) RTSENVHSYLA
(SEQ ID NO: 46) DASNLAD
(SEQ ID NO:47) QQFWSDPYT
(SEQ ID NO:48) GDF15-022 GYTFSDYNID
(SEQ ID NO:41) QINPNNGLIFFNQKFQG
(SEQ ID NO:42) EVITTVGAMDY
(SEQ ID NO:43) RTSQSVHSYLA
(SEQ ID NO: 36) DAKTRAD
(SEQ ID NO:37) QQFSSDPYT
(SEQ ID NO:38) GDF15-100 GYTFSSYNID
(SEQ ID NO:32) QINPNNGLAFYNQKFQG
(SEQ ID NO:33) EAITTVGAMDY
(SEQ ID NO:19) RTSQNVHSYLA
(SEQ ID NO:27) DASTRAD
(SEQ ID NO:28) QQFWSDPWT
(SEQ ID NO:29) GDF15-200 GYTFSSYNIS
(SEQ ID NO:17) GINPINGLAFYNQKFQG
(SEQ ID NO:18) EAITTVGAMDY
(SEQ ID NO:19) RASQSVHSYLA
(SEQ ID NO:7) DASNRAT
(SEQ ID NO:8) QQFWSWPWT
(SEQ ID NO:9)

Potential sites of sequence susceptibility (eg deamidation: NG, isomerization: DG, cleavage: DP) are also underlined.

Example 2 Anti-GDF15 Binding Properties Antibodies: human, cynomolgus and mouse GDF15 binding activity by SPR

The binding affinity of the GDF15_001 antibody (comprising VH containing the amino acid sequence of SEQ ID NO:166 and VL containing the amino acid sequence of SEQ ID NO:163) to human, cynomolgus and mouse GDF15 was determined using a BIAcore T200 instrument (GE Healthcare) at 37°C at a rate of collecting 10 Hz. Mouse human GDF15 Fc (Mu IgG1Fc_Fxa_Hu GDF15; SEQ ID NO:2), mouse GDF15 mouse Fc (Mu IgG1Fc_Fxa_Mu GDF15; SEQ ID NO:5) and mouse GDF15 cynomolgus Fc (Mu IgG1Fc_Fxa_Cyno GDF15; SEQ ID NO: 4 ) are captured on the surface of three different flow cells of the CM4 sensor chip (p/n BR100534, GE Healthcare) using the Mouse Antibody Capture Kit (BR100838, GE Healthcare) according to the manufacturer's protocol. Mobile and sample buffer is 10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% P-20 (HBS-EP+). End capture levels of Mu IgG1Fc_Fxa_Hu GDF15, Mu IgG1Fc_Fxa_Mu GDF15 and Mu IgG1Fc_Fxa_Cyno GDF15 were 40 resonance units (RE), 32 RU and 25 RU, respectively. Flow cell 1 is used as a reference flow cell. A two-fold serial dilution of GDF15_001 at concentrations from 10 nM to 0.625 nM is injected over the touch surface for 120 seconds. Dissociation is monitored for 2.8 hours and the surface is regenerated with 10 mM glycine pH 1.7. Binding affinities and rate constants were determined for GDF15 mice and cynomolgus monkeys by fitting the acquired sensorgram data to a 1:1 Langmuir model in BIAcore T200 Evaluation Version 2.0 (GE Healthcare). Affinity values are determined as shown in Table 6 below.

The binding affinity of several clones of GDF15 binding antibodies to mouse human Fc-GDF15 was also determined using the methodology described herein in Example 2 and as shown in Table 6 below. All clones tested in the divalent format show an apparent KD value below 150 pM, suggesting that they are strong binding agents for human GDF15. Additionally, GDF15_001 clone binding to cynomolgus and mouse GDF15 was measured using the above BIAcore assay (Table 7). Clone GDF15_001 shows strong binding to cynomolgus monkey GDF-15 (apparent KD 8.28 pM) and retains binding to mouse GDF15 (apparent KD 142.3 pM), making clone GDF15_001 suitable for preclinical studies in both species.

Table 6 BIAcore kinetic data for human GDF15-binding antibody clones ligand analyte Ka (1/ms) Kd (1/s) T1/2 min Rmax (PE) %Chi2/Rmax Apparent KD (pM) Mu IgG1Fc_Fxa_Hu GDF15 GDF15_001 1.69E+06 9.22E-06 1252.8 23.2 1.2 <10 GDF15_001 2.03E+06 6.25E-06 1848 70.6 1 <10 GDF15_001 2.18E+06 4.78E-06 2417.3 64.8 0.05 <10 Wed 1.97E+06 6.75E-06 <10 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_003 2.64E+06 7.22E-05 160.04 23.91 0.133 27.4 GDF15_003 3.43E+06 9.19E-05 125.73 17.73 2.51 26.8 Wed 3.03E+06 8.20E-05 27.1±0.3 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_004 2.22E+06 7.14E-05 161.86 45.77 0.291 32.2 1.96E+06 8.20E-05 140.85 37.03 0.51 41.9 Wed 2.09E+06 7.67E-05 37 ± 4.8 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_010 1.31E+06 1.47E-05 783.58 34.1 1.302 11.3 GDF15_010 8.85E+05 1.78E-05 649.24 33.33 1.287 20.1 Wed 1.10E+06 1.63E-05 15.7±4.4 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_013 2.42E+06 6.66E-05 173.35 33.56 1.54 27.5 GDF15_013 7.30E+06 7.51E-05 153.88 25.04 0.57 10.3 Wed 4.86E+06 7.08E-05 18.0±8.6 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_014 1.88E+06 1.79E-05 644.89 29.2 0.13 <10 GDF15_014 1.90E+06 1.48E-05 779.88 23.53 0.13 <10 Wed 1.89E+06 1.64E-05 <10 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_015 1.67E+06 9.93E-05 116.28 23.28 0.42 59.4 GDF15_015 1.81E+06 1.04E-04 111.59 17.68 0.49 57.3 Wed 1.74E+06 1.01E-04 58.4 ± 1.1 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_017 3.24E+06 8.66E-05 133.39 26.35 0.12 26.7 GDF15_017 1.90E+06 8.18E-015 141.22 23.1 0.16 43 Wed 2.57E+06 8.42E-05 34.9 ± 8.2 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_020 9.72E+05 9.46E-05 122.08 22.23 0.1 97.4 GDF15_020 6.86E+05 1.13E-04 102.3 21.57 0.17 165 Wed 8.29E+05 1.04E-04 131.2 ± 33.8 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_021 2.05E+06 2.91E-04 39.64 28.58 0.36 142 GDF15_021 2.14E+06 3.52E-04 32.84 22.75 0.27 165 Wed 2.09E+06 3.22E-04 153.5 ± 11.5 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_022 1.34E+06 1.77E-05 654.39 30.58 0.26 13.2 GDF15_022 8.13E+05 1.45E-05 795.45 38.96 0.33 17.9 Wed 1.08E+06 1.61E-05 15.6±2.4 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_0297 5.54E+06 1.09E-05 1060.61 52.25 0.36 <10 GDF15_0297 5.50E+06 1.29E-05 894.66 53.67 0.39 <10 Wed 5.52E+06 1.19E-05 <10 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_0301 3.36E+06 1.04E-06 11105.77 58.42 1.16 <10 GDF15_0301 5.58E+06 7.08E-06 1632.05 46.84 0.29 <10 GDF15_0301 5.22E+06 1.28E-05 903.76 46.75 0.30 <10 Wed 4.72E+06 6.97E-06 <10 Mu IgG1Fc_Fxa_Hu GDF15 GDF15_0470 1.20E+07 5.21E-05 221.86 43.46 0.17 <10 GDF15_0470 1.30E+07 4.30E-05 268.36 39.94 0.22 <10 Wed 1.25E+07 4.76E-05 <10

Table 7. Kinetic data of BIAcore GDF15_001 with human, mouse, and cynomolgus macaque GDF15 ligand analyte Ka (1/ms) Kd (1/s) T1/2 min Rmax (PE) %Chi2/Rmax Apparent KD (pM) Mu IgG1Fc_Fxa_Hu GDF15 GDF15_001 1.69E+06 9.22E-06 1252.8 23.2 1.2 <10 GDF15_001 2.03E+06 6.25E-06 1848 70.6 1 <10 GDF15_001 2.18E+06 4.78E-06 2417.3 64.8 0.05 <10 Wed 1.97E+06 6.75E-06 <10 Mu IgG1Fc_Fxa_Mu GDF15 GDF15_001 2.55E+06 2.92E-04 39.6 86.7 1.5 114.0 GDF15_001 2.46E+06 4.31E-04 26.8 58.9 1.1 175.0 GDF15_001 2.51E+06 3.47E-04 33.3 54.0 1.5 138.0 Wed 2.51E+06 3.56E-04 142.3 ± 30.7 Mu IgG1Fc_Fxa_Cyno GDF15 GDF15_001 1.47E+06 1.30E-05 886.4 96.0 0.89 8.86 GDF15_001 1.85E+06 1.24E-05 928.5 49.6 0.28 6.71 GDF15_001 1.12E+06 1.03E-05 1119.2 76.50 0.07 9.26 Wed 1.48E+06 1.19E-05 8.28 ± 1.37

Example 3 Anti-GDF15 Antibody Binding Properties: Monomeric Anti-GDF15 Antibody Binding Activity to Human, Cynomolgus and Mouse GDF15 by SPR

To understand the KD value, without avidity effect, the binding of GDF15_001 to GDF15, a monomeric Fc-Fab was prepared and tested in the same assay used in Example 2. The binding affinity of monomeric GDF15_001 to human, cynomolgus, and mouse GDF15 was determined using the BIAcore T200 instrument. (GE Healthcare) at 37°C with 10 Hz acquisition rate. Mu IgG1Fc_Fxa_Hu GDF15, Mu IgG1Fc_Fxa_Mu GDF15, and Mu IgG1Fc_Fxa_Cyno GDF15 capture three different flow cells of the CM4 sensor chip (p/n BR100534, GE Healthcare) on the surface using the Mouse Antibody Capture Kit (BR100838, GE Healthcare) according to the manufacturer's protocol. Mobile and sample buffer is 10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% P-20 (HBS-EP+). End-capture levels of Mu IgG1Fc_Fxa_Hu GDF15, Mu IgG1Fc_Fxa_Mu GDF15 and Mu IgG1Fc_Fxa_Cyno GDF15 were 19 resonance units (RE), 22 RE and 19 RE, respectively. Flow cell 1 is used as a reference flow cell. A two-fold serial dilution of GDF15_001 at concentrations from 10 nM to 1.25 nM is injected over the touch surface for 120 seconds. Dissociation is monitored for 1200 seconds and the surface is regenerated with 10 mM glycine pH 1.7. Binding affinities and rate constants were determined by fitting the acquired sensorgram data to a 1:1 Langmuir model in BIAcore T200 Evaluation Version 2.0 (GE Healthcare). Monomeric clone GDF15_001 shows strong binding to human GDF15 (KD 21.3 pM) and cynomolgus monkey (KD 62.0 pM), with weaker binding to mouse GDF15 (KD 1965.0 pM), as shown in Table 8 below.

Table 8. BIAcore kinetic data of monomeric GDF15_001 with human, mouse, and cynomolgus monkey GDF15 ligand analyte Ka (1/ms) Kd (1/s) T1/2 min Rmax (PE) %Chi2/Rmax Apparent KD (pM) Mu IgG1Fc_Fxa_Hu GDF15 CH23LS-GDF15_001 1.52E+06 2.25E-05 512.65 29.94 0.655 14.9 CH23LS-GDF15_001 1.66E+06 4.58E-05 252.24 24.61 1.601 27.6 Wed 1.59E+06 3.42E-05 21.3 ± 6.4 Mu IgG1Fc_Fxa_Mu GDF15 CH23LS-GDF15_001 1.62E+06 3.41E-03 3.39 32.44 0.364 2110 CH23LS-GDF15_001 1.70E+06 3.10E-03 3.73 26.86 0.521 1820 Wed 1.66E+06 3.26E-03 1965.0±145.0 Mu IgG1Fc_Fxa_Cyno GDF15 GDF15_001 1.63E+06 7.48E-05 154.33 29.65 0.661 45.8 GDF15_001 1.20E+06 9.39E-05 123.07 30.11 0.661 78.2 Wed 1.42E+06 8.43E-05 62.0 ± 16.2

Example 4 Binding Properties of Anti-GDF15 Antibody: Binding Specificity of GDF15_001 to Human GDF15

To assess the binding specificity of GDF15_001 to human GDF15, GDF15_001 binding to additional members of the TGFβ family is tested. The analysis is carried out on an Octet Red instrument.

OctetRED 384 (ForteBio, Menlo Park, CA) is used to evaluate off-target binding of monomeric GDF15_001(CH23LS-GBT-GDF15_001) to ten members of the TGFβ family, including human GDNF (R&D, 212-GD/CF), human inhibin A (R&D, 8506 -AB/CF), Human Activin B (R&D, 659-AB/CF) Human TGFβ-1 (R&D, 240-B/CF) Human BMP2 (R&D, 355-BM/CF), Human BMP3b (R&D, 1543- BP/CF), human BMP6 (R&D, 507-BP/CF), human BMP9 (R&D, 3209-BP/CF), human BMP11 (R&D, 1958-CD/CF), human GDF8 (Pfizer, 41075-201) and control human GDF15 (Mu IgG1Fc_Fxa_Hu GDF15). Members of the TGFβ family are diluted to 10 μg/ml in 10 mM sodium acetate, pH 4.5, and the amine is coupled into AR2G biosensors (catalog # 18-5092, ForteBio) according to the manufacturer's instructions. Monomeric GDF15_001 is diluted to 200 nM in kinetic buffer (ForteBio catalog # 18-5032). Octet assays are performed at room temperature with an association time of 300 seconds and a dissociation time of 180 seconds. Data are presented twice (Myszka, D., J. Mol. Recognit 1999; 279-284) and analyzed using Octet Data Analysis, version 8.1 (ForteBio).

