CN108623685B

CN108623685B - anti-OX 40 antibodies and uses thereof

Info

Publication number: CN108623685B
Application number: CN201710185399.9A
Authority: CN
Inventors: 曾竣玮; H·巴鲁赫; 刘晓林; 陈乘; 刘军建
Original assignee: Innovent Biologics Suzhou Co Ltd
Current assignee: Innovent Biologics Suzhou Co Ltd
Priority date: 2017-03-25
Filing date: 2017-03-25
Publication date: 2022-07-01
Anticipated expiration: 2037-03-25
Also published as: CN108623685A

Abstract

The present invention relates to novel antibodies and antibody fragments that specifically bind to OX40 and compositions containing the antibodies or antibody fragments. Furthermore, the invention relates to nucleic acids encoding said antibodies or antibody fragments thereof and host cells comprising the same, as well as related uses. Furthermore, the invention relates to therapeutic and diagnostic uses of these antibodies and antibody fragments.

Description

anti-OX 40 antibodies and uses thereof

Background

OX40 (also known as CD134, TNFRSF4 and ACT35) WAs originally described as a T cell activation marker on rat CD4T cells (Paterson DJ, Jefferies WA, Green JR, Brandon MR, Corthesy P, Puklavec M, Williams AF. inhibitors of activated T lymphocytes encoding a module of 50,000 Mr protected on CD4positive T blast. mol Immunol. 1987; 24: 1281. packer. TM. MRto CD4positive T blast. mol. 1068) and WAs subsequently shown to be up-regulated in TCR recruitment (Mallett S, Fossum S, cloning. packer. lysis of MRto C40 anti-active CD4positive T lymphocytes J. 1068. EMPLE. 1068). OX40 was identified on CD4+ T cells, CD8+ T cells, NK cells, NKT cells and neutrophils (D.J.Paterson, W.A.Jefferies, J.R.Green et al, "inhibitors of activated Rat T lymphocytes including a module of 50,000M (r) detected only on CD4positive T blasts," Molecular Immunology, vol.24, No.12, pp.1281-1290, 1987). OX40 signaling can promote costimulatory signaling to T cells, leading to enhanced cell proliferation, survival, effector function and migration (Gramaglia I, Weinberg AD, Lemon M, Croft M.Ox-40 ligand: a patent biological specimen for preserving primary CD4T cell responses. J Immunol.1998; 161: 6510. quadrature I, Jember A, Picjig SD, Weinberg AD, Kien N, Croft M.the OX40 biological receptors the modification of CD4 biological regulation primary expression. J.2000; 3: 3050).

The ligand of OX40, OX40L, is expressed primarily on Antigen Presenting Cells (APCs) and its expression can be induced by CD40 and mast cell signaling, toll-like receptors (TLRs), and inflammatory cytokines. In addition to APCs, non-hematopoietic cells such as smooth muscle and vascular endothelial cells may also express OX 40L. In transgenic mice overexpressing OX40L, there was increased T-cell activation and when immunized, these mice produced enhanced T-cell responses (Murata K, Nose M, Ndhlovu LC, Sato T, Sugamura K, Ishii N.structural OX40/OX40 ligand expression. J Immunol.2002; 169: 4628. sup. an. and Sato T, Ishii N, Murata K, Kikuchi K, Nakagawa S, Ndhlovu LC, Sugarura K.sequences of OX40-OX40 ligand expression in yeast cell function: enhanced connectivity strain in OX 40L-3383. sup. J.2002; Eubacterium 3332. sup. J.32). This data indicates that OX40L expression is a limiting factor for OX40 signaling in T cells.

In tumor-bearing mice, in vivo ligation of mouse OX40 (via soluble mouse OX 40L-immunoglobulin fusion protein or mouse OX40L mimetics, such as anti-mouse CD134 specific antibodies) enhanced anti-tumor immunity, resulting in tumor-free survival in mouse models of various mouse malignant tumor cell lines, such as lymphoma, melanoma, sarcoma, colon cancer, breast cancer, and glioma (Sugamura et al Nature Rev Imm 2004; 4: 420-431).

It has been suggested that the immune response to an antigen in mammals can be enhanced by binding OX40 with an OX40 binding agent (WO 99/42585; Weinberg, 2000). Although this document mentions OX40 binding agents in general, the focus is on using OX40L or parts thereof; anti-OX 40 antibodies are disclosed as equivalents to OX 40L. Indeed, when the Weinberg group used this study in a study with non-human primates, they again intentionally selected antibodies that bind to the OX40L binding site and mimic OX40L in general.

Al-Shamkhani et Al (Eur J Chem 1996; 26: 1695-1699) used an anti-OX 40 antibody called OX86, which did not block OX40L binding, to explore differential expression of OX40 on activated mouse T cells; Hirschhom-Cymerman et al (J Exp Med 2009; 206: 1103-. However OX86 was not expected to bind to human OX40 and when selecting antibodies that were effective in humans, one would select antibodies that bind to the binding site of OX40L according to the Weinberg study.

In Severe Combined Immunodeficiency (SCID) mice, in vivo ligation of human OX40 (via anti-human OX40 specific antibodies that interact with the OX40L binding domain on human OX 40; US2009/0214560a1) enhances anti-tumor immunity, which results in tumor growth inhibition of various human malignant tumor cell lines such as lymphoma, prostate, colon, and breast cancers.

In humans, the precise mechanism of the anti-tumor immune response mediated by the human OX40 junction has not been demonstrated, but is thought to be mediated through the OX40 transmembrane signaling pathway stimulated by interaction with OX 40L. This interaction is mediated by binding of trimeric OX40L to OX 40. Trimerized OX40 ligand is recommended as a more potent drug than anti-OX 40 antibody in current anti-cancer treatments (Morris et al. mol Immunol 2007; 44: 3112-3121).

Furthermore, in poorly immunogenic tumors, single anti-OX 40 treatment failed to provide sufficient anti-tumor immunogenicity, and it is therefore also desirable to develop combinations of OX40 with other strategies. It was found that modulating OX40 signaling in combination with other signaling pathways deregulated in tumor cells (e.g., angiogenesis pathway, PD-1 pathway) could further enhance therapeutic efficacy.

However, there remains a need to develop new anti-OX 40 antibodies that avoid blocking the binding of OX40 to OX40L, and thus are better used to treat or delay various cancers, immune-related diseases, and T cell dysfunctional diseases, than anti-OX 40 antibodies known in the art.

Summary of The Invention

Applicants have surprisingly found that, in order to activate T cells, induce T cell mediated anti-tumor activity, use of an antibody or fragment thereof that binds to human OX40, wherein the antibody or fragment thereof is capable of better binding to human OX40 while not blocking the binding of human OX40 to OX40 ligand (OX40L), results in an enhanced immune response.

Preferably, the anti-OX 40 antibodies or fragments thereof of the invention are capable of activating T cells, e.g., enhancing the immunostimulatory/effector function of T effector cells and/or proliferating these cells and/or down regulating the immunosuppressive function of T regulatory cells. More preferably, the antibody is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, the OX40 antibodies of the invention can be used to treat or delay various cancers, immune related diseases, and T cell dysfunctional diseases.

The invention thus provides antibodies or fragments thereof (preferably antigen-binding fragments) that bind to human OX40 or cynomolgus monkey OX40, wherein said antibodies or fragments thereof do not block the binding of human or cynomolgus monkey OX40 to its ligand OX 40L. In preferred embodiments, the antibody or fragment thereof not only does not block binding of human or cynomolgus monkey OX40 to its ligand OX40L, but also increases OX 40L-based T cell activation of OX 40.

In some embodiments, an antibody or fragment thereof of the invention binds to human OX40 or cynomolgus monkey OX 40. In some embodiments, an antibody or fragment thereof of the invention does not bind to murine OX40, e.g., rat OX40 or mouse OX 40.

In some embodiments, the anti-OX 40 antibodies of the invention have agonist activity.

In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention binds to K of human OX40 or cynomolgus monkey OX40_DLess than about 100nM, preferably less than or equal to about 10nM, more preferably less than or equal to about 8nM, more preferably less than or equal to about 7nM, 6nM, 5nM, 4nM, 3nM or 2nM, and most preferably the K is_DLess than or equal to about 1nM or 0.8 nM. In some embodiments, antibody binding affinity is determined using a bio-optical interferometry (e.g., Fortebio affinity measurement) or MSD assay.