GDF15_001 binds to GDF15 as expected. Binding to other members of the TGFβ family (GDF-11, BMP-9, GDF-8, BMP-2, BMP-6, TGFβ-1, activin-B, inhibin-A) was not detected at 200 nM monomeric clone GDF15_001, demonstrating the high specificity of GDF15_001, which does not bind to such other related family members. The high specificity of GDF15_001 minimizes the risk of off-target binding and indicates that GDF15_001 is a potential novel useful therapeutic agent in humans.

Example 5 Anti-GDF15 Antibodies Prevent GDF15 from Binding to GFRAL

The ability of GDF15_001 to prevent human GDF15 from binding to the extracellular domain of GFRAL was evaluated in a competitive assay. The assay is performed using a BIAcore T200 instrument (GE Healthcare, Chicago, IL).

GDF15_001 is titrated in 10 nM human GDF-15 at concentrations ranging from 10 nM to 2.5 nM. Mixtures of GDF-15, GDF15_001 and controls are injected over the extracellular domain of human GFRAL, which is directly immobilized on the CM4 sensor chip. A concentration-dependent inhibition of human GDF15 binding to GFRAL was observed. The binding of human GDF15 to human GFRAL ECD was completely blocked by 7.5 nM GDF15_001.

These data demonstrate that GDF15_001 blocks the interaction between human GDF15 and its cognate receptor, GFRAL, and therefore may interfere with GFRAL signaling. This also demonstrates that GDF15_001 may be a novel, potentially useful therapeutic agent to reduce activity mediated by GDF15 binding to GFRAL.

Example 6 Biophysical Properties of Anti-GDF15 Antibody: Thermal Stability

The thermal stability of the anti-GDF15 antibody was assessed by differential scanning calorimetry (DSC). Proteins are diluted in phosphate buffered saline (PBS) to 0.3 mg/ml in a volume of 400 µl. The FSFB is used as an empty buffer in the reference cell. FGFB contains 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na ₂ HPO ₄ and 1.47 mM KH ₂ PO ₄ , pH 7.2. Samples are distributed in a MicroCal VP-Capillary DSC test tray with automatic dispenser (Malvern Instruments Ltd, Malvern, UK). The samples are equilibrated for 5 minutes at 10°C and then scanned up to 110°C at a rate of 100°C per hour. Select a filter period of 16 seconds. The raw data is corrected for baseline and the protein concentration is normalized. Origin Software 7.0 (OriginLab Corporation, Northampton, MA) is used to fit the data to the MN2-State Model with an appropriate number of hops.

The transition temperatures are shown in Figures 1A, 1B and 1C and are listed in Table 9. T _m 1 represents the temperature at which the C _H 2 antibody is 50% unfolded. T _m 2 represents the temperature at which the Fab of the antibody is 50% unfolded. T _m 3 represents the temperature at which the C _H 3 antibody is 50% unfolded. All clones with a melting point (T _m 1) greater than 65°C are designated as stable clones that will be stable during production and storage.

Table 9. Transition temperatures of anti-GDF15 antibodies Clone T _m 1 (°C) T _m 2 (°C) T _m 3 (°C) GDF15_001 71.67±0.08 84.30±0.25 88.00±0.04 GDF15_002 71.81±0.06 80.51 ± 1.10 82.51±0.21 GDF15_003 71.67 ± 0.10 83.69±0.37 86.66±0.12 GDF15_004 71.57 ± 0.11 84.94±0.29 88.50±0.07 GDF15_005 71.37 ± 0.14 83.30±0.67 85.79±0.18 GDF15_006 71.40±0.09 84.45±0.26 87.92±0.11 GDF15_007 71.41 ± 0.11 85.14±0.23 89.72±0.07 GDF15_008 71.70±0.14 83.05 ± 0.76 85.33±0.24 GDF15_009 71.35±0.15 85.42±0.31 89.88±0.09 GDF15_010 72.14 ± 0.20 81.18±0.64 84.13 ± 0.19 GDF15_100 71.33 ± 0.17 85.45±0.28 90.02±0.07 GDF15_0297 73.01±0.20 74.45±0.07 80.80±0.07 GDF15_0301 76.14±0.06 77.20±0.04 78.82±0.05

Example 7 Biophysical Properties of Anti-GDF15 Antibodies: Size Exclusion Chromatography

Antibody clones are analyzed by analytical size exclusion chromatography (aSEC). Proteins are diluted in phosphate buffered saline (PBS) to 1.0 mg/mL and analyzed aSEC on a YMC-Pack Diol-200, 300×8 mm column with isocratic running buffer containing 20 mM sodium phosphate pH 7.2, 400 mM NaCl. Native SECs are used to determine the relative amounts of high molecular weight (aggregates) and monomeric intact antibody. The proportion of aggregates is calculated as the high molecular weight peak area divided by the total (aggregate and monomer) peak area times 100. The retention time in minutes is recorded and compared with the analytical control. Antibody clones with normal retention time and peak shape from aSEC analysis may suggest minimal stationary phase resin interactions and optimal hydrophobicity, which are useful characteristics and indicate that the antibody may be a potential useful therapeutic agent.

The retention time for each antibody clone tested is shown in Table 10. GDF15_002 shows a delayed retention time and a broad peak shape and is therefore not further investigated.

Table 10. aSEC retention time for anti-GDF15 antibodies Antibody aSEC hold time (min) control mAb 10.40 GDF15_001 10.79 GDF15_002 11.69 GDF15_003 10.22 GDF15_004 10.24 GDF15_005 9.74 GDF15_100 10.30 GDF15_0297 9.96 GDF15_0301 9.91

Example 8 Biophysical Properties of Anti-GDF15 Antibodies: Low pH Stability

After purification of antibodies by capture of Protein A and elution under low pH conditions, anti-GDF15 antibody clones are tested for storage stability at low pH. Antibody at 1.0 mg/ml in PRP, pH 7.2 acidified with glycine pH 3.4 and incubated at 25°C for 5 hours, then neutralized with Tris Base and run on aSEC to determine the number of high molecular weight species (HMMS) and low molecular weight species (LMMS), table 11. All clones tested showed an acceptable amount of increase in HMMS (<5%) after exposure to low pH. This shows that anti-GDF15 antibodies remain stable during purification processes and can potentially be used as therapeutic agents.

Table 11. Amount of HMMS and LMMS after exposure to low pH. GDF15 % HMMS neutral control % increase in HMMS acidified then neutralized %LMMS neutral control % increase in LMMS acidified then neutralized % reduction in monomer peak area acidified then neutralized GDF15_001 2.9 -1.5 0 0 -0.4 GDF15_002 3.0 -0.7 0 0 -0.6 GDF15_003 2.0 -0.3 0 0 1.5 GDF15_004 1.9 -0.3 0 0 1.4 GDF15_005 6.4 -0.6 0 0 1.3 GDF15_100 5.5 -0.3 0 0 3.0

Example 9 Biophysical Properties of Anti-GDF15 Antibodies: Viscosity

The viscosity of GDF15_001 is analyzed on an Anton Paar instrument. GDF15_001 was concentrated to 215 mg/mL using 30 kDa Amicon molecular weight cut-off centrifuge filters (EMD Millipore, Billerica, MA). A dilution series of 46-178 mg/ml is prepared with 20 mM Histidine, 85 g/l sucrose, 0.05 mg/ml EDTA pH 5.8 buffer as diluent. Protein concentrations are determined by 280 nm analysis on the SoloVPE Variable Pathlength System (C Technologies, Inc, Bridgewater, NJ). Viscosity measurements were made using a CP25-1 cone and plate on an MCR-302 rheometer (Anton Paar USA Inc., Ashland, VA) at a constant rotation speed of 150 rpm at 25°C. A total of 10 measurements of 10 seconds each were collected for the sample and the data was analyzed using the Rheoplus software (Anton Paar USA Inc.) V 3.62. Viscosity is presented in centipoise (cP).

The viscosity of GDF15_001 is shown in Figure 2. The data show that the acceptable viscosity limit (20 cps) is reached at about 140 mg/ml, making subcutaneous injection of GDF15_001 realistic, also showing that this antibody is a useful potential therapeutic agent.

Example 10: Immunogenicity of anti-GDF15 antibody

Based on the determination of non-germline T cell epitopes and the calculated adjusted tReg score, the immunogenicity of an anti-GDF15 antibody of the present invention and another anti-GDF15 antibody known in the art as hu01G06 is predicted (the VH and VL sequences of hu01G06 are represented herein as SEQ ID NO:177 and SEQ ID NO:178, respectively (VH, SEQ ID NO:248 of WO2014/100689 and VL, SEQ ID NO:254 of WO2014/100689)). A lower adjusted tReg score predicts a low risk potential for immunogenicity.

Sequences are analyzed using two protocols (protocols 1 and 2 below) to identify epitopes. Any sequences identified by the rules described herein for any protocol are considered an epitope. Sequences are examined at the 9-mer amino acid level.

Protocol 1 - ISPRI/EpiMatrix : Sequences are subjected to EpiMatrix analysis in the ISPRI software package (ISPRI v 1.8.0, EpiVax Inc., Providence, RI (2017); Schafer et al. Vaccine 16(19), 1880-84 (1998 )). The raw results represent a ranking of the binding probability of each 9-mer amino acid fragment against 8 different HLA types. Thus, there are 8 predictions ("observations") for each 9-mer. 9-mer is created starting from each individual position of the linear sequence numbering. It is possible for the same 9-mer to occur more than once in the same sequence. If any 4 observations indicate that 9-mer is in the top 5% of binders, which means that it is expected to be in the top 5% of binders for at least 4 HLA types, 9-mer is considered a predicted epitope. Alternatively, if any 1 of the 8 predictions indicates that the 9-mer is in the top 1% of binders, the 9-mer is also considered the predicted epitope.

Protocol 2 - IEDB Consensus Method : Sequences are analyzed using the MHC-II consensus binding method (Wang et al. BMC Bioinformatics 11, 568 (2010); Wang et al. PLoS Comput Biol. 4(4),e1000048 (2008)) in the immune epitope database (IEDB) (IEDB MHC-II Binding Predictions, http://www.iedb.org; Vita et al., Nucleic Acids Res. Jan 28 (43), D405-12 (2015). Out program results are classified by 15-mer A consensus score and percentile ranking is presented for each combination of 15-mer and HLA type However, the individual scores from which each 15-mer consensus is derived are the ranks of the specific 9-mer found in the 15-mer : each method for consensus represents the percentile rank for the 9-mer in the 15-mer, and the consensus taken as the value for the entire 15-mer is the prediction for the 9-mer having the median score.The 9-mer is classified as an epitope if (a) it is selected as a 15-mer consensus type, and (b) it has a percentile rank in the top 10% of binders for the HLA type in question and if criteria (a) and (b) are present for three or more distinct HLA types for one and the same 9-mer (i.e. three observations). The HLA types in question are DRB1*01, 1*03, 1*04, 1*07, 1*08, 1*11, 1*13, and 1*15, which are the same HLA types in the standard ISPRI/EpiMatrix report. For ease of comparison with protocol 1, the data is reinterpreted to produce a list of predicted 9-mer epitopes, although the primary output of the consensus method is a 15-mer ranking.

Each epitope is classified as a germline or non-germline epitope. For antibodies, each epitope is then classified based on its location in the antibody (eg, CDR or non-CDR). Human V domain sequences obtained from IMGT (www.imgt.org) are filtered to remove germlines annotated as pseudogenes or open reading frames. Any predicted 9-mer epitope found in the remaining sequences is considered a germline epitope. Epitopes found in the J or C regions (including IgG1, IgG2, IgG3 and IgG4) or connections between these regions are also classified as germline epitopes. Otherwise, the epitope is classified as a non-germline epitope.

CDR definitions are based on the Cabot method, where CDRs are defined to include the following residues: HCDR-1 (H26-H35 including inserts such as H35A up to but not including H36), HCDR-2 (H50-H65 inclusive), HCDR -3 (H95-H102 included) LCDR-1 (L24-L34 included), LCDR-2 (L54-L56 included), LCDR-3 (L89-L97 included). A predicted 9-mer epitope is defined as a CDR epitope if any of its amino acids are part of a CDR region.

Overall Sequence Score (Adjusted tReg Score): For a single strand, or for a pairing of the VH and VL domain of an antibody, the overall score can be calculated by summing each of the 9-mer constituents as described below.

All individual combinations of 9-mer and HLA type ("observations") are tested whether or not the 9-mer is an epitope. If a particular observation indicates that the peptide is in the top 5% binders for that HLA type, the EpiMatrix Z-score for that observation is added to the subtotal associated with the entire protein sequence. The total number of observations verified is also recorded. The one exception is that all 9-mer observations identified by ISPRI as "T-regiotopes" (amino acid sequences of the framework region of a monoclonal antibody that can potentially activate natural regulatory T cells and reduce unwanted immune responses) are accepted as having EpiMatrix scores of zero.

In the subtotal, a base score of 0.05 * 2.2248 is subtracted from each observation (including T-regitopes). The final score is calculated as follows: Adjusted score tReg = (subtotal)*1000 / (number of observations)

The calculated scores are listed in Table 12. As indicated above, a lower score indicates the lowest predicted immunogenic potential. Anti-GDF15 antibodies of the present invention have scores lower than hu01G06. Clones GDF15_001, GDF15_004, GDF15_005 and GDF15_013 have the lowest scores and therefore have the lowest potential predicted risk of inducing immunogenic responses. It also shows that the antibodies of the invention are potential useful therapeutic agents.

Table 12. Immunogenicity search prediction for GDF15 mAb, adjusted tReg score Clone # Rating (adjusted tReg) GDF15_001 -41.58 GDF15_002 -39.34 GDF15_003 -26.99 GDF15_004 -42.06 GDF15_005 -50.36 GDF15_006 -34.21 GDF15_007 -39.6 GDF15_008 -24.95 GDF15_009 -29.30 GDF15_010 -34.08 GDF15_012 -38.79 GDF15_013 -42.09 GDF15_014 -34.36 GDF15_015 -34.18 GDF15_017 -38.82 GDF15_018 -33.79 GDF15_020 -33.64 GDF15_021 -31.56 GDF15_022 -33.16 GDF15_100 -36.41 GDF15_200 -26.01 hu01G06 -20.36

The predicted T-cell epitopes of GDF15_001 and hu01G06 are also compared based on the in silico methods described above. As shown in Table 13 below, hu01G06 has two predicted T cell epitopes in the heavy chain and one predicted T cell epitope in the light chain, although GDF15_001 has no predicted T cell epitopes. This means that GDF15_001 also has the potential risk of inducing immunogenic responses compared to hu01G06. This also means that GDF15_001 is a potentially useful new therapeutic agent with improved performance.

Table 13. Predicted T cell epitopes of GDF15_001 and hu01G06 Name heavy chain light chain Hu01G06 2 1 GDF15_001 0 0

Example 11 Inhibition of Human and Mouse GDF15 in Healthy Mice

The ability of GDF15_001 to inhibit human GDF15 activity was evaluated in healthy C57Bl6N mice treated with human adeno-associated virus (AAV)-GDF15. Two weeks after treatment with human AAV-GDF15, circulating levels of human GDF15 are elevated to about 17 ng/mL (Figure 3) and body weight is reduced by 15% (Figure 4). The introduction of GDF15_001 rapidly reverses the loss of body weight (figure 4), fat (figure 5) and muscle mass (figure 6) in mice treated with human AAV-GDF15, relative to IgG control. GDF15_001 has no effect on mice treated with the AAV control vector.

The ability of GDF15_001 to inhibit mouse GDF15 activity was also evaluated in healthy C57Bl6N mice treated with mouse AAV-GDF15. Eleven days after administration of mouse AAV-GDF15, circulating mouse GDF15 levels are elevated to about 3 ng/mL (Figure 7) and body weight is reduced by about 10% (Figure 8). Administration of GDF15_001 rapidly reversed body weight loss (Figure 8) and increased food intake (Figure 9) in mice treated with mouse AAV-GDF15 relative to IgG control. GDF15_001 has no effect on mice treated with the AAV control vector.

These data demonstrate that GDF15_001 reverses body weight loss due to loss of muscle and fat mass, and increases food intake in healthy mice further in the presence of elevated levels of GDF15.

Example 12 Inhibition of Human GDF15 in Mice Having Fibrosarcoma (HT-1080)

To determine whether GDF15_001 can reverse cachexia and prolong survival in tumor-bearing mice, HT-1080 human fibrosarcoma cells are subcutaneously implanted in severely immunocompromised (SCID) mice. Two weeks after tumor implantation, body weight is reduced by approximately 10% (figure 10). GDF15_001 administration rapidly reversed body weight loss (FIG. 10), fat mass loss (FIG. 11), and muscle loss (FIG. 12) compared to the IgG control. In addition to improving body composition, GDF15_001 improves survival (Figure 13) compared to the IgG control.

To confirm the metabolic benefit of GDF15_001 in HT-1080 tumor-bearing mice under thermoneutral conditions, in which basal metabolic rate under cold stress conditions is excluded, the study was repeated when the mice were housed at 86°F. The level of GDF15 in the human circulation rises to ~5 ng/ml (figure 14). Introduction GDF15_001 retains a rapid reversal of body weight loss (Figure 15) compared to IgG control. GDF15_001 had no effect on any of these parameters in otherwise similar but tumor-free mice.

These data demonstrate that GDF15_001 improves survival and reverses cachexia in tumor-bearing mice. Thus, these results also indicate that GDF15_001 is a potential new human therapeutic agent.

Example 13 Inhibition of Human GDF15 in Mice Having a Pancreatic Tumor

To further evaluate whether GDF15_001 can reverse cachexia and prolong survival in tumor-bearing mice, SCID mice are subcutaneously implanted with PA-0165 tumor tissue fragments derived from liver metastases of a pancreatic tumor. The level of GDF15 in the human circulation rises to ~2 ng/ml (figure 16). Four weeks after tumor implantation, body weight is reduced by approximately 10% (figure 17). The introduction of GDF15_001 completely prevents the loss of body weight (figure 17) compared with the IgG control. In addition to improving body weight, GDF15_001 improves survival (Figure 18) compared to the IgG control.

These data also demonstrate that GDF15_001 improves survival and reverses cachexia in tumor-bearing mice. Thus, these results also indicate that GDF15_001 is a potential new human therapeutic agent.

Example 14 Inhibition of Human GDF15 in Tumor Bearing Mice Treated with Anticancer Agents

To determine whether GDF15_001 can reverse cachexia and prolong survival in tumor-bearing mice treated with anti-cancer agents, BALB/c mice are implanted with RENCA (mouse kidney adenocarcinoma) cells. The circulating level of mouse GDF15 is increased by ~8 ng/ml (Figure 19). When the tumor size reaches 100 mg, treatment with the anticancer agent sorafenib is started, followed by addition of GDF15_001 or IgG control when 10% body weight loss is achieved (Figure 20). Administration of GDF15_001 to sorafenib-treated mice completely reversed body weight loss (Figure 20). Together with the improvement in body weight, the combination of sorafenib+GDF15_001 improves survival compared to sorafenib+IgG control (Figure 21).

The ability of GDF15_001 to improve body weight is also being investigated in tumor-bearing mice treated with the anti-cancer agent cisplatin. SCID mice are implanted subcutaneously with NSX-26115 tumor tissue fragments (derived from human non-small cell lung carcinoma adenocarcinoma). The circulating level of mouse GDF15 is increased by ~4.5 ng/ml (Figure 22). When the tumor size reaches 100 mg, a 10% body weight loss is achieved and treatment with the anti-cancer agent cisplatin is started along with GDF15_001 or an IgG control (Figure 23). GDF15_001 or IgG control is also given without cisplatin. GDF15_001 administration reversed body weight loss when administered with or without cisplatin compared to the IgG control (Figure 23). Together with the improvement in body weight, the combination of cisplatin+GDF15_001 improves survival (Figure 24) compared to cisplatin+IgG control.

These data also demonstrate that GDF15_001 improves survival and reverses cachexia in tumor bearing mice treated with anticancer agents. Thus, these results further demonstrate that GDF15_001 is a novel potential human therapeutic agent.

Example 15 Anti-GDF15 Antibody Dosing

There are problems associated with determining effective doses and dosing regimens of anti-GDF15 antibodies for the treatment of cachexia. The total effective dose depends on the initial concentration of GDF15 in the patient's bloodstream, which is known to be variable. In addition, free GDF15 levels in patients with cachexia decrease at different rates in response to therapy. Thus, an effective dose can be defined for a given dosing interval as the amount administered to a 70 kg patient that reduces free GDF15 to less than 0.5 ng/mL during the steady state dosing interval (i.e., after such a number of doses that the maximum concentration after the dose is essentially the same as the maximum concentration after the previous dose). This level of free GDF15 is chosen based on data described for healthy subjects (Wollert et al, 2017, J Appl Lab Med 1(5):510-521). Thus, the effective dose described herein is described as a function of the initial level of free GDF15 in the affected subject and reducing this level to less than 0.5 ng/ml. The level of free GDF in a sample can be measured, for example, using an electrochemiluminescence immunoassay such as the ELECSYS GDF15 assay (Roche Diagnostics). The ELECSYS GDF15 assay uses biotin-streptavidin technology and is based on the principles of a sandwich immunoassay (see Wollert et al, 2017). Other assays that can measure the amount of free GDF15 in a sample, which are known in the art, can also be used to determine the level of free GDF15 in a patient.

To overcome the problems of predicting effective doses and dosing regimens for the antibody described herein, a two-component pharmacokinetic model was developed. Subcutaneous administration of anti-GDF15 antibodies is modeled by dosing a certain amount into a depot, which is then absorbed into the central compartment at a given rate k _a . The antibody is distributed into the peripheral compartment and then cleared out of the central compartment. As used herein, the term "depot" refers to the site where the drug is deposited (e.g., subcutaneous space), the term "central compartment" refers to the vascular space and highly perfused organs (e.g., liver, kidneys, and lungs), and the term ""peripheralcompartment" refers to tissues with less perfusion (eg, fat and muscle).

The parameters for GDF15_001 are determined by scaling from the parameters in the pharmacokinetic study of GDF15_001 in mice expressing human FcRn. Briefly, 6 mice are injected with 1 mg/kg GDF15_001 intravenously and observed for 1000 hours; the resulting antibody pharmacokinetics are fitted to a two-compartment pharmacokinetic model. Parameters for other antibodies described herein can be determined in a similar manner. GDF15_001 is produced in the central compartment, distributed to the peripheral compartment, and cleared from both spaces. The volumes and distribution rates are assumed to be the same as those determined for GDF15_001 in the pharmacokinetic study described above. Turnover is chosen to give a half-life of GDF15_001 of 16.6 hours based on an experiment to quantify the incorporation of stable isotope-labelled amino acids into GDF15 blood flow in two healthy volunteers (experimental methodology is basically as presented in Lassman et al., 2014, Clin Chem 60(9):1217-1224). The rate of GDF15 synthesis is chosen so as to obtain a certain level of GDF15 in the bloodstream. This circulating level may represent the highest level described for a given disease (to treat all patients with the condition), a lower threshold but still higher than the values for the specified proportion of subjects (to control the maximum dose while allowing treatment for the majority of patients), or a specific level determined for a particular subject in order to provide a more accurate individual dose. For ranges of GDF15 levels associated with human disease, see, for example, Mutlu et al, 2015, Inflammation 38(5):1805-1813; Montero et al, 2016, PLoS One 11(2):e0148709; Kempf and Wollert, 2009, Heart Fail Clin 5(4):537-547; and Lerner et al, 2016, Oncol Lett 12(5): 4219-4223.

In the central and peripheral compartments, GDF15 can reversibly bind to an anti-GDF15 antibody based on the association and dissociation rate constants for the antibody. For GDF15_001, these parameters are defined as 1.59×10 ⁶ M ^-1 s ^-1 (k _a ) and 3.42×10 ^-5 s ^-1 (k _d ), respectively (equating to an equilibrium dissociation constant of 21.3 pM ), based on Biacore measurements (see Table 8 above). Similar studies can be carried out for other antibodies described herein, or the parameters can be used as input parameters to study the effect of changes in affinity on dose. Without wishing to be bound by any particular theory, the antibody/GDF15 complex partitions between the two compartments, and is cleared from the central compartment using the same pharmacokinetic parameters as determined for the antibody itself. The parameters for the two-compartment dosing model are shown in Table 14.

Table 14. Parameters of the two-compartment dosing model GDF15_001 Parameter Meaning Units Source V1 49 ml/kg Scaled from pharmacokinetic parameters determined in mice expressing hFcRn using state-of-the-art optimal methodology V2 46 ml/kg Q 0.42 ml/h/kg CL 0.24 ml/h/kg Body mass 70 kg Type person t _1/2,GDF15 16.6 h Determined from stable isotope administration in human GDF15 in healthy subjects k _deg 0.0418 Turnover, ln(2)/t _1,2,GDF15 k _on 1.59×10 ⁶ M ^-1 s ^-1 Measured SPR k _off 3.42×10 ^-5 from ^-1 Measured SPR _KD 21.3 pM k _off / k _on [GDF15] _base 0.5 ng/ml ELECSYS GDF15 Analytical Insert (Roche Diagnostics) Dose Variable mg Input specification, or defined to obtain a reduction in the target level of free GDF15 k _a 0.0108 h ^-1 Typical MW _GDF15 25 kDa Derived from gene deposits at Uniprot http://www.uniprot.org/uniprot/Q99988 MM antibodies 150 kDa Typical Bioavailability 0.5 dimensionless Estimated

Predicted therapeutically effective subcutaneous doses for GDF15_001 as a function of starting free GDF15 level for the subject are shown in Tables 15, 16, and 17 and Figures 25, 26, and 27. Predicted therapeutically effective doses for weekly, biweekly, and approximately monthly dosing are shown.

The dosing data presented herein suggests that GDF15_001 can be administered in amounts and dosing intervals that allow a potentially therapeutic level of free GDF15 reduction to be achieved. These results demonstrate that GDF15_001 dosing can be achieved and maintained on a dosing schedule that is feasible and will ensure patient compliance.

Table 15 Potential weekly dosing for subcutaneous GDF15_001 to reduce GDF15 levels Initial free GDF15 (ng/ml) Weekly subcutaneous dose (mg) 5 2 10 5 15 7 20 10 25 12 thirty 15 50 25 75 40 100 50

Table 16 Potential biweekly dosing for subcutaneous GDF15_001 to reduce GDF15 levels Initial free GDF15 (ng/ml) Subcutaneous dose every other week (mg) 5 5 10 12 15 20 20 25 25 thirty thirty 40 50 60 75 90 100 125

Table 17 Potential dosage for approximately monthly subcutaneous administration of GDF15_001 to reduce GDF15 levels Initial free GDF15 (ng/ml) Subcutaneous dose every four weeks (mg) 5 15 10 40 15 60 20 75 25 100 thirty 115 50 200 75 300 100 385

Example 16 Combination Treatment with Anti-PD-1 (F2) Antibody and Anti-GDF15 (GDF15-297) in a Subcutaneous RCC Model

The data disclosed herein demonstrate the synergistic therapeutic effect of combination therapy with an anti-GDF15 antibody and an anti-PD1 antibody in the RENCA model of metastatic renal cell carcinoma (RCC) in mice.

Tumor bearing mice are prepared as follows: Six 6-8 week old female BALB/c mice are purchased from Jackson Laboratories. All animals are housed in a pathogen-free CID vivarium and experiments are performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC). The RENCA cell line is purchased from the American Type Culture Collection (ATCC). Cells are cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine at 37°C in 5% carbon dioxide (CO ₂ ) and tested for pathogens by IMPACT at Research Animal Diagnostic Laboratory (RADIL) (Columbia, MO). Pathogenic-free cells grown in the exponential growth phase are harvested and used for tumor inoculation. BALB/c mice are subcutaneously inoculated in the right flank with 0.2 x 10 ⁶ RENCA in 0.1 ml serum-free DMEM.