In some embodiments, binding of an antibody or fragment thereof of the invention to human OX40 or cynomolgus monkey OX40 is determined using a flow cytometry (e.g., FACS) assay. In some embodiments, binding to human OX40 or cynomolgus monkey OX40 has an EC50 of less than or equal to about 10nM, 9nM, 8nM, or 7 nM. In some embodiments, binding to human OX40 or cynomolgus monkey OX40 has an EC50 of less than or equal to about 6nM or about 5nM or about 4nM or about 3 nM. In some embodiments, in an assay performed with a flow cytometry assay, an antibody or fragment thereof of the invention binds to OX40 expressed on a cell with an MFI value greater than 1000-fold difference, preferably greater than 1100-fold difference, 1200-fold difference, 1300-fold difference, 1400-fold difference, 1500-fold difference, 1600-fold difference, 1700-fold difference, 1800-fold difference, 1900-fold difference, 2000-fold difference, compared to a corresponding control cell that does not express OX 40.

In another aspect, the invention provides anti-OX 40 antibodies or fragments thereof having agonist activity that are capable of activating T cells (e.g., CD4+ T cells).

In some embodiments, agonist activity of the anti-OX 40 antibody is assessed by the level of cytokine released upon T cell activation. Accordingly, the invention provides anti-OX 40 antibodies or fragments thereof that increase cytokine production by CD4+ T cells as compared to cytokine production by CD4+ T cells treated with an IgG control antibody. In some embodiments, the cytokine is an inflammatory cytokine, such as a gamma interferon (e.g., IFNg) or an interleukin (e.g., IL-2).

Preferably, an anti-OX 40 antibody or fragment thereof of the invention is capable of increasing the level of IL-2 secreted by CD4+ T cells by up to about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold or more compared to a corresponding control IgG. In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention are capable of increasing the level of IFNg secreted by CD4+ T cells by up to about 1-fold, 2-fold, or 3-fold or more compared to a corresponding control IgG. In some embodiments, the level of cytokine secretion by T cells is determined by ELISA.

In some embodiments, agonist activity of an anti-OX 40 antibody is assessed by OX40 signaling (e.g., monitoring NFkB downstream signaling). Accordingly, the invention provides anti-OX 40 antibodies or fragments thereof that increase the level of NF κ B-mediated transcriptional activity compared to control antibodies with IgG. Preferably, an anti-OX 40 antibody or fragment thereof of the invention is capable of increasing the level of NF-. kappa.B-mediated transcriptional activity by about 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more compared to a corresponding control IgG.

In some preferred embodiments, the anti-OX 40 antibodies or fragments thereof of the invention are capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention function in need of antibody cross-linking. In some embodiments, the anti-OX 40 antibody or fragment thereof of the invention functions as one or more of the following: increase CD4+ effector T cell proliferation and/or cytokine production, increase CD4+ memory T cell proliferation and/or cytokine production, and/or deplete cells via ADCC.

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention have better anti-tumor activity than known anti-OX 40 antibodies, e.g., the anti-OX 40 antibodies or fragments thereof of the invention are capable of reducing tumor volume in a subject, preferably while not affecting the subject's body weight, compared to an IgG control or known anti-OX 40 antibodies.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Heavy Chain Variable Region (HCVR), wherein the HCVR comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 30. 1, 2 and 3, or an amino acid sequence having or consisting of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to an amino acid sequence selected from SEQ ID NO: 31. 4, 5,6 and 7 has or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:8, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Light Chain Variable Region (LCVR), wherein the LCVR comprises Complementarity Determining Regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises a Light Chain Variable Region (LCVR) that differs from the light chain variable region of SEQ ID NO:9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to the amino acid sequence set forth in SEQ ID NO:10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to the amino acid sequence set forth in SEQ ID NO:11, or an amino acid sequence consisting of or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the HCVR comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3 and the LCVR Comprises (CDRs) LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the HCVR comprises a Heavy Chain Variable Region (HCVR) comprising a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR) comprising a Heavy Chain Variable Region (HCVR) and a light chain variable region (LCDR 3538) comprising a Heavy Chain Variable Region (HCVR) selected from the group consisting of SEQ ID NOs: 30. 1, 2 and 3, or an amino acid sequence having or consisting of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to an amino acid sequence selected from SEQ ID NO: 31. 4, 5,6 and 7 has or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:8, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity; wherein LCDR1 comprises a nucleotide sequence identical to SEQ ID NO:9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to the amino acid sequence set forth in SEQ ID NO:10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to the amino acid sequence set forth in SEQ ID NO:11, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Heavy Chain Variable Region (HCVR), wherein the HCVR comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30. 1, 2 and 3, or consists of said amino acid sequence; HCDR2 comprises a sequence selected from SEQ ID NO: 31. 4, 5,6 and 7, or consists of said amino acid sequence; HCDR3 comprises SEQ ID NO:8 or consists of said amino acid sequence.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Light Chain Variable Region (LCVR), wherein the LCVR comprises Complementarity Determining Regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO:9 or consists of said amino acid sequence; LCDR2 comprises SEQ ID NO:10 or consists of said amino acid sequence; LCDR3 comprises SEQ ID NO:11 or consists of said amino acid sequence.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the HCVR comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3 and the LCVR Comprises (CDRs) LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises a Heavy Chain Variable Region (HCVR) comprising a Heavy Chain Variable Region (HCVR) selected from the group consisting of SEQ ID NO: 30. 1, 2 and 3, or consists of said amino acid sequence; HCDR2 comprises a sequence selected from SEQ ID NO: 31. 4, 5,6 and 7, or consists of said amino acid sequence; HCDR3 comprises SEQ ID NO:8 or consists of said amino acid sequence; LCDR1 comprises SEQ ID NO:9 or consists of said amino acid sequence; LCDR2 comprises SEQ ID NO:10 or consists of said amino acid sequence; LCDR3 comprises SEQ ID NO:11 or consists of said amino acid sequence.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen-binding fragment thereof comprising a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the HCVR comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR Comprises (CDRs) LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 30, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31 and HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8 or consists of the amino acid sequence shown in 8; wherein LCDR1 comprises SEQ ID NO:9, LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:10, LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:11 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen-binding fragment thereof comprising a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the HCVR comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR Comprises (CDRs) LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:1, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:4 and HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8 or consists of the amino acid sequence shown in 8; wherein LCDR1 comprises SEQ ID NO:9, LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:10, and LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:11 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen-binding fragment thereof comprising a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the HCVR comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR Comprises (CDRs) LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:2 or consists of the amino acid sequence shown in the specification; HCDR2 comprises SEQ ID NO:5 or consists thereof; HCDR3 comprises SEQ ID NO:8 or consists of the amino acid sequence shown in 8; LCDR1 comprises SEQ ID NO:9 or consists thereof; LCDR2 comprises SEQ ID NO:10 or consists thereof; and LCDR3 comprises SEQ ID NO:11 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen-binding fragment thereof comprising a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the HCVR comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR Comprises (CDRs) LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:1 or consists thereof; HCDR2 comprises SEQ ID NO:6 or consists thereof; HCDR3 comprises SEQ ID NO:8 or consists of the amino acid sequence shown in 8; LCDR1 comprises SEQ ID NO:9 or consists thereof; LCDR2 comprises SEQ ID NO:10 or consists thereof; and LCDR3 comprises SEQ ID NO:11 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen-binding fragment thereof comprising a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the HCVR comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR Comprises (CDRs) LCDR1, LCDR2 and LCDR3, wherein the HCDR1 comprises SEQ ID NO:3 or consists thereof; HCDR2 comprises SEQ ID NO:7 or consists thereof; HCDR3 comprises SEQ ID NO:8 or consists of the amino acid sequence shown in 8; LCDR1 comprises SEQ ID NO:9 or consists thereof; LCDR2 comprises SEQ ID NO:10 or consists thereof; and LCDR3 comprises SEQ ID NO:11 or consists thereof.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region HCVR comprising an amino acid sequence identical to a sequence selected from SEQ ID NOs: 16. 17, 18 and 19 has or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region LCVR comprising a sequence identical to SEQ ID NO:21, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the heavy chain variable region HCVR comprises an amino acid sequence that is identical to a sequence selected from SEQ ID NOs: 16. 17, 18 and 19, or an amino acid sequence consisting of or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity; the light chain variable region LCVR comprises an amino acid sequence substantially identical to SEQ ID NO:21, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16. 17, 18 and 19 or consists thereof.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region LCVR comprising the amino acid sequence of SEQ ID NO:21 or consists thereof.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the heavy chain variable region HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16. 17, 18 and 19 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:21 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen-binding fragment thereof comprising a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the heavy chain variable region HCVR comprises the amino acid sequence of SEQ ID NO:16 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:21 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen-binding fragment thereof comprising a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the heavy chain variable region HCVR comprises the amino acid sequence of SEQ ID NO:17 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:21 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen-binding fragment thereof comprising a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the heavy chain variable region HCVR comprises the amino acid sequence of SEQ ID NO:18 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:21 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen-binding fragment thereof comprising a Heavy Chain Variable Region (HCVR) and a Light Chain Variable Region (LCVR), wherein the heavy chain variable region HCVR comprises the amino acid sequence of SEQ ID NO:19 or consists thereof; the light chain variable region LCVR comprises SEQ ID NO:21 or consists thereof.