Tumor volume analysis is performed as follows: when tumors reach the target size, mice are randomly assigned to treatment groups. Treatment begins on the day of distribution, ie. day 0. The size of the tumor is measured twice a week in 2 sizes using a caliper, and the volume is expressed in cubic millimeters using the formula: V=0.5 L × W ² where L is the largest diameter of the tumor, and W is the diameter perpendicular to L. Body weight is recorded twice a week.

Therapeutic antibodies used in this study are anti-mouse PD1 F2 mouse (mouse immunoglobulin G1 [mIgG1]) and anti-mouse GDF15_0297 containing mouse IgG1 with null effector function D265A, i.e., from asparagine (Asp, D) to alanine (Ala, A) at amino acid residue number 265), a mutation in Fc. The GDF15_0297 antibody is essentially a surrogate mouse GDF15_001 antibody that similarly binds to this antibody. Also, F2 is a mouse surrogate antibody that binds similarly to sasanlimab (RN888). Anti-PD1 antibody and anti-GDF15 antibody were each diluted to 2 mg/mL in phosphate buffered saline (PBS) (Life Technologies) and dosed at 10 mg/kg per mouse ip on treatment day 0 ( d0). One-way or 2-way ANOVA is used to compare statistical differences among multiple groups with respect to isotype control (negative mouse IgG1 control) or other treatment groups. An unpaired t-test is used to compare the statistical difference between two groups. P < 0.05 is considered a significant difference.

The RENCA model is used to evaluate the therapeutic efficacy of anti-GDF15 in combination with anti-PD1 in the rRCC model, the average initial tumor size is from 25 to 38 mm ³ (n=10 animals per group). Animals are observed for two weeks after the start of treatment. Treatment, i.e. v.b. the introduction of anti-GDF15 and anti-PD1, start 11 days after transplantation. Combination treatment with anti-GDF15 antibody and anti-PD1 antibody significantly inhibited the progression of subcutaneous tumor RENCA rRCC compared to the isotype control group. Anti-GDF15 monotherapy and anti-PD1 monotherapy were not effective in inhibiting tumor progression under these conditions (Table 18 and Table 19).

Table 18: Tumor measurements (mean ± SOS) of subcutaneous RENCA renal carcinoma over time Group 1. Isotype control day of treatment Average tumor size (mm ³ ) SOS N 0 24.8179 18.48923 10 3 41.10472 16.01365 10 7 118.3017 38.05085 10 eleven 316.4947 87.86244 10 14 458.5579 180.3841 10 Group 2 Anti-GDF15 antibody (10 mg/kg) Days after tumor inoculation Average tumor size (mm ³ ) SOS N 0 38.6903 23.46297 10 3 74.46177 55.75148 10 7 118.0962 76.10373 10 eleven 242.452 131.6799 10 14 421.239 250.0879 10 Group 3 Anti-PD1 antibody (10 mg/kg) Days after tumor inoculation Average tumor size (mm ³ ) SEM N 0 30.47708 19.57651 10 3 46.06364 33.32501 10 7 81.07916 48.32548 10 eleven 188.6607 104.1654 10 14 279.6987 159.2461 10 Group 4 Anti-PD1 and anti-GDF15 antibody (10 mg/kg each) Days after tumor inoculation Average tumor size (mm ³ ) SOS N 0 31.10019 20.06108 10 3 58.65684 27.55265 10 7 100.9666 49.4239 10 eleven 179.1596 73.56688 10 14 367.1608 165.8177 10

Tumor volume is expressed in mm ³ . N=number of animals in each group; SOS=standard error of the mean.

Table 19: Statistical comparison of subcutaneous RENCA tumor measurements of renal carcinoma over time versus controls Day Anti-GDF15
(10 mg/kg) Anti-PD1
(10 mg/kg) Anti-GDF15 (10mg/kg) + Anti-PD1 (10mg/kg) 0 0.9995 >0.9999 >0.9999 3 0.9721 0.9986 >0.9999 7 >0.9999 0.9987 0.9554 eleven 0.5250 0.0205 0.0394 14 0.9549 0.2835 0.0007

The results of the statistical analysis presented in Table 19 demonstrate that anti-GDF15 antibody treatment in combination with anti-PD1 has a synergistic effect in the early stages of tumor progression in the RCC cancer model.

Example 17 Treatment of Pancreatic Cancer with a Combination of an Anti-PD1 Antibody and an Anti-GDF15 Antibody

This example illustrates the synergistic therapeutic effect of combination therapy with an anti-GDF15 antibody and an anti-PD1 antibody in a mouse model of metastatic pancreatic adenocarcinoma, ie the Pan02 model. To facilitate identification of tumor growth in the orthotopic model, Pan02 (NHI/NCI) cells are infected with a firefly luciferase construct generating Pan02-luciferase cells.

Six 8-week-old female B6(Cg)-Tyrc-2J/J (B6 albino) mice were purchased from Jackson Laboratories. All animals are housed in a pathogen-free CID vivarium and experiments are performed according to protocols in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC).

The Pan02 cell line was obtained from NHI/NCI. Cells are cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine at 37°C in 5% carbon dioxide (CO ₂ ) and tested for pathogens by IMPACT at Research Animal Diagnostic Laboratory (RADIL) (Columbia, MO). Pathogenic-free cells grown in the exponential growth phase are harvested and used for tumor transplantation. Albino B6 mice are injected into the pancreas with 20 μl of tumor cells: matrigel mixture containing 0.2 x 10 ⁶ ml of Pan02-luciferase in serum-free DMEM and matrigel (Corning, ref 356237). Four days after transplantation, the tumor mass is assessed by the level of luminescence. The animals are divided into groups and the test begins. To determine luciferase emission, animals are injected with 0.1 ml of 30 mg/ml D-luciferin monosodium salt in saline (Pierce, # 88292). Luminescence levels are determined on IVIS Spectrum CT (Caliper Life Science) after 1-15 sec treatment at medium sorting, 1 F/stop. Images are analyzed with LivingImage 4.5.1. Tumor mass is calculated as photon/second in ROI (region of interest) after background values have been subtracted. In one example, the average emission at the time of inclusion in the study is 4.8*10 ⁸ photons/second. In another example, the average emission at the time of inclusion in the study is 1.8*10 ⁸ photons/second.

Mouse anti-mouse PD1 F2 antibody (mouse immunoglobulin G1 [mIgG1]) and mouse anti-mouse GDF15_0297 antibody are as described in Example 16. Anti-PD1 antibody and anti-GDF15 antibody are each diluted to a concentration of 2 mg/mL in saline. phosphate buffered solution (PBS) (Life Technologies) and dosed at 10 mg/kg per mouse intraperitoneally (ip). In another example, anti-PD1 antibody is diluted to a concentration of 1 mg/ml and dosed at 5 mg/kg per mouse. In each case, the PD-1 antibody and the GDF15 antibody are administered on the same day (treatment day 0).

Tumor progression is monitored weekly based on the level of luminescence in treated animals. Luminescence levels are determined as described above. Body weight is recorded weekly.

On day 35 after transplantation, the change in tumor mass for animals from the isotype antibody, anti-GDF15 antibody, anti-PD1 antibody and anti-GDF15 antibody + anti-PD1 combination groups is determined by the formula: change in tumor mass = (photon flux in d35 - flux photons in q4)/photon flux on day 4 . Treatment with anti-GDF15 antibody + anti-PD1 antibody significantly reduced the tumor mass of animals transplanted with Pan02 tumors, where 8 out of 12 animals showed signs of regression compared to anti-PD1, anti-GDF15 and isotype groups (4/12; 1/12 and 0/12, respectively). The results are summarized in Table 20 below. Table 20 shows the individual results for each of the twelve animals per group.

Table 20 Changes in tumor mass in Pan02 transplanted animals 35 days after transplantation Isotype (10 mg/kg)
(n=12) Anti-GDF15 (10 mg/kg)
(n=12) Anti-PD1 (10 mg/kg)
(n=12) Anti-GDF15 (10mg/kg) + Anti-PD1 (10mg/kg)
(n=12) 388650.8 238089.09 51810.29 121462.31 202783.4 42471.88 5095.89 729.61 146446 40946.73 1997.97 509.41 20687.27 35690.5 1435.5 117.08 19259.32 17367.64 390.93 -35.58 11767.25 12343.34 192.99 -53.22 11056.08 4468.03 145.6 -79.94 4223.002 1331.72 48.91 -82.36 1969.562 127.73 -5.73 -98.16 1357.696 70.79 -81.18 -99.36 561.6385 38.24 -94.2 -99.63 477.8538 -64.21 -99.55 -99.67

These changes in tumor mass correspond to an increase in the survival of animals treated with anti-GDF15 antibody + anti-PD1 antibody, with a mean survival of up to 90 days compared to a survival of 66 days (anti-PD1), 52.5 days (anti-PD1). GDF15) and 45 days (isotype control). When compared with the p-value for the isotype control group at the end of the experiment (day 145), the p-values were p<0.0001 (anti-GDF15+anti-PD1), p=0.0010 (anti-PD1) and not statistically significant ( ns) (anti-GDF15). At day 79, there was a statistically significant difference between the survival of animals treated with anti-GDF15+anti-PD1 combination and anti-PD1 monotherapy, which was reflected in p=0.05, established by the Matel-Cox test, and p=0.042, by the Gehan-Breslow test. -Wilcoxon.

In another example, in a Pan02 model of the pancreas with an average tumor mass estimated through luminescence emission at study entry of 1.8*10 ⁸ photons/sec (n=13 animals per group), the therapeutic efficacy of 10 mg/kg of anti- GDF15 antibody in combination with 5 mg/kg anti-PD1 antibody. Treatment starts four days after transplantation. On day 39, tumor volume in the anti-GDF15+anti-PD1 treated group fell below the detection limit in 38% of the animals. PD1 monotherapy resulted in tumor shrinkage in only 15% of the animals, while both isotype control and anti-GDF15 monotherapy showed no therapeutic effect under these conditions.

These results demonstrate that anti-GDF15 antibody treatment in combination with anti-PD1 provides a synergistic antitumor effect greater than the additive effects of each treatment given alone on progression in an orthotopic model of pancreatic cancer.

Thus, these data demonstrate that combination therapy of an anti-GDF15 and a PD-1 axis antagonist could be a useful new synergistic therapy for cancer treatment.

Example 18 Selection of Synergistic Mouse Models for Testing Efficacy in Cancer Immunotherapy.

To select an appropriate model to determine the therapeutic efficacy of anti-GDF15 therapy, the following mouse syngeneic cells are tested: MC38 (courtesy of Dr. Antoni Ribas at University of California, Los Angeles, CA), Pan02 (NHI/NCI), 4T1 (ATCC), RENCA (ATCC), CT26 (ATCC), ID8 (University of Kansas and University of Kansas Medical Center), LL2 (ATCC), B16F10 (ATCC) and GL261 (NHI/NCI). GDF15 levels in the conditioned medium are analyzed using the GDF15 ELISA mouse kit (R&D Systems, MGD 150). Cells were seeded three times at 0.3×10 ⁶ cells/well in a 6-well cell culture dish (353224, Corning) and cultured for forty-eight hours. The supernatants are collected, centrifuged for 5-10 min at 2000 rpm, transferred to fresh tubes and stored at +4°C. The number of live/dead cells was assessed by triptan blue exclusion methods on a BioRad TC 20 cell counter. The volume and number of cells for each sample were recorded. The results are presented in table 21.

Table 21 cell line pg/ml SO Replicate 4T1 0 0.33 3 B16-F10 12.8 0.71 3 CT26 0 0.25 3 GL261 14.72744 2.38 3 ID8 6.375451 0.47 3 LL2 4.20784 1.35 3 MC38 55.65824 3.56 3 PAN02 38.44435 16.32 3 RENCA 342.397 8.08 3

To select a suitable in vivo model, blood from tumor-bearing animals is collected retroorbitally and transferred to Microtainer blood collection tubes (365967, BD). Tumor volume is recorded and adjusted to 200 mm ³ if applicable. Serum from animals with orthotopic Pan02 tumors is collected from animals at the final stage (35-40 days after transplantation). Serum from animals that do not have tumors and match the genetic background, is collected as a control. Mouse GDF15 levels are determined using the GDF15 mouse ELISA kit and are presented in Table 22.

Table 22. Strain/Model pg/ml SO Replicate BalbC 31.10779804 6.632974 3 C57Bl6 74.55406 2.443727 2 FVB/N 38.96494373 13.71892286 3 RENCA 3460.438 1398.909 3 4T1 32.45177948 8.056048 3 Pan02 1880.244884 1095.055333 3 MC-38 44.13618956 21.41318 3 PyMT 146.4433483 27.91198329 3 B16_F10 136.8055 193.4721 3

The metastatic renal RCC (RENCA) model and the pancreatic cancer model (Pan02) are identified as the highest in vivo producers of GDF15 and are further used in in vivo studies to determine the therapeutic potential of anti-GDF15 therapies in combination with immuno-oncological therapies.

Example 19 Combination Treatment with Anti-CD40 Antibody and Anti-GDF15 Antibody

This example illustrates the therapeutic activity of combination therapy with an anti-GDF15 antibody and an anti-CD40 antibody in a mouse model of metastatic RCC RENCA.

Six 6-8 week old female BALB/c mice are purchased from Jackson Laboratories. All animals are housed in a pathogen-free CID vivarium and experiments are performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC).

The RENCA cell line is purchased from the American Type Culture Collection (ATCC). Cells are cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine at 37°C in 5% carbon dioxide (CO ₂ ) and tested for pathogens by IMPACT at Research Animal Diagnostic Laboratory (RADIL) (Columbia, MO). Pathogenic-free cells grown in the exponential growth phase are harvested and used for tumor inoculation. BalbC mice are subcutaneously inoculated in the right flank with 0.2 x 10 ⁶ RENCA in 0.1 ml serum-free DMEM.

Anti-mouse CD40 antibody FGK45 (mouse IgG1) and anti-mouse GDF15 antibody GDF_0297 (containing mouse IgG1 Fc with null effector function containing a substitution at amino acid residue number 265 from aspartic acid to alanine (D265A)) are diluted at concentrations of 2 mg/ml phosphate buffered saline (PBS) (Life Technologies) and dosed at 10 mg/kg per mouse intraperitoneally (ip). In another example, anti-CD40 is diluted to 1 mg/ml and dosed at 5 mg/kg per mouse.

When tumors reach the target size, mice are randomly assigned to treatment groups. Treatment begins on the day of distribution. The size of the tumor is measured twice a week in two sizes using a caliper and the volume is expressed in cubic millimeters using the formula: V=0.5 L×W ² where L is the largest diameter of the tumor and W is the diameter perpendicular to L. Mass bodies are recorded twice a week.

To analyze the effect of combined treatment on the status of tumor infiltration and leukocytes in the spleen, the following analysis is performed. Tumors are disseminated to single cell suspensions using a soft MACS™ and Miltenyi Mouse Dissociation Kit (Miltenyi Biotec) according to the manufacturer's protocol with modification. Ammonium potassium chloride (ACK) lysis buffer (Life Technologies) is used to remove red blood cells. Cells are washed twice with FACS staining buffer (PBS with 10% PBS) and finally resuspended in FACS staining buffer. To determine leukocytes in the spleen, excised spleens are sprayed with 3 ml digest solution containing 0.2 mg/ml Liberase TL (5401020001, Roche) and 20 µl/ml DNase I Roche 4716728001, Roche). The spleens are incubated for 15 min at 37°C with gentle shaking. At the end of the incubation, the enzyme activity is quenched by adding 1 ml of PBS, the spleens are cut into small pieces and strained into 50 ml conical tubes, the cells are washed in PBS, centrifuged at 1500 rpm for 5 minutes, and the resulting pellet is resuspended in cold MACS buffer . Lysis Buffer ACK is used to remove red blood cells.

An aliquot of cells is preincubated with 0.5 μg/10 ⁶ mouse BD Fc Block cells (Biolegend, 101310) in 10% rat serum in PBS for 10 minutes, before phenotyping, the mAb is added to specifically stained immune cells. Cell surface antigens are labeled by incubating the cells at 4° C. for 30 minutes. After removal of unbound mAbs, cells are washed twice with FACS staining buffer, fixed in fixing buffer (FBS + 1% paraformaldehyde) and stored at 4° C. in the dark until flow cytometric analysis. Intracellular staining was performed using the Foxp3/Transcription Factor Staining Buffer kit (eBioscience) according to the manufacturer's protocol. Flow cytometry data is collected using Fortessa™ SORP (BD Biosciences) and analyzed using FlowJo™ (TreeStar Inc.).

Antibodies used for cell surface or intracellular staining are purchased from BD Biosciences, eBioscience, Invitrogen, or Biolegend. They are rat anti-mouse CD45-PerCP-Cy5.5 (clone 30-F11, BD Biosciences), Texas Red rat anti-mouse CD11b (M1/70, BD Biosciences), rat anti-mouse CD25-BUV395 (PC61, BD Biosciences), rat anti-mouse Ly6G-FITC (1A8, BD Biosciences), rat anti-mouse CD90.2-BV786 (53-2.1, BD Biosciences), rat anti-mouse CD8-BV650 (53-6, 7, BD Biosciences), rat anti-mouse Ly6C-BV421 (AL-21, BD Biosciences), rat anti-mouse F4/80-PECy7 (BM8, Invitrogen™), rat anti-mouse CD86-BV605 (GL1, BD Biosciences ), Armenian hamster anti-mouse CD49a-APC (HMA1, BioLegend), rat anti-mouse MHCII (IA-IE)-Alexa Fluor 700 (M5/114,15,2, BioLegend), rat anti-mouse CD4-V450 ( RM4-5, BD Biosciences), rat anti-mouse Foxp3-PE (FJK-16s, eBiosciences). To avoid staining artifacts, samples were stored in Brilliant Stain Buffer (BD Biosciences). Live cells are separated from dead cells using the LIVE/DEAD Fixable eFluor™ 780 Dead Cell Stain Kit (Invitrogen).

The results are expressed as mean±SOS. Statistical analysis is carried out using GraphPad Prism 6.0. One-way or 2-way ANOVA is used to compare the static difference between multiple isotype control or other treatment groups. An unpaired t-test is used to compare the statistical difference between two groups. P < 0.05 is considered a significant difference.

The RENCA model is used to evaluate the therapeutic efficacy of anti-GDF15 in combination with anti-CD40 in the rRCC model, mean initial tumor size 25-38 mm ³ (n=10 animals per group). Animals are sacrificed when the tumor volume reaches 2000 mm ³ or 20% loss of body weight. The average survival of an animal with tumors for the isotype, only anti-GDF15 antibody and only anti-CD40 antibody, and the combination of anti-GDF15 antibody+anti-CD40 antibody is 26, 25.5, 32.5 and 34 days, respectively. The anti-CD40 antibody + anti-GDF15 antibody group also produced 30% complete responders, compared to 0% in the isotype group, only anti-GDF15 antibody and only anti-CD40 antibody.

In a separate study, the therapeutic efficacy of an anti-GDF15 antibody in combination with two different doses of an anti-CD40 antibody (10 mg/kg and 5 mg/kg) was evaluated in the RENCA model of recurrent renal cell carcinoma (rRCC), with a mean initial tumor volume of 47 -54 mm ³ (n=10 animals per group). The results are summarized in Table 23 and Table 24.

Table 23. Tumor measurements (mean ± SOS) of subcutaneous renal carcinoma Renca over time Group 1. Isotype control Days after tumor inoculation Average tumor size (mm ³ ) SOS N 10 53.90874 14.3443 10 13 130.8436 28.27738 10 17 353.1065 85.68442 10 20 552.0961 137.3508 10 25 858.6344 162.7132 10 27 1195.16 292.976 10 Group 2 Anti-GDF15 antibody (10 mg/kg) Days after tumor inoculation Average tumor size (mm ³ ) SOS N 10 53.24435 13.3418 10 13 118.301 31.60418 10 17 347.7124 117.5708 10 20 602.2936 247.2447 10 25 1207.494 545.9653 9 27 1455.983 309.5726 8 Group 3. Anti-CD40 antibody (10 mg/kg) Days after tumor inoculation Average tumor size (mm ³ ) SOS N 10 53.44178 13.94953 10 13 110.619 34.34105 10 17 224.5232 70.38568 10 20 338.2354 74.07587 10 25 532.1088 155.9947 10 27 715.2596 193.4371 10 Group 4. Anti-CD40 antibody (5 mg/kg) Days after tumor inoculation Average tumor size (mm ³ ) SOS N 10 53.09616 13.09756 10 13 96.98625 33.47341 10 17 254.366 86.80886 10 20 376.4508 110.6125 10 25 648.832 170.2004 10 27 795.9384 357.4954 10 Group 5. Anti-GDF15 antibody + anti-CD40 antibody (10 mg/kg) Days after tumor inoculation 10 46.99903 11.86423 10 13 106.4694 43.02584 10 17 186.6874 88.81804 10 20 233.0179 132.9253 10 25 349.6296 234.2788 10 27 458.9107 371.8595 10 Group 5. Anti-GDF15 antibody + anti-CD40 antibody (5 mg/kg) Days after tumor inoculation Average tumor size (mm ³ ) SEM N 10 53.78749 14.79445 10 13 97.36322 37.22007 10 17 232.1165 95.47443 10 20 315.4536 157.8604 10 25 565.9923 322.4584 9 27 584.3638 417.7759 9

Tumor volume is expressed in mm ³ . N=number of animals in each group; SOS=standard error of the mean.

Table 24: Statistical comparison between tumor measurements in RENCA subcutaneous renal carcinoma over time versus isotype controls Day Anti-CD40 antibody (10 mg) Anti-CD40 antibody (5 mg) Anti-GDF15 antibody Anti-GDF15 antibody + anti-CD40 antibody (10 mg) Anti-GDF15 antibody + anti-CD40 antibody (10 mg) 10 >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 13 0.9997 0.9983 0.9999 0.9997 0.9983 17 0.6932 0.8611 >0.9999 0.4581 0.7398 20 0.2271 0.4057 0.9911 0.0256 0.152 25 0.0213 0.243 0.0152 <0.0001 0.0571 27 0.0002 0.0028 0.1278 <0.0001 <0.0001

Average survival of animals with tumors for the isotype, anti-GDF15 antibodies only, anti-CD40 antibodies (5 mg/kg), anti-CD40 antibodies (10 mg/kg) and combinations of anti-GDF15 antibodies (10 mg/kg) + anti- CD40 antibody (5 mg/kg) and combination of anti-GDF15 antibody (10 mg/kg) + anti-CD40 antibody (10 mg/kg) is 29.5, 29.5, 36, 36, 38 and 44.5 days , respectively.

To determine the contribution of tumor infiltrating lymphocytes (TILs) to the anti-GDF15-mediated antitumor response, TILs were isolated from RENCA tumors twenty-four hours after the last treatment and analyzed for markers associated with the antitumor immune response. The results are summarized in FIG. 29 (p-value determined by unpaired t-test; p=0.0026).

Combination treatment [anti-GDF15 antibody (10 mg/kg) + anti-CD40 antibody (10 mg/kg)] increases the activation status (MHCII MFI) of macrophages gated as CD11b+; F4/80+; Ly6C cells in tumors by an average of 187% compared with the isotype-treated group (FIG. 29). Conversely, anti-CD40 monotherapy increases the MFI of MHCII by 67% (FIG. 29). Monotherapy with anti-GDF15 does not increase the activation status of macrophages (FIG. 29).

The ratio of CD8+ T cells/regulatory T cells (Treg) in the isotype and anti-GDF15 groups is 2.4 and 2.1, respectively (FIG. 30, solid circles and solid triangles). The CD8+ T cell/Treg ratio increases to 17 when treated with anti-CD40 antibody alone (FIG. 30, open circles). Conversely, combined anti-GDF15 antibody plus anti-CD40 antibody treatment resulted in a CD8+ T cell/Treg ratio of 84 (FIG. 30, open triangles).

These results demonstrate that anti-CDF15 based therapy is effective in increasing the antitumor efficacy of anti-CD40 therapy.

In addition to enhancing the antitumor efficacy of anti-CD40 therapy in the RRC model, anti-GDF15-based therapy attenuates anti-CD40-induced toxicity manifested by weight loss in treated animals. Although anti-CD40-based therapy resulted in a 4.43% weight loss in treated animals, the combined anti-GDF15 antibody+anti-CD40 antibody group showed a 1.5% increase in body weight compared to a body weight gain of 1.5%. 1.2% in the isotype control treated group (n=20 animals per group, anti-CD40 antibody vs. anti-CD40 antibody + anti-GDF15 antibody, p=0.0006 unpaired t-test). Restoration of body weight in the anti-CD40-antibody+anti-GDF15-antibody group is accompanied by a decrease in cytokine expression, usually associated with cytokine-mediated toxicity in the serum of treated animals.

Serum samples were collected from treated animals as described in Example 1. Cytokine levels were analyzed with the V-PLEX® Proinflammatory Panel 1 (mouse) Kit from Meso Scale Discover® (Mescoscale® K15048D). Each sample is diluted 1 to 4 with Diluent 41 (Mescoscale® R50AH-1) and analyzed in triplicate according to the manufacturer's protocol. The results are analyzed on a Meso Scale Discovery® Quickplex® SQ 120 machine and using the Discovery software. Analyze the expression of the following cytokines: IFNγ, IL10, IL12p70, IL1 beta, IL2, IL4, IL5, IL6, KC/GRO, TNFα. As shown in FIG. 31, expression levels of multiple pro-inflammatory cytokines (IFNγ (IFNg), TNFα (TNFa), IL6, IL10, KC/GRO) are significantly reduced in the anti-CD40-treated group after anti-GDF15 treatment (n=5, anti-CD40 vs. -CD40+anti-GDF15, p=0.0044 according to two-way ANOVA).

These results demonstrate that anti-GDF15 antibody treatment in combination with anti-CD40 has a synergistic antitumor effect accompanied by increased levels of CD11b+ activation; F4/80+ cells and an increased ratio of CD8+ T cells/regulatory T cells (Treg) at the tumor site. Moreover, anti-GDF15 therapy alleviates the clinical signs of anti-CD40 therapy toxicity associated with increased cytokine production.

Example 20 Treatment of Pancreatic Cancer with Anti-CD40 Antibody and Anti-GDF15 Antibody

This example illustrates the therapeutic activity of combination therapy with an anti-GDF15 antibody and an anti-CD40 antibody in a murine pancreatic adenocarcinoma (Pan02) model. Tumor-bearing animals are prepared as described in Example 19. Tumor mass is calculated as photon/second per ROI (region of interest) after background values have been subtracted. The average emission at the time of entry into the study is 4.8 * 10 ⁸ photons/second.

Anti-mouse anti-mouse FGK45 antibody FGK45 (mouse immunoglobulin G1 [mIgG1]) (10 µg/kg) and anti-mouse GDF15_0297 mouse (mouse IgG1 with null effector function D265A mutation) (10 µg/kg) .

The mean survival of animals treated with anti-GDF15 antibody + anti-CD40 antibody was 126.5 days, compared with 106 days in the group treated with only anti-CD40 antibody, 52.5 days in the group treated with only anti-GDF15 antibody, and 45 days in the isotype control group.

These results demonstrate that anti-GDF15 antibody treatment in combination with anti-CD40 has a synergistic antitumor effect on the progression of pancreatic cancer.

Example 21 GDF15 Neutralizing Antibodies Reverse GDF15-Mediated Inhibition of Macrophage-Mediated Cell Killing

This example illustrates the function of GDF15 in modulating macrophage tumor-killing activity.