In some embodiments, the anti-OX 40 antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain, wherein the heavy chain comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 32. 33, 35, 36, 37, 38, 39 or 40, or an amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical.

In some embodiments, the anti-OX 40 antibodies or antigen-binding fragments thereof of the invention comprise a light chain, wherein the light chain comprises an amino acid sequence identical to SEQ ID NO:34, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 32. 33, 35, 36, 37, 38, 39 or 40, or an amino acid sequence consisting of or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity; the light chain comprises a sequence identical to SEQ ID NO:34, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity.

In some embodiments, the anti-OX 40 antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32. 33, 35, 36, 37, 38, 39 or 40, or a combination thereof.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a light chain, wherein the light chain comprises SEQ ID NO:34 or consists thereof.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32. 33, 35, 36, 37, 38, 39 or 40 or consists thereof; the light chain comprises SEQ ID NO:34 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:32 or consists thereof; the light chain comprises SEQ ID NO:34 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:33 or consists thereof; the light chain comprises SEQ ID NO:34 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:35 or consists thereof; the light chain comprises SEQ ID NO:34 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:36 or consists thereof; the light chain comprises SEQ ID NO:34 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:37 or consists of the amino acid sequence shown in seq id no; the light chain comprises SEQ ID NO:34 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:38 or consists thereof; the light chain comprises SEQ ID NO:34 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:39 or consists thereof; the light chain comprises SEQ ID NO:34 or consists thereof.

In a preferred embodiment, the invention provides an anti-OX 40 antibody or antigen binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:40 or consists thereof; the light chain comprises SEQ ID NO:34 or consists thereof.

In some embodiments, the heavy and/or light chain of an anti-OX 40 antibody or fragment thereof of the invention further comprises a signal peptide sequence, e.g., METDTLLLWVLLLWVPGSTG (SEQ ID NO: 46).

In some embodiments, the antibodies of the invention also encompass variants of the amino acid sequence of the anti-OX 40 antibody, as well as antibodies that bind the same epitope as any of the antibodies described above.

In certain embodiments, antibodies or antibody fragments (preferably antigen-binding fragments) that bind to OX40 or fragments thereof are provided, wherein the antibodies bind to an epitope within a fragment of OX 40.

In some embodiments, an anti-OX 40 antibody of the invention is an antibody in the form of IgG1 or an antibody in the form of IgG2 or an antibody in the form of IgG 4. In some embodiments, the anti-OX 40 antibody is a monoclonal antibody. In some embodiments, the anti-OX 40 antibody is humanized. In some embodiments, the anti-OX 40 antibody is a human antibody. In some embodiments, at least a portion of the framework sequence of the anti-OX 40 antibody is a human consensus framework sequence. In one embodiment, the anti-OX 40 antibodies of the invention also encompass antibody fragments thereof, preferably antibody fragments selected from the group consisting of: fab, Fab '-SH, Fv, scFv or (Fab')₂And (3) fragment.

In one aspect, the invention provides a nucleic acid encoding any of the above anti-OX 40 antibodies or fragments thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell (e.g., a CHO cell or 293 cell), or other cell suitable for production of an antibody or antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic.

In one embodiment, the invention provides a method of making an anti-OX 40 antibody or fragment thereof (preferably antigen-binding fragment), wherein the method comprises culturing the host cell under conditions suitable for expression of a nucleic acid encoding the antibody or fragment thereof (preferably antigen-binding fragment), and optionally isolating the antibody or fragment thereof (preferably antigen-binding fragment). In a certain embodiment, the method further comprises recovering the anti-OX 40 antibody or fragment thereof (preferably an antigen-binding fragment) from the host cell.

In some embodiments, the invention provides a composition comprising any of the anti-OX 40 antibodies or fragments thereof (preferably antigen-binding fragments thereof) described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is for treating cancer, preferably lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like.

In some embodiments, the invention provides immunoconjugates comprising any of the anti-OX 40 antibodies provided herein and a cytotoxic agent. In some embodiments, the immunoconjugate is for use in the treatment of cancer, preferably lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like.

In one aspect, the invention relates to a method of activating T cells or inducing a T cell-mediated anti-tumor activity or enhancing an immune response in a subject, comprising administering to the subject an effective amount of any of the anti-OX 40 antibodies or fragments thereof described herein. The invention also relates to the use of any of the anti-OX 40 antibodies or fragments thereof described herein to prepare a composition or medicament for activating T cells or inducing T cell-mediated anti-tumor activity or enhancing an immune response in a subject.

In another aspect, the invention relates to a method of treating cancer in a subject, comprising administering to the subject an effective amount of any of the anti-OX 40 antibodies or fragments thereof described herein. In one embodiment, the cancer is lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like. In another aspect, the invention also relates to the use of any of the anti-OX 40 antibodies or fragments thereof described herein for the preparation of a medicament for treating cancer in a subject. In one embodiment, the cancer is lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like.

In some embodiments, the methods described herein further comprise co-administering to the subject an effective amount of a second drug or active agent, wherein the anti-OX 40 antibody or fragment thereof described herein is the first drug. In some embodiments, the second drug or active agent is selected from chemotherapeutic agents. In some embodiments, the second drug or active agent is selected from a PD-1 axis binding antagonist (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody) or an anti-angiogenic agent (e.g., bevacizumab).

In some embodiments, the subject or individual is a mammal, preferably a human.

In one aspect, the invention relates to a method of detecting OX40 in a sample, the method comprising (a) contacting the sample with any of the anti-OX 40 antibodies or fragments thereof described herein; and (b) detecting the formation of a complex between the anti-OX 40 antibody or fragment thereof and OX 40. In one embodiment, the anti-OX 40 antibody is detectably labeled.

In some embodiments, the invention relates to a kit or article of manufacture comprising any of the anti-OX 40 antibodies or fragments thereof described herein. In some embodiments, the kits or articles of manufacture comprise an anti-OX 40 antibody or fragment thereof described herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the kit or article of manufacture further comprises instructions for administering the medicament to treat cancer.

The invention also encompasses any combination of any of the embodiments described herein. Any of the embodiments described herein or any combination thereof are applicable to any and all of the anti-OX 40 antibodies or fragments, methods, and uses thereof of the inventions described herein.

Drawings

FIG. 1 shows the blocking of the binding of OX40L to OX40 expressed on CHO cells with increasing concentration of an antibody of the invention produced in CHO cells (ADI-20057 in the form of IgG2, expressed in CHO cells) as determined by flow cytometry.

FIG. 2 shows IL-2 secretion upon T cell activation in PBMCs activated with PHA plus antibodies of the invention in the form of IgG1 and IgG2 produced in CHO cells.

FIG. 3 shows the detection of luciferase reporter genes in Jurkat stably transfected with human OX40 and NFkB promoter-luc by anti-CD 3 and anti-CD 28 stimulation plus IgG1 or IgG2 forms of the antibody of the invention produced in CHO cells, where Jurkat is mixed with Raji cells.

FIG. 4 shows the detection of luciferase reporter in Jurkat stably transfected with human OX40 and NFkB promoter-luc by anti-CD 3 and anti-CD 28 stimulation plus an antibody of the invention in the form of IgG1 produced in CHO cells, where Jurkat was mixed with Raji cells.

FIG. 5 shows a DC co-culture assay with SEE (1ng/ml) and a form of IgG1 or IgG2 of the OX40 antibody of the invention produced in CHO cells.

FIG. 6 shows luciferase reporter assay in Jurkat cells stably transfected with human OX40 and NFkB promoter-luc upon mixing Raji cells, stimulated with anti-CD 3 and anti-CD 28 and recombinant OX40L plus antibodies of the invention in the form of IgG1 or IgG2 produced in CHO cells or controls (IgG4, Pogalizumab, OX 40L).

Figure 7 shows ADCC activity of antibodies of the invention in the form of IgG1 produced in HEK293 cells against CHO cells expressing human OX40 in a luciferase reporter based format.

Figure 8 shows ADCC activity of antibodies of the invention in the form of IgG2 produced in HEK293 cells against CHO cells expressing human OX40 in a luciferase reporter based format.

FIG. 9 shows tumor growth curves for individual mice in a Hu-PBMC LoVo tumor mouse model administered in NOG mice with an IgG1 form of the OX40 antibody ADI-20057-g1 produced in CHO cells (where each curve represents data for one mouse).

FIG. 10 shows tumor growth curves for individual mice in a Hu-PBMC LoVo tumor mouse model administered in NOG mice with the OX40 antibody ADI-20057-g2 as IgG2 produced in CHO cells (where each curve represents data for one mouse).

FIG. 11 shows a Hu-PBMC LoVo tumor mouse model of administration of IgG1 and IgG2 forms of the OX40 antibodies ADI-20057-g1 and ADI-20057-g2 produced in CHO cells in NOG mice. Final tumor volume measurements at day 28 post tumor implantation are shown.

FIG. 12 shows a Hu-PBMC LoVo tumor mouse model of administration of IgG1 and IgG2 forms of the OX40 antibodies ADI-20057-g1 and ADI-20057-g2 produced in CHO cells in NOG mice. The change in body weight of the mice after administration is shown.

Detailed Description

Definition of

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

For the purpose of interpreting this specification, the following definitions will be used, and where appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is intended to encompass the numerical value within a range having a lower limit that is 5% less than the stated numerical value and an upper limit that is 5% greater than the stated numerical value.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K)_D) To express. Affinity can be measured by common methods known in the art, including those known in the art and described herein.

As used herein, the terms "anti-OX 40 antibody," "anti-OX 40," "OX 40 antibody," or "an antibody that binds to OX 40" refer to an antibody that is capable of binding to a human or cynomolgus monkey OX40 protein or fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent in targeting human or cynomolgus monkey OX 40. In one embodiment, the anti-OX 40 antibody binds to a non-human or cynomolgus monkey OX40 protein to a lesser extent than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of the binding of the antibody to human or cynomolgus monkey OX40 as measured, for example, by Radioimmunoassay (RIA) or biophotonic interferometry or MSD assay. In some embodiments, the anti-OX 40 antibody binds to the equilibrium dissociation constant (K) of human or cynomolgus monkey OX40_D) 100nM or less, 10nM or less, or 1nM (e.g., 10 nM) or less^-7M or less, e.g. 10^-7M to 10^-10M, e.g. 10^-8M to 10^-9M)。

As used herein, "monoclonal antibody" or "mAb" refers to an antibody derived from a single copy or clone, e.g., of a eukaryote, prokaryote, or phage clone, and not to the method of its production. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques such as CDR grafting, or combinations of these or other techniques known in the art.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is an heterotetrameric glycan protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has one variable region (VH), also called variable or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a Constant Light (CL) domain. Antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences. A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-a and a allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4Fc region; and naturally occurring variants thereof.

An "antibody fragment" refers to a molecule distinct from an intact antibody that comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab ' -SH, F (ab ') 2; a diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of 50% or more of the reference antibody to its antigen in a competition assay, whereas a reference antibody blocks binding of 50% or more of the antibody to its antigen in a competition assay.

Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG and IgM, and several of these antibodies may be further divided into subclasses (isotypes), e.g., IgG₁，IgG₂，IgG₃，IgG₄，IgA₁And IgA₂. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively.

An "IgG-form antibody" refers to the IgG form to which the heavy chain constant region of an antibody belongs. The heavy chain constant regions are the same for all antibodies of the same type, and differ between antibodies of different types. For example, an antibody in the form of IgG1 refers to a heavy chain constant region, Ig, domain that is the Ig domain of IgG 1.

The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation. As used herein, the term "dysfunction" also includes an inability to sense or respond to antigen recognition, in particular, an impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., gamma interferon) and/or target cell killing.

The expression "does not block the binding of human OX40 to OX40 ligand/OX 40L" as used herein means that the antibodies of the invention do not affect the binding of human OX40 to OX 40L. In some embodiments, the degree of binding of OX40 to OX40L is not reduced in the presence of an anti-OX 40 antibody of the invention as compared to the degree of binding of OX40 to OX40L in the presence of a negative control (e.g., an IgG antibody). In some embodiments, the degree of binding of OX40 to OX40L is reduced by up to 10% in the presence of an anti-OX 40 antibody of the invention as compared to the degree of binding of OX40 to OX40L in the presence of a negative control (e.g., an IgG antibody). In some embodiments, the degree of binding of OX40 to OX40L is determined by a luciferase reporter gene. In some embodiments, the degree of binding of OX40 to OX40L is determined by flow cytometry.

By "activating T cells" is meant inducing, causing or stimulating effector or memory T cells to have renewed, sustained or amplified biological function. Examples of enhancing T cell function include: elevated levels from CD8 relative to pre-intervention such levels⁺Secretion of gamma-interferon (e.g., IFNg) or interleukin (e.g., IL-2) by effector T cells, elevated levels of interferon-gamma from CD4⁺Increased secretion of interferon-gamma (e.g., IFNg) or interleukins (e.g., IL-2) by memory and/or effector T cells, increased CD4⁺Increased CD8 for effector and/or memory T cell proliferation⁺Effector T cells proliferate, increased antigen responsiveness (e.g., clearance). In one embodiment, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold. The manner of measuring this enhancement is known to those of ordinary skill in the art.

"tumor immunity" refers to the process by which a tumor evades immune recognition and clearance. As such, as a therapeutic concept, tumor immunity is "treated" when such evasion is diminished, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance.

"immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic and enhancing tumor immunogenicity helps to eliminate tumor cells by an immune response.

As used herein, "agonist activity of an antibody" refers to the biological activity of an antibody that activates the antigen to which it binds.

"anti-angiogenic agent" refers to a compound that blocks or interferes to some extent with vascular development. The anti-angiogenic agent can be, for example, a small molecule or antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis. In one embodiment, the anti-angiogenic agent is an antibody that binds Vascular Endothelial Growth Factor (VEGF), such as bevacizumab (AVASTIN).

The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of the PD-1 axis binding partner with one or more of its binding partners, thereby removing T cell dysfunction resulting from signaling on the PD-1 signaling axis-one outcome is restoration or enhancement of T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists (e.g., anti-PD-1 antibodies), PD-L1 binding antagonists (e.g., anti-PD-L1 antibodies), and PD-L2 binding antagonists (e.g., anti-PD-L2 antibodies).

The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2). In some embodiments, the PD-1 binding antagonist is a molecule that inhibits binding of PD-1 to one or more of its binding partners. In a particular aspect, the PD-1 binding antagonist inhibits PD-1 from binding to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1 binding antagonist reduces negative co-stimulatory signals (mediated signaling via PD-1) mediated by or via cell surface proteins expressed on T lymphocytes, thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific embodiment, the PD-1 binding antagonist is MDX-1106(nivolumab), MK-3475(pembrolizumab), CT-011(pidilizumab), or AMP-224 as disclosed in WO 2015/095423.

The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L1 with one 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 binding of PD-L1 to its binding partner. In a particular aspect, the PD-L1 binding antagonist inhibits PD-L1 from binding to PD-1 and/or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of 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 negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes (signal transduction mediated via PD-L1), thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In a particular aspect, the anti-PD-L1 antibody is yw243.55.s70, MDX-1105, MPDL3280A or MEDI4736 disclosed in WO 2015/095423.

The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates or interferes with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits PD-L2 from binding to one or more of its binding partners. In a particular 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 reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from 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 negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes (signaling is mediated via PD-L2), thereby rendering dysfunctional T cells less dysfunctional (e.g., enhancing effector response to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted immunoglobulins that bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells, followed by killing of the target cells with cytotoxins. The main cell mediating ADCC, NK cells, expresses only Fc γ RIII, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravatch and Kinet, annu.rev.immunol.9: 457-92(1991) 464 Page table 3 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. Pat. No.5,500,362 or 5,821, 337 or U.S. Pat. No.6,737,056 (Presta). Effector cells useful in such assays include PBMC and NK cells. Alternatively/additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models, such as Clynes et al, pnas (usa) 95: 652-. An exemplary assay for assessing ADCC activity is provided in the examples herein.

As used herein, the term "OX 40" refers to any native OX40 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed OX40 as well as any form of OX40 that results from processing in a cell. The term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants.

"OX 40 activation" refers to the activation of the OX40 receptor. Generally, OX40 activation results in signal transduction.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Examples of cytotoxic agents are disclosed in WO 2015/153513.

"chemotherapeutic agents" include chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents are disclosed in WO2015/153513, including erlotinib (erlotinib) ((ll-A))

Genentech/OSI Pharm), bortezomib (bortezomib), (b), (d

Millennium Pharm.), disulfiram (disulfiram), alkylating agents (alkylating agents), such as thiotepa (thiotepa); alkyl sulfonates (alkyl sulfonates); aziridines (aziridines), and the like; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

Chemotherapeutic agents also include (i) anti-hormonal agents that act to modulate or inhibit the action of hormones on tumors, such as anti-estrogens and Selective Estrogen Receptor Modulators (SERMs), including, for example, tamoxifen (including

Tamoxifen citrate); (ii) aromatase inhibitors which inhibit aromatase which regulates estrogen production in the adrenal glands, such as, for example, 4(5) -imidazole, aminoglutethimide; (iii) anti-androgens such as flutamide (flutamide), nilutamide (nilutamide); (iv) protein kinase inhibitors; (v) a lipid kinase inhibitor; (vi) antisense oligonucleotides, particularly antisense oligonucleotides that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC- α, Ralf, and H-Ras; (vii) ribozymes, such as VEGF expression inhibitors (e.g.