Peritoneal macrophages were isolated from C57Bl6 mice five days after an ip injection of 1 ml of 3% Brewer Thioglycollate medium (B2551, Sigma). Macrophages were seeded at 0.35×10 ⁶ cells/well in a 24-well cell culture plate in complete macrophage medium (RPMI 1640 supplemented with 10% heat-inactivated PBS, 2 mM L-glutamine, 100 U/ml penicillin, 0 1 mg/mL streptomycin, 10 µM sodium pyruvate, 100 µM non-essential amino acids, 25 mM HEPES [pH 7.4], 50 µM β-mercaptoethanol) alone or in the presence of tested cytokines and antibodies. The following cytokines and antibodies were used in the study: recombinant mouse GDF15 (purified E. coli , RD systems 8944-GD), anti-mouse GDF15_0297 antibody "297" and anti-GDF15_001. After three hours of incubation with recombinant mouse GDF15 (10 nM) and antibodies (100 nM each), cells are treated with LPS (10 ng/mL, L2654, Sigma) and IFNγ (100 U/mL, 485M100/CF, R&D Systems) to achieve macrophage activation. The next day, Pan02 cells are labeled with CSFE (C34554, Invitrogen) according to manufacturer's instructions and co-plated with activated macrophages at a 1:4 ratio. Twenty-four hours later, sacrophages and tumor cells are harvested after incubation with TrypLE Express™ and analyzed by FACS as described in Example 17. Myeloid cells are identified with rat anti-mouse CD11b BUV395 (M1/70, BD Biosciences), tumor cells are identified as CSFE-positive/ CD11b negative population, live cells are separated from dead cells using DAPI (4',6-diamidino-2-phenylindole) (62247, Thermo Scientific).

As shown in FIG. 32, recombinant GDF15 inhibits macrophage-mediated tumor cell killing (solid triangles). This GDF15-mediated inhibition was abolished by anti-GDF15 antibody treatment (open circles and empty triangles).

These results demonstrate that treatment with anti-GDF15 antibodies is effective in stimulating the anti-carcinogenic activity of immune cells.

While the concepts disclosed have been described with reference to various uses, methods, kits, and compositions, it should be understood that various changes and modifications may be made without departing from the teachings herein and the claims below. The above examples are provided to better illustrate the ideas disclosed and are not intended to limit the scope of the ideas presented herein. While the present ideas have been described in terms of these exemplary embodiments, one of ordinary skill in the art will readily appreciate that numerous variations and modifications to these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present teachings.

All references cited herein, including patents, patent applications, articles, textbooks, and the like, as well as references cited herein, to the extent not already indicated, are incorporated herein by reference in their entirety. In the event that one or more of the included literature and the like differ from or contradict this application, including, but not limited to, certain terms, use of terms, methods described, and the like, this application shall govern.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope and spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the description and practice of the invention as described herein. The description and examples are intended to be regarded as illustrative only, with the true scope and spirit of the invention being indicated in the following claims.

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SEQUENCE LISTS

<110> Pfizer Inc.

BEAUMONT, Kevin Charles

BRIN, Danna M.

LAMBERT, Matthew Allister

CHABOT, Jeffrey Raymond

HE, Tao

SCHORS, Ksenia

APGAR, James R.

<120> ANTI-GDF15 ANTIBODIES, COMPOSITIONS AND METHODS OF USE

<130> PC72348A

<150> 62/765289

<151> 2018-08-20

<150> 62/750479

<151> 2018-10-25

<150> 62/750393

<151> 2018-10-25

<150> 62/881064

<151> 2019-07-31

<160> 221

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130 135 140

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

145 150 155 160

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Asn Ser Thr Leu Thr

165 170 175

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

180 185 190

Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu

195 200 205

Ser Pro Lys Gly Ser Glu Asn Leu Tyr Phe Gln Gly Ala Arg Asn Gly

210 215 220

Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val

225 230 235 240

Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro

245 250 255

Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe

260 265 270

Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser Leu His Arg Leu

275 280 285

Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn

290 295 300

Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr

305 310 315 320

Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys Ile

325 330

<210> 4

<211> 345

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 4

Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe

1 5 10 15

Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro

20 25 30

Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val

35 40 45

Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr

50 55 60

Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu

65 70 75 80

Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys

85 90 95

Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser

100 105 110

Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro

115 120 125

Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile

130 135 140

Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly

145 150 155 160

Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp

165 170 175

Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp

180 185 190

Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His

195 200 205

Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys Ile Glu

210 215 220

Gly Arg Met Asp Gly Gly Gly Gly Ser Ala Arg Asn Gly Asp Arg Cys

225 230 235 240

Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val His Ala Ser

245 250 255

Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val

260 265 270

Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Glu Ala

275 280 285

Asn Met His Ala Gln Ile Lys Met Asn Leu His Arg Leu Lys Pro Asp

290 295 300

Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val

305 310 315 320

Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp

325 330 335

Leu Leu Ala Lys Asp Cys His Cys Val

340 345

<210> 5

<211> 345

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 5

Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe

1 5 10 15

Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro

20 25 30

Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val

35 40 45

Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr

50 55 60

Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu

65 70 75 80

Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys

85 90 95

Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser

100 105 110

Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro

115 120 125

Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile

130 135 140

Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly

145 150 155 160

Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp

165 170 175

Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp

180 185 190

Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His

195 200 205

Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys Ile Glu

210 215 220

Gly Arg Met Asp Gly Gly Gly Gly Ser Ala Arg Asn Gly Asp His Cys

225 230 235 240

Pro Leu Gly Pro Gly Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser

245 250 255

Leu Glu Asp Leu Gly Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val

260 265 270

Gln Val Thr Met Cys Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala

275 280 285

Asn Met His Ala Gln Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp

290 295 300

Thr Val Pro Ala Pro Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val

305 310 315 320

Leu Ile Gln Lys Thr Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp

325 330 335

Leu Leu Ala Lys Asp Cys His Cys Ile

340 345

<210> 6

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 6

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 7

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 7

Arg Ala Ser Gln Ser Val His Ser Tyr Leu Ala

1 5 10

<210> 8

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 8

Asp Ala Ser Asn Arg Ala Thr

15

<210> 9

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 9

Gln Gln Phe Trp Ser Trp Pro Trp Thr

15

<210> 10

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 10

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 11

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 11

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 12

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 12

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

1 5 10

<210> 13

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 13

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 14

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 14

Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

100 105 110

<210> 15

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 15

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

100 105

<210> 16

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 16

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ile Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 17

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 17

Gly Tyr Thr Phe Ser Ser Tyr Asn Ile Ser

1 5 10

<210> 18

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 18

Gly Ile Asn Pro Ile Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 19

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 19

Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr

1 5 10

<210> 20

<211> 97

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 20

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys

<210> 21

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ile Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser

20 25

<210> 23

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 23

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10

<210> 24

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 24

Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 25

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 25

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 26

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 26

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 27

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 27

Arg Thr Ser Gln Asn Val His Ser Tyr Leu Ala

1 5 10

<210> 28

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 28

Asp Ala Ser Thr Arg Ala Asp

15

<210> 29

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 29

Gln Gln Phe Trp Ser Asp Pro Trp Thr

15

<210> 30

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 30

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 31

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 31

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 32

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 32

Gly Tyr Thr Phe Ser Ser Tyr Asn Ile Asp

1 5 10

<210> 33

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 33

Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 34

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 34

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 35

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 35

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Ser Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 36

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 36

Arg Thr Ser Gln Ser Val His Ser Tyr Leu Ala

1 5 10

<210> 37

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 37

Asp Ala Lys Thr Arg Ala Asp

15

<210> 38

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 38

Gln Gln Phe Ser Ser Asp Pro Tyr Thr

15

<210> 39

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 39

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Ser Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 40

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 40

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ile Phe Phe Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 41

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 41

Gly Tyr Thr Phe Ser Asp Tyr Asn Ile Asp

1 5 10

<210> 42

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 42

Gln Ile Asn Pro Asn Asn Gly Leu Ile Phe Phe Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 43

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 43

Glu Val Ile Thr Thr Val Gly Ala Met Asp Tyr

1 5 10

<210> 44

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 44

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ile Phe Phe Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 45

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 45

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 46

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 46

Arg Thr Ser Glu Asn Val His Ser Tyr Leu Ala

1 5 10

<210> 47

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 47

Asp Ala Ser Asn Leu Ala Asp

15

<210> 48

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 48

Gln Gln Phe Trp Ser Asp Pro Tyr Thr

15

<210> 49

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 49

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 50

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 50

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ile Asn Gly Leu Ile Phe Phe Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp His Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 51

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 51

Gly Ile Asn Pro Ile Asn Gly Leu Ile Phe Phe Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 52

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 52

Glu Ala Ile Thr Thr Val Gly Ala Met Asp His

1 5 10

<210> 53

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 53

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ile Asn Gly Leu Ile Phe Phe Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp His Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 54

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 54

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Leu His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 55

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 55

Arg Ala Ser Gln Asn Leu His Ser Tyr Leu Ala

1 5 10

<210> 56

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 56

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Leu His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 57

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 57

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 58

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 58

Gly Tyr Thr Phe Ser Asp Tyr Asn Met Asp

1 5 10

<210> 59

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 59

Gln Ile Asn Pro Asn Asn Gly Leu Ala Asn Tyr Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 60

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 60

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Asn Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 61

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 61

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Asn Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 62

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 62

Arg Ala Ser Gln Asn Val His Ser Tyr Leu Ala

1 5 10

<210> 63

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 63

Gln Gln Phe Trp Asn Asp Pro Tyr Thr

15

<210> 64

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 64

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Asn Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 65

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 65

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ile Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 66

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 66

Gly Tyr Thr Phe Thr Asp Tyr Asn Ile Asp

1 5 10

<210> 67

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 67

Gln Ile Asn Pro Asn Asn Gly Leu Ile Phe Tyr Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 68

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 68

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ile Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 69

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 69

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Ser Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 70

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 70

Asp Ala Lys Thr Arg Ala Thr

15

<210> 71

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 71

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Ser Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 72

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 72

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Asn Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 73

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 73

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Asn Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 74

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 74

Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Asn Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 75

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 75

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Ser Asn Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 76

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 76

Gln Gln Phe Ser Asn Asp Pro Trp Thr

15

<210> 77

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 77

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Ser Asn Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 78

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 78

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Thr Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 79

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 79

Glu Ala Ile Thr Thr Val Gly Ala Thr Asp Tyr

1 5 10

<210> 80

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 80

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Thr Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 81

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 81

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Asn Val His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 82

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 82

Arg Thr Ser Gln Asn Val His Asn Tyr Leu Ala

1 5 10

<210> 83

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 83

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Asn Val His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 84

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 84

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Ile Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 85

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 85

Gln Ile Asn Pro Ile Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 86

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 86

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Ile Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 87

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 87

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Ser Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Asn Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 88

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 88

Arg Thr Ser Glu Ser Val His Ser Tyr Leu Ala

1 5 10

<210> 89

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 89

Gln Gln Phe Trp Asn Trp Pro Trp Thr

15

<210> 90

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 90

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Ser Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Asn Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 91

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 91

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 92

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 92

Gly Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 93

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 93

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 94

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 94

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Ser Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 95

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 95

Arg Thr Ser Gln Ser Val His Asn Tyr Leu Ala

1 5 10

<210> 96

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 96

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Ser Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 97

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 97

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Ile Phe Gly Leu Ala Phe Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 98

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 98

Gln Ile Asn Pro Ile Phe Gly Leu Ala Phe Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 99

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 99

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Ile Phe Gly Leu Ala Phe Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 100

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 100

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Leu His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Asn Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 101

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 101

Arg Thr Ser Gln Ser Leu His Ser Tyr Leu Ala

1 5 10

<210> 102

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 102

Gln Gln Phe Trp Asn Asp Pro Trp Thr

15

<210> 103

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 103

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Leu His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Asn Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 104

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 104

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Asn Asn Gly Leu Ala Phe Phe Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 105

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 105

Gly Ile Asn Pro Asn Asn Gly Leu Ala Phe Phe Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 106

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 106

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Asn Asn Gly Leu Ala Phe Phe Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 107

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 107

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Asn Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 108

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 108

Asp Ala Lys Asn Arg Ala Asp

15

<210> 109

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 109

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Asn Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 110

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 110

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Glu Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 111

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 111

Glu Ala Ile Thr Thr Val Gly Ala Met Glu Tyr

1 5 10

<210> 112

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 112

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Glu Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 113

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 113

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Asn Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 114

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 114

Asp Ala Ser Asn Arg Ala Asp

15

<210> 115

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 115

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Asn Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 116

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 116

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ile Phe Phe Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Gln Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 117

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 117

Gly Tyr Thr Phe Thr Ser Tyr Asn Ile Ser

1 5 10

<210> 118

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 118

Gln Ile Asn Pro Asn Asn Gly Leu Ile Phe Phe Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 119

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 119

Glu Ala Ile Thr Thr Val Gly Ala Met Asp Gln

1 5 10

<210> 120

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 120

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ile Phe Phe Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Gln Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 121

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 121

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Leu Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 122

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 122

Asp Ala Ser Thr Leu Ala Thr

15

<210> 123

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 123

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Asn Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Leu Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 124

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 124

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ile Phe Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 125

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 125

Gly Tyr Thr Phe Ser Asp Tyr Asn Ile Ser

1 5 10

<210> 126

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 126

Gly Ile Asn Pro Ile Phe Gly Leu Ala Phe Tyr Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 127

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 127

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ile Phe Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 128

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 128

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val Ser Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Asn Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 129

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 129

Arg Thr Ser Gln Ser Val Ser Asn Tyr Leu Ala

1 5 10

<210> 130

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 130

Asp Ala Lys Asn Arg Ala Thr

15

<210> 131

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 131

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val Ser Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Asn Asp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 132

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 132

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Phe Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 133

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 133

Gly Tyr Thr Phe Thr Asp Tyr Asn Ile Ser

1 5 10

<210> 134

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 134

Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 135

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 135

Glu Phe Ile Thr Thr Val Gly Ala Met Asp Tyr

1 5 10

<210> 136

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 136

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Phe Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 137

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 137

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 138

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 138

Arg Thr Ser Glu Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 139

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 139

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 140

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 140

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Asn Asn Gly Thr Ala Phe Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Gln Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 141

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 141

Gly Ile Asn Pro Asn Asn Gly Thr Ala Phe Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 142