And inhibitors of HER2 expression; (viii) vaccines, such as gene therapy vaccines, e.g.

To know

rIL-2; topoisomerase 1 inhibitors, such as

rmRH; and (ix) and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

Chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab (bevacizumab), (b) and (c)

Genentech), and the like, and antibody drug conjugates, gemtuzumab ozogamicin (gemtuzumab ozogamicin), (b), (c), (d

Wyeth). Further humanized monoclonal antibodies having therapeutic potential as agents in combination with the compounds of the invention are e.g. aprezumab (apolizumab), ustekumab (ustekinumab), vislizumab (visilizumab), and anti-interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories), which is a recombinant proprietary human sequence full-length IgG1 λ antibody genetically modified to recognize interleukin-12 p40 protein.

Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, which are otherwise also referred to as "EGFR antagonists. Examples of such agents include antibodies and small molecules that bind EGFR. Examples of antibodies that bind EGFR include MAb 579(ATCC CRL HB 8506); antibodies that bind type II mutant EGFR (U.S. Pat. No.5,212,290); humanized and chimeric antibodies that bind EGFR as described in U.S. patent No.5,891,996; and human antibodies that bind to EGFR, such as ABX-EGF or Panitumumab (Panitumumab) (see WO98/50433, Abgenix/Amgen); human EGFR antibody, HuMax-EGFR (GenMab). anti-EGFR antibodies can be conjugated to cytotoxic agents, thereby producing immunoconjugates (see, e.g., EP 659,439a2, Merck Patent GmbH). EGFR antagonists include small molecules such as those described in U.S. patent nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, and PCT publications WO98/14451, WO98/50038, WO99/09016, and WO 99/24037. Specific small molecule EGFR antagonists include OSI-774(CP-358774, erlotinib, etc.).

Chemotherapeutic agents also include "tyrosine kinase inhibitors" including the EGFR-targeting drugs mentioned in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; rapamycin (a compound of formula I) is rapamycin (sirolimus,

) Etc.; or any of the following patent publications: U.S. patent nos. 5,804,396; WO1999/09016(American Cyanamid); WO1998/43960(American Cyanamid); WO1997/38983(Warner Lambert); WO1999/06378(Wamer Lambert); WO1999/06396 (WarnerLambert); WO1996/30347(Pfizer, Inc); WO1996/33978 (Zeneca); WO1996/3397(Zeneca) and WO 1996/33980 (Zeneca).

Chemotherapeutic agents also include dexamethasone (dexamethasone), hydrocortisone (hydrocortisone), interferon, bevacizumab (bevacizumab), bexarotene (bexarotene), and the like, and pharmaceutically acceptable salts thereof.

Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs having analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include aspirin (aspirin). NSAIDs may be indicated for symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory joint disease, ankylosing spondylitis, psoriatic arthritis, reiter's syndrome, acute gout, dysmenorrhea, bone metastasis pain, headache and migraine, post-operative pain, mild to moderate pain due to inflammation and tissue injury, fever, ileus, and renal colic.

For a detailed listing and description of the various chemotherapeutic agents described above, see those disclosed in PCT International application WO2015/153513, which is incorporated herein by reference in its entirety.

The term "cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines; interleukins (IL), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factors such as TNF- α or TNF- β; and other polypeptide factors, including LIF and Kit Ligand (KL) and interferon gamma. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the natural sequence cytokines, including small molecule entities produced by artificial synthesis, and pharmaceutically acceptable derivatives and salts thereof.

The term "diabodies" refers to antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to pair between two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain to create two antigen binding sites. Diabodies may be bivalent or bispecific. Diabodies are more fully described in e.g. EP 404,097; WO 1993/01161; hudson et al, nat. med.9: 129-134 (2003); and Hollinger et al, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.usa) 90: 6444- > 6448 (1993). Triabodies and tetrabodies are also described in Hudson et al, nat. med.9: 129-134 (2003).

A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q combinations; CDC; fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions generally require that the Fc region be associated with a binding domain (e.g., an antibody variable domain) and can be evaluated using a variety of assays, such as those disclosed herein.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: clq binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"human effector cells" refer to leukocytes which express one or more fcrs and which exert effector function. In certain embodiments, the cell expresses at least Fc γ RIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. The effector cells may be isolated from their natural source, e.g., blood.

The term "effective amount" refers to an amount or dose of an antibody or fragment of the invention that produces the desired effect in the treated patient upon administration to the patient in a single or multiple dose. An effective amount can be readily determined by the attending physician, as one skilled in the art, by considering a number of factors: species such as mammals; its size, age and general health; the specific diseases involved; the degree or severity of the disease; the response of the individual patient; the specific antibody administered; a mode of administration; bioavailability characteristics of the administered formulation; a selected dosing regimen; and the use of any concomitant therapies.

Suitable "antibodies and antigen-binding fragments thereof" for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterohybrid, chimeric, humanized (particularly CDR grafted), deimmunizedEpidemic or human antibodies, Fab fragments, Fab 'fragments, F (ab')₂Fragments, fragments produced by Fab expression libraries, Fd, Fv, disulfide linked Fv (dsfv), single chain antibodies (e.g., scFv), diabodies or tetrabodies (Holliger p. et al (1993) proc. natl. acad. sci. u.s.a.90(14), 6444-6448), nanobodies (also known as single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies directed against an antibody of the invention), and epitope-binding fragments of any of the foregoing.

The "Fab" fragment includes the heavy and light chain variable domains, and also includes the constant domain of the light chain and the first constant domain of the heavy chain (CHl). Fab' fragments differ from Fab fragments by the addition of residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residue of the constant domain carries a free thiol group. F (ab')₂Antibody fragments were originally produced as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, with each domain comprising four conserved Framework Regions (FRs) and three Complementarity Determining Regions (CDRs). (Ginseng radixSee, for example, Kindt et al Kuby Immunology, 6^thed., page 2007, w.h.freeman and co.91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a particular antigen can be used to isolate antibodies that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al, j.immunol.150: 880- & ltwbr & gt 887 & gt (1993); clarkson et al, Nature 352: 624-628(1991).

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) (e.g., complementarity determining region) residues. The FRs of a variable domain typically consist of four FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the following sequences of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

Unless otherwise indicated, the numbering of residues in each domain of an antibody is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The terms "full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, a two-chain Fv species consists of dimers of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In the single chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain may be covalently linked by a flexible peptide linker so that the light and heavy chains may associate in a "dimeric" structure similar to that of a two-chain Fv species. In this configuration, the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. In summary, six HVRs confer antigen binding specificity to antibodies. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with less affinity than the entire binding site. For reviews on scFv see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 113, edited by Rosenburg and Moore, (Springer-Verlag, New York, 1994), pp.269-315.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid is introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primarily transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected in the originally transformed cell.

"human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or other human antibody coding sequence. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

"human consensus framework" refers to a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. In general, a subtype of this sequence is that disclosed in Kabat et al (Sequences of Proteins of Immunological Interest, fifth edition, NIH Publication 91-3242, Bethesda MD (1991), Vol.1-3). In one embodiment, for VL, this subtype is subtype kappa I as in Kabat et al (supra). In one embodiment, for the VH, this subtype is subtype III as in Kabat et al (supra).

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have been humanized.

The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo malignant melanoma, acromegaly melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with scarring nevus (phakomatases), edema (such as associated with brain tumors) and Meigs (Meigs) syndrome, brain tumors and cancers, as well as head and neck cancers, and associated metastases. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer, including metastatic forms of those cancers.

The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

The term "tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents).

An "individual" or "subject" includes a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al, j.chromatogr.b848: 79-87(2007).

An "isolated" nucleic acid is a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

An "isolated nucleic acid encoding an anti-OX 40 antibody or antigen-binding fragment thereof" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or antigen-binding fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are the same as the amino acid residues in the reference polypeptide sequence, after the sequences are aligned (and gaps introduced, if necessary) to obtain the maximum percent sequence identity, and no conservative substitutions are considered as part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared.

When referring to percentages of sequence identity in the present application, these percentages are calculated over the full length of the longer sequence, unless otherwise specifically indicated. The full length calculation relative to the longer sequence applies to both nucleic acid and polypeptide sequences.

The term "pharmaceutical composition" refers to a formulation that is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the formulation is administered.

The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (e.g., freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered.

As used herein, "treating" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

The term "vector" when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors which are self-replicating nucleic acid structures as well as vectors which are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

"subject/patient sample" refers to a collection of cells or fluids obtained from a cancer patient or cancer subject. The source of the tissue or cell sample may be a solid tissue, like from a fresh, frozen and/or preserved organ or tissue sample or biopsy sample or punch sample; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time of pregnancy or development. Tissue samples may contain compounds that are not naturally intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage, pleural fluid (pleural fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.

The term "package insert" is used to refer to instructions for use typically contained in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings relating to the use of such therapeutic products.

Antibodies of the invention

In one aspect, the invention provides an isolated antibody or fragment thereof (preferably an antigen-binding fragment) that binds to human OX40, wherein the antibody or fragment thereof is capable of binding to human OX40 while not blocking the binding of human OX40 to OX40 ligand (OX 40L).

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention are capable of activating T cells, e.g., enhancing the immunostimulatory/effector functions of T effector cells and/or proliferating these cells.

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention are capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC).

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be used to treat or delay various cancers, immune-related diseases, and T cell dysfunctional diseases.

In some embodiments, binding of an antibody or fragment thereof of the invention to human OX40 or cynomolgus monkey OX40 is determined using a flow cytometry (e.g., FACS) assay. In some embodiments, binding to human OX40 or cynomolgus monkey OX40 has an EC50 of less than or equal to about 10nM, 9nM, 8nM, or 7 nM. In some embodiments, binding to human OX40 or cynomolgus monkey OX40 has an EC50 of less than or equal to about 6nM or about 5nM or about 4nM or about 3 nM. In some embodiments, an antibody or fragment thereof of the invention binds to OX40 expressed on a cell in an assay performed with a flow cytometry assay, and the MFI value is greater than 1000-fold difference, preferably greater than 1100-fold difference, 1200-fold difference, 1300-fold difference, 1400-fold difference, 1500-fold difference, 1600-fold difference, 1700-fold difference, 1800-fold difference, 1900-fold difference, 2000-fold difference compared to a corresponding control cell that does not express OX 40.

In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention has a functional Fc region. In some embodiments, the effector function of the functional Fc region is ADCC. In some embodiments, the Fc region is human IgG 1.

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention are capable of activating T cells.

In some embodiments, the anti-OX 40 antibodies of the invention are capable of not only not blocking binding of human OX40 to its ligand OX40L, but also increasing OX 40L-based T cell activation of OX 40.

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention enhance CD4+ effector T cell function, e.g., by increasing CD4+ effector T cell proliferation and/or increasing gamma-interferon production by CD4+ effector T cells (e.g., as compared to proliferation and/or cytokine production prior to treatment with the anti-OX 40 antibodies or fragments thereof of the invention, or as compared to proliferation and/or cytokine production by CD4+ effector T cells treated with a control antibody (e.g., an IgG antibody)). In some embodiments, the cytokine is a gamma interferon, such as IFNg or an interleukin, such as IL-2.

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention increase the number of (infiltrating) CD4+ effector T cells (e.g., the total number of CD4+ effector T cells, or the percentage of CD4+ cells in, e.g., CD45+ cells) within the tumor, e.g., as compared to the number of (infiltrating) CD4+ T cells within the tumor prior to treatment with the anti-OX 40 antibodies or fragments thereof of the invention (or after treatment with a control antibody (e.g., an IgG antibody)). In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention increase the number of gamma-interferon expressing intratumoral (infiltrating) CD4+ effector T cells (e.g., the total number of gamma-interferon expressing CD4+ cells, or e.g., the percentage of gamma-interferon expressing CD4+ cells in the total CD4+ cells), e.g., as compared to the number of gamma-interferon expressing intratumoral (infiltrating) CD4+ T cells prior to treatment with the anti-OX 40 antibodies or fragments thereof of the invention (or after treatment with a control antibody (e.g., an IgG antibody)).

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention increase the number of (infiltrating) CD8+ effector T cells (e.g., the total number of CD8+ effector T cells, or the percentage of CD8+ in, e.g., CD45+ cells) within the tumor, e.g., as compared to the number of (infiltrating) CD8+ effector cells within the tumor prior to treatment with the anti-OX 40 antibodies or fragments thereof of the invention (or after treatment with a control antibody (e.g., an IgG antibody)). In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention increase the number of gamma-interferon expressing intratumoral (infiltrating) CD8+ effector T cells (e.g., the percentage of gamma-interferon expressing CD8+ cells out of total CD8+ cells), e.g., as compared to the number of gamma-interferon expressing intratumoral (infiltrating) CD8+ T cells prior to treatment with the anti-OX 40 antibodies or fragments thereof of the invention (or after treatment with a control antibody, e.g., an IgG antibody).

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention enhance memory T cell function, e.g., by increasing memory T cell proliferation and/or increasing cytokine production by memory cells. In some embodiments, the cytokine is a gamma interferon or an interleukin.

In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention increases OX40 signaling in a target cell expressing OX 40. In some embodiments, OX40 signaling is detected by monitoring NFkB downstream signaling.

In some embodiments, the agonist activity of an anti-OX 40 antibody or fragment thereof of the invention is assessed by the level of cytokine released upon T cell activation. Accordingly, the invention provides anti-OX 40 antibodies or fragments thereof that increase cytokine production by CD4+ T cells as compared to cytokine production by CD4+ T cells treated with an IgG control. In some embodiments, the cytokine is an inflammatory cytokine, such as a gamma interferon (e.g., IFNg) or an interleukin (e.g., IL-2).

In some embodiments, agonist activity of an anti-OX 40 antibody is assessed by OX40 signaling (e.g., monitoring NFkB downstream signaling). Accordingly, the invention provides anti-OX 40 antibodies or fragments thereof that increase the level of NF κ B-mediated transcriptional activity compared to control antibodies with IgG. Preferably, the anti-OX 40 antibodies or fragments thereof of the invention are capable of increasing the level of NF κ B-mediated transcriptional activity by about 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more compared to a corresponding control IgG.

In some preferred embodiments, the anti-OX 40 antibodies or fragments thereof of the invention are capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention bind to human effector cells, e.g., bind to Fc γ R (e.g., activating Fc γ R) expressed by human effector cells. In some embodiments, the human effector cell performs (is capable of performing) ADCC effector function.

In some embodiments, the anti-OX 40 antibody or fragment thereof of the invention functions in need of antibody cross-linking. In some embodiments, the function is to stimulate CD4+ effector T cell proliferation. In some embodiments, antibody cross-linking is determined by providing an anti-human OX40 agonist antibody that adheres to a solid surface (e.g., a cell culture plate). In some embodiments, antibody cross-linking is determined by introducing mutations (e.g., amino acid sequence mutations) in the IgG Fc portion of the antibody and testing the mutant antibody for function. In some embodiments, antibody cross-linking is achieved by FcgRIIb.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Heavy Chain Variable Region (HCVR), wherein the HCVR comprises Complementarity Determining Regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 30. 1, 2 and 3, or an amino acid sequence having or consisting of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to an amino acid sequence selected from SEQ ID NO: 31. 4, 5,6 and 7, and HCDR3 comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to the amino acid sequence of SEQ ID NO:8, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity. In certain embodiments, the amino acid sequence of a CDR of an HCVR of an anti-OX 40 antibody or fragment thereof (e.g., a CDR that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to a reference sequence) comprises one or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to the corresponding reference sequence, but an anti-OX 40 antibody or fragment thereof comprising the CDR still has the ability to bind OX40 and does not block the binding of OX40 to its ligand OX 40L.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Light Chain Variable Region (LCVR), wherein the LCVR comprises Complementarity Determining Regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises a Light Chain Variable Region (LCVR) that differs from the light chain variable region of SEQ ID NO:9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to the amino acid sequence set forth in SEQ ID NO:10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity to the amino acid sequence set forth in SEQ ID NO:11, or an amino acid sequence consisting of or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity. In certain embodiments, the amino acid sequence of a CDR of an LCVR of an anti-OX 40 antibody or fragment thereof (e.g., a CDR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or 100% identity to a reference sequence) comprises one or more substitutions (e.g., conservative substitutions), insertions, or deletions relative to the corresponding reference sequence, but an anti-OX 40 antibody or fragment thereof comprising the CDR still has the ability to bind OX40 and does not block the binding of OX40 to its ligand OX 40L.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Heavy Chain Variable Region (HCVR), wherein the heavy chain variable region HCVR comprises an amino acid sequence that is identical to a sequence selected from SEQ ID NOs: 16. 17, 18 and 19 has or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity. In some embodiments, the heavy chain variable region HCVR of the anti-OX 40 antibody comprises a sequence identical to a sequence selected from SEQ ID NOs: 16. 17, 18 and 19 have one or more substitutions (e.g., conservative substitutions), insertions or deletions compared to the amino acid sequence of said HCVR, but an anti-OX 40 antibody or fragment thereof comprising said HCVR still has the ability to bind OX40 and does not block the binding of OX40 to its ligand OX 40L.