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 142

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Asn Asn Gly Thr Ala Phe Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Gln Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 143

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 143

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Ser Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 144

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 144

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Glu Ser Val His Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 145

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 145

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Ile Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 146

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 146

Gln Ile Asn Pro Asn Asn Gly Leu Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 147

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 147

Glu Ala Ile Thr Thr Ile Gly Ala Met Asp Tyr

1 5 10

<210> 148

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 148

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Ile Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 149

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 149

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Asn Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Ser Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 150

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 150

Arg Ala Ser Gln Ser Leu Ser Ser Tyr Leu Ala

1 5 10

<210> 151

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 151

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Asn Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Ser Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 152

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 152

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gln Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 153

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 153

Gly Tyr Thr Phe Thr Ser Tyr Asn Ile Asp

1 5 10

<210> 154

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 154

Glu Gln Ile Thr Val Gly Ala Met Asp Tyr

1 5 10

<210> 155

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 155

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gln Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 156

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 156

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 157

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 157

Arg Ala Ser Gln Asn Val His Asn Tyr Leu Ala

1 5 10

<210> 158

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 158

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asn Val His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 159

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 159

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ile Phe Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 160

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 160

Glu Ala Ile Thr Thr Val Gly Ala Met Asp Pro

1 5 10

<210> 161

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 161

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ile Phe Gly Leu Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 162

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 162

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 163

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 163

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Thr Arg Ala Asp Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Trp Ser Trp Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 164

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 164

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ile Phe Gly Thr Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp His Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

gly

<210> 165

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 165

Gly Ile Asn Pro Ile Phe Gly Thr Ala Phe Tyr Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> 166

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 166

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Asn Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ile Phe Gly Thr Ala Phe Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp His Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 167

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 167

gaaattgtgc tgacccagag cccggcgacc ctgagcctga gcccggggcga acgcgcgacc

60

ctgagctgcc gcaccagcca gagcgttcat aactatctgg cgtggtatca gcagaaaccg

120

ggccaggcgc cgcgcctgct gatttatgat gcgagcaccc gtgcggatgg cattccggca

180

cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctggaaccg

240

gaagattttg cggtgtatta ttgccagcag ttttggagct ggccgtggac ctttggccag

300

ggcaccaaag tggaaattaa a

321

<210> 168

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 168

caggtgcagc tggtgcagag cggcgcggaa gtgaaaaaac cgggcagcag cgtgaaagtg

60

agctgcaaag cgagcggcta tacctttagc agctataaca ttgattgggt gcgccaggcg

120

ccggggccagg gcctggaatg gatgggcggt attaacccga tttttggcac cgcattttat

180

aaccagaaat ttcagggccg cgtgaccatt accgcggatg aaagcaccag caccgcgtat

240

atggaactga gcagcctgcg cagcgaagat accgcggtgt attattgcgc acgcgaagcg

300

attaccaccg tgggcgcgat ggatcattgg ggccagggca ccctggtgac cgtgagcagc

360

<210> 169

<211> 642

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 169

gaaattgtgc tgacccagag cccggcgacc ctgagcctga gcccggggcga acgcgcgacc

60

ctgagctgcc gcaccagcca gagcgttcat aactatctgg cgtggtatca gcagaaaccg

120

ggccaggcgc cgcgcctgct gatttatgat gcgagcaccc gtgcggatgg cattccggca

180

cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctggaaccg

240

gaagattttg cggtgtatta ttgccagcag ttttggagct ggccgtggac ctttggccag

300

ggcaccaaag tggaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca

360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat

420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag

480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg

540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcaggggc

600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt

642

<210> 170

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 170

caggtgcagc tggtgcagag cggcgcggaa gtgaaaaaac cgggcagcag cgtgaaagtg

60

agctgcaaag cgagcggcta tacctttagc agctataaca ttgattgggt gcgccaggcg

120

ccggggccagg gcctggaatg gatgggcggt attaacccga tttttggcac cgcattttat

180

aaccagaaat ttcagggccg cgtgaccatt accgcggatg aaagcaccag caccgcgtat

240

atggaactga gcagcctgcg cagcgaagat accgcggtgt attattgcgc acgcgaagcg

300

attaccaccg tgggcgcgat ggatcattgg ggccagggca ccctggtgac cgtgagcagc

360

gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag caccctctggg

420

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg

480

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca

540

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc

600

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc

660

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgctggggca

720

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct

780

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg

840

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac

900

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag

960

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc

1020

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag

1080

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc

1140

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg

1200

ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg

1260

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg

1320

cagaagagcc tctccctgtc ccccggga

1347

<210> 171

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OTHER_INDICATOR

<222> (5)..(5)

<223> Xaa can be S or T

<220>

<221> OTHER_INDICATOR

<222>(6)..(6)

<223> Xaa can be S or D

<400> 171

Gly Tyr Thr Phe Xaa Xaa Tyr Asn Ile Asp

1 5 10

<210> 172

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OTHER_INDICATOR

<222> (1)..(1)

<223> Xaa can be G or Q

<220>

<221> OTHER_INDICATOR

<222> (5)..(5)

<223> Xaa can be I or N

<220>

<221> OTHER_INDICATOR

<222>(6)..(6)

<223> Xaa can be F or N

<220>

<221> OTHER_INDICATOR

<222>(8)..(8)

<223> Xaa can be T or L

<220>

<221> OTHER_INDICATOR

<222> (10)..(10)

<223> Xaa can be F or N

<220>

<221> OTHER_INDICATOR

<222> (11)..(11)

<223> Xaa can be Y or F

<220>

<221> OTHER_INDICATOR

<222> (12)..(12)

<223> Xaa can be N or A

<400> 172

Xaa Ile Asn Pro Xaa Xaa Gly Xaa Ala Xaa Xaa Xaa Gln Lys Phe Gln

1 5 10 15

gly

<210> 173

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OTHER_INDICATOR

<222> (2)..(2)

<223> Xaa can be A or Q

<220>

<221> OTHER_INDICATOR

<222>(6)..(6)

<223> Xaa can be V or I

<220>

<221> OTHER_INDICATOR

<222> (11)..(11)

<223> Xaa can be H or Y

<400> 173

Glu Xaa Ile Thr Thr Xaa Gly Ala Met Asp Xaa

1 5 10

<210> 174

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OTHER_INDICATOR

<222> (2)..(2)

<223> Xaa can be T or A

<220>

<221> OTHER_INDICATOR

<222> (5)..(5)

<223> Xaa can be S or N

<220>

<221> OTHER_INDICATOR

<222>(6)..(6)

<223> Xaa can be V or L

<220>

<221> OTHER_INDICATOR

<222>(7)..(7)

<223> Xaa can be H or S

<220>

<221> OTHER_INDICATOR

<222>(8)..(8)

<223> Xaa can be N or S

<400> 174

Arg Xaa Ser Gln Xaa Xaa Xaa Xaa Tyr Leu Ala

1 5 10

<210> 175

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OTHER_INDICATOR

<222> (3)..(3)

<223> Xaa can be S or K

<220>

<221> OTHER_INDICATOR

<222> (4)..(4)

<223> Xaa can be T or N

<220>

<221> OTHER_INDICATOR

<222>(7)..(7)

<223> Xaa can be D or T

<400> 175

Asp Ala Xaa Xaa Arg Ala Xaa

15

<210> 176

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<220>

<221> OTHER_INDICATOR

<222> (4)..(4)

<223> Xaa can be W or S

<220>

<221> OTHER_INDICATOR

<222>(5)..(5)

<223> Xaa can be S or N

<220>

<221> OTHER_INDICATOR

<222>(6)..(6)

<223> Xaa can be W or D

<220>

<221> OTHER_INDICATOR

<222>(8)..(8)

<223> Xaa can be W or Y

<400> 176

Gln Gln Phe Xaa Xaa Xaa Pro Xaa Thr

15

<210> 177

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 177

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met Asp Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Met

35 40 45

Gly Gln Ile Asn Pro Asn Asn Gly Leu Ile Phe Phe Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Ala Ile Thr Thr Val Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 178

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 178

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Leu His Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Ser Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 179

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 179

Gly Tyr Pro Phe Glu Gly Trp Tyr Ile His

1 5 10

<210> 180

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 180

Trp Asn Asn Pro Arg Thr Gly Leu Thr Asn His Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 181

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 181

Gly Val Gly Ala Asp Ala Ala Phe Asp Ile

1 5 10

<210> 182

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 182

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Pro Phe Glu Gly Trp

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Asn Asn Pro Arg Thr Gly Leu Thr Asn His Ala Gln Lys Phe

50 55 60

Gln Gly Lys Val Thr Met Thr Arg Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Gly Ala Asp Ala Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 183

<211> 443

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 183

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Pro Phe Glu Gly Trp

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Asn Asn Pro Arg Thr Gly Leu Thr Asn His Ala Gln Lys Phe

50 55 60

Gln Gly Lys Val Thr Met Thr Arg Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Gly Ala Asp Ala Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Ser Val Tyr

115 120 125

Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu

130 135 140

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp

145 150 155 160

Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser

180 185 190

Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser

195 200 205

Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys

210 215 220

Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr

245 250 255

Cys Val Val Val Ala Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser

260 265 270

Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile

290 295 300

Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

305 310 315 320

Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu

340 345 350

Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe

355 360 365

Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

370 375 380

Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

385 390 395 400

Phe Ile Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly

405 410 415

Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His

420 425 430

Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 184

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 184

Arg Ser Ser Gln Ser Leu Leu Trp Lys His Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 185

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 185

Leu Asp Arg Asn Arg Ala His

15

<210> 186

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 186

Met Gln Ser Phe Glu Thr Pro Ile Thr

15

<210> 187

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 187

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Arg Ser Ser Gln Ser Leu Leu Trp Lys

20 25 30

His Gly Tyr Asn Tyr Leu Asp Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Leu Asp Arg Asn Arg Ala His Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Met Gln Ser

85 90 95

Phe Glu Thr Pro Ile Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 188

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 188

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Arg Ser Ser Gln Ser Leu Leu Trp Lys

20 25 30

His Gly Tyr Asn Tyr Leu Asp Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Leu Asp Arg Asn Arg Ala His Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Met Gln Ser

85 90 95

Phe Glu Thr Pro Ile Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

115 120 125

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

130 135 140

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

145 150 155 160

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

180 185 190

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

195 200 205

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 189

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 189

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Pro Phe Glu Gly Trp

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Asn Asn Pro Arg Thr Gly Leu Thr Asn His Ala Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Gly Ala Asp Ala Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 190

<211> 443

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 190

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Pro Phe Glu Gly Trp

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Asn Asn Pro Arg Thr Gly Leu Thr Asn His Ala Gln Lys Phe

50 55 60

Gln Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Gly Ala Asp Ala Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Ser Val Tyr

115 120 125

Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu

130 135 140

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp

145 150 155 160

Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser

180 185 190

Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser

195 200 205

Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys

210 215 220

Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr

245 250 255

Cys Val Val Val Ala Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser

260 265 270

Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile

290 295 300

Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

305 310 315 320

Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu

340 345 350

Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe

355 360 365

Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

370 375 380

Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

385 390 395 400

Phe Ile Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly

405 410 415

Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His

420 425 430

Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 191

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 191

Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Arg Ser Ser Gln Ser Leu Leu Trp Lys

20 25 30

His Gly Tyr Asn Tyr Leu Asp Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Leu Asp Arg Asn Arg Ala His Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Met Gln Ser

85 90 95

Phe Glu Thr Pro Ile Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 192

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 192

Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Arg Ser Ser Gln Ser Leu Leu Trp Lys

20 25 30

His Gly Tyr Asn Tyr Leu Asp Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Leu Asp Arg Asn Arg Ala His Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Met Gln Ser

85 90 95

Phe Glu Thr Pro Ile Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

115 120 125

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

130 135 140

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

145 150 155 160

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

180 185 190

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

195 200 205

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 193

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 193

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Phe Glu Gly Trp

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Asn Asn Pro Arg Thr Gly Leu Thr Asn His Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Gly Ala Asp Ala Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 194

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 194

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Pro Phe Glu Gly Trp

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Asn Asn Pro Arg Thr Gly Leu Thr Asn His Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Gly Ala Asp Ala Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 195

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 195

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Leu Trp Lys

20 25 30

His Gly Tyr Asn Tyr Leu Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg Leu Leu Ile Tyr Leu Asp Arg Asn Arg Ala His Gly Ile Pro

50 55 60

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Met Gln Ser

85 90 95

Phe Glu Thr Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 196

<211> 219

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 196

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Leu Trp Lys

20 25 30

His Gly Tyr Asn Tyr Leu Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ala

35 40 45

Pro Arg Leu Leu Ile Tyr Leu Asp Arg Asn Arg Ala His Gly Ile Pro

50 55 60

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Met Gln Ser

85 90 95

Phe Glu Thr Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 197

<211> 444

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 197

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Gly Ser Ser Leu Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Ser Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 198

<211> 443

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 198

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Gly Ser Ser Leu Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Ser Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 199

<211> 221

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 199

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Trp Asp Ser

20 25 30

Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Thr Ser Tyr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro His Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Gly Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

115 120 125

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

130 135 140

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

145 150 155 160

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

165 170 175

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

180 185 190

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

195 200 205

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 200

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 200

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Gly Ser Ser Leu Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Ser Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 201

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 201

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Trp Pro Gly Ser Ser Leu Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Leu Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 202

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 202

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Trp Asp Ser

20 25 30

Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Thr Ser Tyr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro His Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 203