In some embodiments, an anti-OX 40 antibody or antigen-binding fragment thereof of the invention comprises a Light Chain Variable Region (LCVR), wherein the light chain variable region LCVR comprises an amino acid sequence identical to SEQ ID NO:21, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity. In some embodiments, the light chain variable region LCVR of the anti-OX 40 antibody comprises a sequence identical to SEQ ID NO:21, but an anti-OX 40 antibody or fragment thereof comprising the LCVR still has the ability to bind OX40 and does not block the binding of OX40 to its ligand OX 40L.

In some embodiments, the anti-OX 40 antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain, wherein the heavy chain comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 32. 33, 35, 36, 37, 38, 39 or 40, or an amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical. In some embodiments, the heavy chain of the anti-OX 40 antibody comprises a heavy chain that differs from a heavy chain selected from SEQ ID NOs: 32. 33, 35, 36, 37, 38, 39, or 40 has one or more substitutions (e.g., conservative substitutions), insertions, or deletions compared to the amino acid sequence of seq id no, but an anti-OX 40 antibody or fragment thereof comprising said heavy chain still has the ability to bind OX40 and does not block the binding of OX40 to its ligand OX 40L.

In some embodiments, the anti-OX 40 antibodies or antigen-binding fragments thereof of the invention comprise a light chain, wherein the light chain comprises an amino acid sequence identical to SEQ ID NO:34, or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or 100% identity. In some embodiments, the light chain of the anti-OX 40 antibody comprises a heavy chain that differs from SEQ ID NO:34, but an anti-OX 40 antibody or fragment thereof comprising said light chain still has the ability to bind OX40 and does not block the binding of OX40 to its ligand OX 40L. In preferred embodiments, the substitution, insertion or deletion occurs in a region outside the CDRs (e.g., in the FRs). Optionally, the anti-OX 40 antibodies of the invention include post-translational modifications to the light chain variable region, the heavy chain variable region, the light chain, or the heavy chain.

In some embodiments, the substitution is a conservative substitution. Conservative substitutions are those where one amino acid is substituted with another within the same class, for example, where one acidic amino acid is substituted with another acidic amino acid, one basic amino acid is substituted with another basic amino acid, or one neutral amino acid is substituted with another neutral amino acid. Exemplary substitutions are shown in table a below:

TABLE A

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence so as to create or remove one or more glycosylation sites. In some applications, modifications to remove unwanted glycosylation sites may be useful, or for example, to remove fucose moieties to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al (2002) JBC 277: 26733). In other applications, galactosylation modifications may be made to modify Complement Dependent Cytotoxicity (CDC).

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate Fc region variants, in order to enhance the effectiveness of the antibody, for example, in treating cancer or cell proliferative diseases. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3, or IgG4Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, it may be desirable to generate cysteine engineered antibodies, such as "thio mabs," in which one or more residues of the antibody are replaced with a cysteine residue.

In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties known and readily available in the art. Suitable antibody-derived moieties include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

In some embodiments, the invention encompasses fragments of anti-OX 40 antibodies. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂Diabodies, linear antibodies, single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, eachFrom having a single antigen binding site, and a residual "Fc" fragment, the name reflects its ability to crystallize readily. Pepsin treatment to yield F (ab')₂A fragment having two antigen binding sites and still being capable of cross-linking antigens.

In some embodiments, the anti-OX 40 antibodies of the invention are humanized antibodies. Different methods for humanizing antibodies are known to the skilled artisan, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J (2008) Frontiers in bioscience 13: 1619-1633). Almagro & Fransson distinguishes between rational and empirical approaches. Rational approach is characterized by generating a few engineered antibody variants and evaluating their binding or any other property of interest. If the design variation does not produce the expected result, a new round of design and integration evaluation is initiated. Rational approaches include CDR grafting, Resurfacing (Resurfacing), super-humanization (superfuranization), and herringbone string content Optimization (Human StringContent Optimization). In contrast, empirical approaches are based on generating large libraries of humanized variants and selecting the best clones using enrichment techniques or high throughput screening. Thus, empirical approaches rely on reliable selection and/or screening systems that can search for a large number of antibody variants. In vitro display techniques such as phage display and ribosome display fulfill these requirements and are well known to the skilled person. Empirical approaches include FR library, Guided selection (Guided selection), Framework shuffling (Framework-shuffling), and Humaneering.

In some embodiments, the anti-OX 40 antibodies of the invention are human antibodies. Human antibodies can be made using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, curr. 368-74(2001) and Lonberg, curr. opin. immunol 20: 450-459(2008).

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries for antibodies with desired binding characteristics. These Methods are described, for example, in Hoogenboom et al, Methods in Molecular Biology 178: 1-37 (compiled by O' Brien et al, human Press, Totowa, NJ, 2001), and further to the methods described in, for example, McCafferty et al, Nature 348: 552 and 554; clackso et al, Nature 352: 624-628 (1991); marks et al, j.mol.biol.222: 581-597 (1992); marks and Bradbury, Methods in Molecular Biology 248: 161-175(Lo eds., Human Press, Totowa, NJ, 2003); sidhu et al, j.mol.biol.338 (2): 299-310 (2004); lee et al, j. mol. biol340 (5): 1073-1093 (2004); fellouse, proc.natl.acad.sci.usa 101 (34): 12467-12472 (2004); and Lee et al, j.immunol.methods284 (1-2): 119, and 132 (2004).

In some embodiments, the invention also encompasses anti-OX 40 monoclonal antibodies ("immunoconjugates") conjugated to a therapeutic moiety, such as a cytotoxic or immunosuppressive agent. Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art, see, e.g., WO 05/103081. For example, cytotoxic agents include, but are not limited to: radioisotope (e.g., At)²¹¹，I¹³¹，I¹²⁵，Y⁹⁰，Re¹⁸⁶，Re¹⁸⁸，Sm¹⁵³，Bi²¹²，P³²，pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate (methotrexate), doxorubicin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorambucil), daunorubicin (daunorubicin), or other intercalating agents); a growth inhibitor; enzymes and fragments thereof such as nucleic acid hydrolases; (ii) an antibiotic; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various known antitumor or anticancer agents.

Exemplary cytotoxic agents may be selected from the group consisting of antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, inhibitors of LDH-a, inhibitors of fatty acid biosynthesis, inhibitors of cell cycle signaling, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism.

In one embodiment, the cytotoxic agent is selected from the group consisting of antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotics, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, pro-apoptotic agents, inhibitors of LDH-a, inhibitors of fatty acid biosynthesis, inhibitors of cell cycle signaling, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism. In one embodiment, the cytotoxic agent is a taxane (taxane). In one embodiment, the taxane is paclitaxel (paclitaxel) or docetaxel (docetaxel). In one embodiment, the cytotoxic agent is a platinum agent. In one embodiment, the cytotoxic agent is an antagonist of EGFR. In one embodiment, the antagonist of EGFR is N- (3-ethynylphenyl) -6, 7-bis (2-methoxyethoxy) quinazolin-4-amine (e.g., erlotinib). In one embodiment, the cytotoxic agent is a RAF inhibitor. In one embodiment, the RAF inhibitor is a BRAF and/or CRAF inhibitor. In one embodiment, the RAF inhibitor is vemurafenib (vemurafenib). In one embodiment, the cytotoxic agent is a PI3K inhibitor.

In some embodiments, the antibodies of the invention may be monospecific, bispecific, or multispecific. Multispecific mabs may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al (1991) j.immunol.147: 60-69. The anti-OX 40 monoclonal antibody can be linked to or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecules, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second or more binding specificities.

Nucleic acids of the invention and host cells comprising the same

In one aspect, the invention provides a nucleic acid encoding any of the above anti-OX 40 antibodies or fragments thereof. The nucleic acid may encode an amino acid sequence comprising a light chain variable region and/or a heavy chain variable region of an antibody, or an amino acid sequence comprising a light chain and/or a heavy chain of an antibody. Exemplary nucleic acid sequences encoding the variable region of the antibody heavy chain include sequences substantially identical to the sequences selected from SEQ ID NOs: 12, 13, 14 or 15, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a nucleic acid sequence selected from SEQ ID NOs: 12, 13, 14 or 15. Exemplary nucleic acid sequences encoding the variable region of the antibody light chain include sequences substantially identical to SEQ ID NO: 20, or a nucleic acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleic acid sequence set forth in SEQ ID NO: 20, or a nucleic acid sequence as set forth in seq id no.

In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector.

In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199 and 5,840,523, and also Charlton, Methods in Molecular Biology, Vol.248 (B.K.C.Lo, eds., Humana Press, Totowa, NJ, 2003), page 245-. After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, or other cell suitable for use in the production of an antibody or antigen-binding fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains, whose glycosylation pathways have been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, nat. biotech.22: 1409-: 210-215(2006). Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for growth in suspension may be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (293HEK or 293 cells, as described, e.g., in Graham et al, J.Gen Virol.36: 59 (1977)), and the like. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B.K.C.Lo, ed., Humana Press, Totowa, NJ), pp.255-268 (2003).