<211> 432

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 203

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Trp Gln Pro Gly Arg Ser

1 5 10 15

Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser Gly

20 25 30

Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

35 40 45

Val Arg Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

115 120 125

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Asp Tyr Phe

130 135 140

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

145 150 155 160

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

165 170 175

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Thr Tyr Thr

180 185 190

Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Arg Val Glu

195 200 205

Ser Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

210 215 220

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

225 230 235 240

Ile Ser Arg Thr Pro Glu Val Thr Cys Trp Val Asp Val Ser Gln Glu

245 250 255

Asp Pro Glu Val Gln Phe Asn Trp Tyr Tyr Asp Gly Val Glu Val His

260 265 270

Asn Ala Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

275 280 285

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

290 295 300

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

305 310 315 320

Thr Ile Ser Lys Ala Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

325 330 335

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

340 345 350

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

355 360 365

Asn Gly Gln Pro Glu Lys Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

370 375 380

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

385 390 395 400

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

405 410 415

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

420 425 430

<210> 204

<211> 208

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 204

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Ser Gly Thr Ala Ser

115 120 125

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Val Gln Trp

130 135 140

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

145 150 155 160

Glu Gln Asp Ser Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu

165 170 175

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr

180 185 190

His Gln Gly Leu Ser Ser Pro Val Thr Ser Phe Asn Arg Gly Glu Cys

195 200 205

<210> 205

<211> 447

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 205

Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 206

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 206

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 207

<211> 432

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 207

Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly

1 5 10 15

Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn

20 25 30

Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser

35 40 45

Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly

50 55 60

Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln

65 70 75 80

Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu

85 90 95

Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu

100 105 110

Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly

115 120 125

Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn

130 135 140

Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val

145 150 155 160

Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe Trp

165 170 175

Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser

180 185 190

Gln Met Glu Pro Arg Thr His Pro Thr Trp Glu Pro Lys Ser Cys Asp

195 200 205

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

210 215 220

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

225 230 235 240

Ser Arg Thr Pro Glu Val Thr Cys Trp Val Asp Val Ser His Glu Asp

245 250 255

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

260 265 270

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Trp

275 280 285

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

290 295 300

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

305 310 315 320

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

325 330 335

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

340 345 350

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

355 360 365

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

370 375 380

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

385 390 395 400

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

405 410 415

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

420 425 430

<210> 208

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 208

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 209

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 209

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 210

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 210

Gly Tyr Thr Phe Thr Ser Tyr

15

<210> 211

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 211

Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn

1 5 10

<210> 212

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 212

Ser Tyr Trp Ile Asn

15

<210> 213

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 213

Asn Ile Tyr Pro Gly Ser Ser Leu

15

<210> 214

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 214

Asn Ile Tyr Pro Gly Ser Ser Leu Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

<210> 215

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 215

Leu Ser Thr Gly Thr Phe Ala Tyr

15

<210> 216

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 216

Lys Ser Ser Gln Ser Leu Trp Asp Ser Gly Asn Gln Lys Asn Phe Leu

1 5 10 15

Thr

<210> 217

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 217

Trp Thr Ser Tyr Arg Glu Ser

15

<210> 218

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 218

Gln Asn Asp Tyr Phe Tyr Pro His Thr

15

<210> 219

<211> 117

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 219

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Gly Ser Ser Leu Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Ser Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 220

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 220

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Trp Asp Ser

20 25 30

Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Thr Ser Tyr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro His Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 221

<211> 290

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 221

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Asn Glu Arg Thr His

225 230 235 240

Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr

245 250 255

Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys

260 265 270

Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu

275 280 285

Glu Thr

290

<---

Claims (73)

1. An antibody, or antigen-binding fragment thereof, that specifically binds to human growth and differentiation factor 15 (GDF15), containing at least one of the following (a)-(d): (a) light chain complementarity determining region 1 (LCDR-1) comprising the amino acid sequence of SEQ ID NO:95, LCDR-2 containing the amino acid sequence of SEQ ID NO:28, LCDR-3 containing the amino acid sequence of SEQ ID NO:9, heavy chain complementarity determining region 1 (HCDR-1) comprising the amino acid sequence of SEQ ID NO:32, HCDR-2 containing the amino acid sequence of SEQ ID NO:165, and HCDR-3 containing the amino acid sequence of SEQ ID NO:52; (b) the amino acid sequences of HCDR-1, HCDR-2 and HCDR-3 as indicated in the amino acid sequence of SEQ ID NO:166, and the amino acid sequences of LCDR-1, LCDR-2 and LCDR-3 as indicated in the amino acid sequence of SEQ ID NO:163; (c) a heavy chain variable domain (VH) containing an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:166, and HCDR-1 containing the amino acid sequence of SEQ ID NO:32, HCDR-2 containing the amino acid the sequence of SEQ ID NO:165, and HCDR-3 containing the amino acid sequence of SEQ ID NO:52; And light chain variable domain (LH) containing an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:163, and LCDR-1 containing the amino acid sequence of SEQ ID NO:95, LCDR-2 containing the amino acid sequence of SEQ ID NO:28, and LCDR-3 containing the amino acid sequence of SEQ ID NO:9; or (d) a VH containing the amino acid sequence of SEQ ID NO:166, and VL containing the amino acid sequence of SEQ ID NO:163. 2. The antibody or antigen-binding fragment according to claim 1, containing a human Fc domain selected from the group consisting of the Fc domain of IgA ₁ IgA ₂ , IgD, IgE, IgM, IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ . 3. An antibody or its antigen-binding fragment according to any one of claims 1-2, containing: (a) a heavy chain (HC) containing an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:164, and a light chain (LC) containing an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:162; (b) HC containing the amino acid sequence of SEQ ID NO:164, and LC containing the amino acid sequence of SEQ ID NO:162. 4. An antibody or antigen-binding fragment according to any one of paragraphs. 1-3, wherein the antibody, or antigen-binding fragment thereof, binds to human or cynomolgus monkey GDF15 with a K _D of about or less than a value selected from the group consisting of about 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM . 5. Selected nucleic acid encoding the antibody or antigennegative part, according to any one of paragraphs. 1-4. 6. An isolated nucleic acid encoding an antibody or its antigen-binding portion, containing: a) the nucleic acid sequence of SEQ ID NO:167 and the nucleic acid sequence of SEQ ID NO:168, or b) the nucleic acid sequence of SEQ ID NO:169 and the nucleic acid sequence of SEQ ID NO:170. 7. An expression vector containing a nucleic acid according to claim 5 or claim 6. 8. The host cell to obtain an antibody or antigennegative fragment according to any one of paragraphs. 1-4, where the cell contains the vector according to claim 7. 9. The host cell of claim 8, wherein said host cell is a mammalian cell selected from the group consisting of a CHO cell, COS cell, HEK-293 cell, NS0 cell, PER.C6® cell, or Sp2.0 cell. 10. A method for producing an antibody or antigen-binding fragment thereof, comprising culturing a host cell according to claim 8 under conditions under which said antibody or antigen-binding fragment thereof is expressed by said host cell. 11. Pharmaceutical composition for the treatment of a medical condition, disease or disorder mediated or associated with the expression of GDF15, where the composition contains an antibody or antigen-binding fragment according to any one of paragraphs. 1-4 and a pharmaceutically acceptable carrier or excipient. 12. A method of treating a medical condition, disease, or disorder mediated by or associated with GDF15 expression in a patient, wherein said method comprises administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-4 or a pharmaceutical composition according to claim 11. 13. The method of claim 12, wherein the condition is cachexia associated with cancer, chemotherapy, chemotherapy in combination with immuno-oncological therapy, chronic obstructive pulmonary disease, chronic kidney disease, chronic heart failure, congestive heart failure, or sarcopenia. 14. A method of reducing the level of free GDF15 in a patient, wherein said method comprises administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1-4, or a pharmaceutical composition according to claim 11, wherein the level of free GDF15 is reduced. 15. A method for reducing GDF15 activity in a patient, wherein the method comprises administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-4, or a pharmaceutical composition according to claim 11. 16. The method of claim 15, wherein the GDF15 activity is at least one selected from the group consisting of: (a) reduce food intake; (b) decreased appetite; (c) weight loss; (d) increase in body weight loss; (e) decrease in fat mass; (f) decrease in muscle mass; (g) increasing fat loss; (h) increasing muscle loss, (i) binding to GFRAL; (j) amplifying downstream signaling mediated by RET; (k) increasing ERK phosphorylation; (l) increasing phosphorylation of the S6 ribosomal protein; (m) enhancing RET-mediated activation of the MAPK signaling pathway; (n) enhancing activation of the RET signaling pathway of AKT; And (o) enhancing activation of the PLC-γ1 signaling pathway. 17. A method of treating cancer in a patient, comprising administering to the patient a combination therapy that provides a synergistic therapeutic effect, where the method includes administering a synergistic therapeutically effective amount of an antibody or antigen-binding fragment according to any one of paragraphs. 1-4, or a pharmaceutical composition according to claim 11, and (a) a synergistic therapeutically effective amount of an anti-CD40 antagonist antibody; or (b) a synergistic therapeutically effective amount of a PD-1 binding antagonist, wherein the PD-1 binding antagonist is not avelumab. 18. The method of claim 17 wherein the PD-1 axis binding antagonist is an anti-PD-1 antibody selected from the group consisting of nivolumab, pembrolizumab, spartalizumab, pidilizumab, tiselizumab, AMP-224, AMP-514, cemiplimab, and sasanlimab (PF-06801591). 19. The method of claim 17, wherein the PD-1 axis binding antagonist is an anti-PD-L1 antibody selected from the group consisting of atezolizumab, durvalumab, BMS-936559, MEDI4736, and MPDL3280A, and wherein the anti-PD-L1 antibody is not is avelumab. 20. The method according to any one of paragraphs. 18-19, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, renal cell carcinoma, Merkel cell carcinoma, ovarian cancer, breast cancer, pancreatic cancer, urothelial cancer, and castration resistant prostate cancer. 21. The method of claim 17 wherein the cancer is selected from the group consisting of stomach cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, head and neck squamous cell carcinoma, thymus cancer, epithelial cancer, saliva cancer, liver cancer , gastric cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, cancer colon, oral cancer, skin cancer and melanoma. 22. A set for the treatment of cancer, containing a synergistic therapeutically effective amount of anti-GDF15 antibodies according to any one of paragraphs. 1-4 and a synergistic therapeutically effective amount of an anti-PD-1 antibody. 23. Set for the treatment of cancer, containing a therapeutically effective amount of anti-GDF15 antibodies according to any one of paragraphs. 1-4 and a therapeutically effective amount of anti-CD40 antibody. 24. A method for improving the therapeutic effect of an immunomodulator administered to a patient for the treatment of cancer, wherein the method comprises administering to a patient receiving the immunomodulator an effective amount of an anti-GDF15 antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-4, or a pharmaceutical composition according to claim 11. 25. The method of claim 24, wherein the cancer is selected from the group consisting of breast cancer, gastric cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, glioma, glioblastoma, kidney cancer, endometrial cancer, and rectal cancer. intestines. 26. A method of reducing or inhibiting toxicity in a patient experiencing cytokine release syndrome (CRS) or cytokine storm, or susceptible to CRS or cytokine storm, wherein the method comprises administering to the patient an effective amount of an anti-GDF15 antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-4, or a pharmaceutical composition according to claim 11. 27. The method of claim 26, wherein the patient is undergoing cancer therapy and said method does not reduce the effectiveness of the cancer therapy. 28. The method of claim 27 wherein the cancer therapy comprises an immunomodulator such as an anti-CD40 antibody, anti-CD47 antibody, anti-CTLA4 antibody, anti-4-1BB antibody, IL-12 or IL-15. 29. The method according to any one of paragraphs. 26-28, where the cause of the CRS or cytokine storm includes an infectious stimulus, condition, or syndrome, or where the cause of said cytokine release syndrome or cytokine storm includes a non-infectious stimulus, condition, or syndrome, or any combination thereof. 30. The method of claim 29, wherein said infectious stimulus, condition, or syndrome includes influenza, avian influenza, severe acute respiratory syndrome (SARS), Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (HLH), sepsis, gram-negative sepsis, malaria, Ebola virus, variola virus, systemic gram-negative bacterial infection, or Jarisch-Herxheimer syndrome, or where said non-infectious stimulus, condition, or syndrome includes hemophagocytic lymphohistiocytosis (HLH), sporadic HLH, macrophage activation syndrome (MAS), chronic arthritis, systemic juvenile idiopathic arthritis (sJIA), Still's disease, cryopyrin-associated periodic syndrome (CAPS), familial cold autoinflammatory syndrome (FCAS), familial cold urticaria (FCU), Muckle-Wale syndrome (MWS), chronic childhood neurological skin and joint syndrome (CINCA) , cryopyrinopathy containing inherited or de novo functional mutations in the NLRP3 gene, hereditary autoinflammatory disorder, acute pancreatitis, severe burns, trauma, acute respiratory distress syndrome, immunotherapy, monoclonal antibody therapy, secondary to drug use, secondary to inhalation toxins, lipopolysaccharide (LPS), gram-positive toxins, fungal toxins, glycosylphosphatidylinositol (GPI), or modulation of RIG-1 gene expression. 31. The use of a therapeutically effective amount of an antibody or antigen-binding fragment, according to any one of paragraphs. 1-4, or a pharmaceutical composition according to claim 11, for the treatment of a medical condition, disease, or disorder mediated or associated with GDF15 expression in a patient. 32. The use of a therapeutically effective amount of an antibody or antigen-binding fragment, according to any one of paragraphs. 1-4, or a pharmaceutical composition according to claim 11, to reduce the level of free GDF15 in a patient. 33. The use of a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of paragraphs. 1-4, or a pharmaceutical composition according to claim 11, to reduce the activity of GDF15 in a patient. 34. The use of a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of paragraphs. 1-4, or a pharmaceutical composition according to claim 11, for the treatment of cancer in a patient, where the antibody or antigen-binding fragment or pharmaceutical composition is administered in combination with: (a) a synergistic therapeutically effective amount of an anti-CD40 antagonist antibody; or (b) a synergistic therapeutically effective amount of a PD-1 binding antagonist, wherein the PD-1 binding antagonist is not avelumab. 35. The use of an effective amount of an antibody or antigen-binding fragment according to any one of paragraphs. 1-4, or a pharmaceutical composition according to claim 11, to improve the therapeutic effect of an immunomodulator administered to a patient for the treatment of cancer. 36. The use of an effective amount of an antibody or antigen-binding fragment according to any one of paragraphs. 1-4, or a pharmaceutical composition according to claim 11, for reducing or inhibiting cytotoxicity in a patient experiencing cytokine release syndrome (CRS) or cytokine storm, or susceptible to CRS or cytokine storm.

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