In one embodiment, a method of making an anti-OX 40 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally recovering the antibody from the host cell (or host cell culture medium). For recombinant production of anti-OX 40 antibodies, nucleic acids encoding antibodies (e.g., the antibodies described above) are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Assay method

The anti-OX 40 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art. In one aspect, antibodies of the invention are tested for antigen binding activity, for example, by known methods such as ELISA, Western blot, and the like. OX40 binding can be determined using methods known in the art, exemplary methods are disclosed herein. In some embodiments, a biophotonic interferometry (e.g., Fortebio affinity measurement) or MSD assay is used.

In another aspect, a competition assay can be used to identify antibodies that compete for binding to OX40 with any of the anti-OX 40 antibodies disclosed herein. In certain embodiments, such competitive antibodies bind to the same epitope (e.g., a linear or conformational epitope) as any of the anti-OX 40 antibodies disclosed herein bind to. A detailed exemplary method for locating epitopes bound by antibodies is described in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol.66(Humana Press, Totowa, NJ).

The invention also provides assays for identifying anti-OX 40 antibodies that have biological activity. Biological activities may include, for example, binding to OX40 (e.g., binding to human and/or cynomolgus monkey OX40), increasing OX 40-mediated signal transduction (e.g., increasing NFkB-mediated transcription), depleting cells expressing human OX40 by ADCC, enhancing T effector cell function (e.g., CD4+ effector T cells) (e.g., by increasing effector T cell proliferation and/or increasing cytokine production by effector T cells (e.g., gamma interferon or interleukins)), enhancing memory T cell function (e.g., CD4+ memory T cells) (e.g., by increasing memory T cell proliferation and/or increasing cytokine production by memory T cells (e.g., gamma interferon or interleukins)), binding to human effector cells. Also provided are antibodies having such biological activity in vivo and/or in vitro.

In certain embodiments, antibodies of the invention are tested for such biological activity.

T cell activation can be determined using methods known in the art. For example, by the level of cytokines, such as gamma interferon or interleukins, released upon activation of the T cells. T cell activation can also be determined using methods known in the art to determine OX40 signaling. In one embodiment, transgenic cells are generated that express human OX40 and a reporter gene comprising an NFkB promoter fused to a reporter gene (e.g., β luciferase). Addition of anti-OX 40 antibody to cells resulted in increased NFkB transcription, which was detected using an assay directed to a reporter gene (e.g., luciferase reporter gene assay).

Cells for use in any of the above in vitro assays include cells or cell lines that naturally express OX40 or that are engineered to express OX 40. Such cells include activated T cells that naturally express OX40, Treg cells, and activated memory T cells. Such cells also include cell lines that express OX40 and cell lines that do not normally express OX40 but have been transfected with a nucleic acid encoding OX 40.

It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to an anti-OX 40 antibody.

It will be appreciated that any of the above assays can be performed using anti-OX 40 antibodies and other therapeutic agents.

Pharmaceutical composition and pharmaceutical preparation

The invention also includes compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising anti-OX 40 antibodies and compositions comprising polynucleotides encoding anti-OX 40 antibodies. In certain embodiments, the compositions comprise one or more antibodies or fragments thereof that bind OX40 or one or more polynucleotides encoding one or more antibodies or fragments thereof that bind OX 40. These compositions may also contain suitable pharmaceutically acceptable carriers such as pharmaceutically acceptable excipients known in the art, including buffers.

Pharmaceutical carriers suitable for use in the present invention may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. For the use of excipients and their use, see also "Handbook of pharmaceutical excipients", fifth edition, r.c. rowe, p.j.seskey and s.c. owen, pharmaceutical press, London, Chicago. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

Pharmaceutical formulations comprising the anti-OX 40 antibodies described herein can be prepared by mixing the anti-OX 40 antibodies of the invention having the desired purity with one or more optional Pharmaceutical carriers (Remington's Pharmaceutical Sciences, 16 th edition, Osol, A. eds. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.

Exemplary lyophilized antibody formulations are described in U.S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulation including histidine-acetate buffer.

The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient as required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other anti-cancer active ingredients, such as chemotherapeutic agents, PD-1 axis binding antagonists (e.g., anti-PD-1 antibodies or anti-PD-L1 antibodies or anti-PD-L2 antibodies), or anti-angiogenic agents (e.g., bevacizumab). The active ingredients are suitably present in combination in an amount effective for the intended use.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

For pharmaceutical formulations comprising the antibodies of the invention, see also those disclosed in WO 2015/153513.

Antibody treatment methods and uses

In one aspect, the invention relates to a method of activating T cells or inducing a T cell-mediated anti-tumor activity or enhancing an immune response in a subject, the method comprising administering to the subject an effective amount of any of the anti-OX 40 antibodies or fragments thereof described herein.

In another aspect, the invention relates to a method of treating cancer in a subject, comprising administering to the subject an effective amount of any of the anti-OX 40 antibodies or fragments thereof described herein. In one embodiment, the cancer is lung cancer (e.g., non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like.

In another aspect, the invention relates to a method of inducing antibody-dependent cell-mediated cytotoxicity in a subject, comprising administering to the subject an effective amount of any of the anti-OX 40 antibodies or fragments thereof described herein.

In another aspect, the invention relates to methods of treating or delaying various cancers, immune-related diseases, and T cell dysfunctional diseases in a subject, comprising administering to the subject an effective amount of any of the anti-OX 40 antibodies or fragments thereof described herein.

In some embodiments, the subject or individual is a mammal, preferably a human.

In other aspects, the invention provides the use of an anti-OX 40 antibody or fragment thereof in the manufacture or preparation of a medicament for the treatment of a related disease or disorder as mentioned above.

In some embodiments, an antibody or antibody fragment of the invention delays onset of a disorder and/or symptoms associated with a disorder.

In some embodiments, examples of cancer further include, but are not limited to, B-cell lymphomas (including low grade/follicular non-Hodgkin's lymphoma (NHL), Small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunocytogenic NHL, high grade lymphoblastic NHL, high grade small non-nucleated NHL, storage disease (bulk disease) NHL, mantle cell lymphoma, AIDS related lymphoma, and Waldenstrom's macroglobulinemia), Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with scarring nevus (phakomatoses), edema (such as associated with brain tumors), B cell proliferative disorders, and Meigs' syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, anterior (front line) low grade NHL, stage III/IV NHL, chemotherapy-resistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, small lymphocytic lymphoma, B cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B cell prolymphocytic lymphoma, immune cell tumor and/or lymphoplasmacytic (lymphoplasmacytic) lymphoma, lymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone (extranodal marginal zone) -MALT lymphoma, nodal marginal zone (nodal marginal zone) lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell myeloma, low grade/follicular lymphoma, intermediate grade/follicular NHL, mantle cell lymphoma, follicular central lymphoma (follicular), intermediate grade diffuse NHL, diffuse large B-cell lymphoma, aggressive (aggressive) NHL (including aggressive frontline NHL and aggressive relapsed NHL), relapsed or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, advanced immunoblastic NHL, advanced lymphoblastic NHL, advanced small anucleate NHL, deposit disease (bulky disease) NHL, Burkitt's (Burkitt) lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, cutaneous lymphoma, anaplastic large cell lymphoma, angiocentral lymphoma.

In some embodiments, examples of cancer further include, but are not limited to, B cell proliferative disorders, which further include, but are not limited to, lymphomas (e.g., B cell non-hodgkin's lymphoma (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, a) follicular Lymphoma, B) Small Non-nucleated lymphomas (Small Non-cleared Cell Lymphoma)/Burkitt's (Burkitt) lymphomas (including endemic Burkitt's Lymphoma, sporadic Burkitt's Lymphoma, and Non-Burkitt's Lymphoma), c) marginal zone lymphomas (including extranodal marginal zone B-Cell Lymphoma (mucosa-associated lymphoid tissue Lymphoma, MALT), nodal marginal zone B-Cell Lymphoma, and splenic marginal zone Lymphoma), d) Mantle Cell Lymphoma (MCL), e) large Cell Lymphoma (including B-Cell Diffuse Large Cell Lymphoma (DLCL), diffuse mixed Cell Lymphoma, immunoblastic Lymphoma, primary mediastinal B Cell Lymphoma, angiocentric Lymphoma-lung B Cell Lymphoma), f) hairy Cell leukemia, g) lymphocytic lymphoma, waldenstrom's macroglobulinemia, h) Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma, and/or j) hodgkin's disease.

In some embodiments of any of the methods, the cancer is a B cell proliferative disorder. In some embodiments, the B cell proliferative disorder is lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed indolent NHL, refractory indolent NHL, Chronic Lymphocytic Leukemia (CLL), small lymphocytic lymphoma, leukemia, Hairy Cell Leukemia (HCL), Acute Lymphocytic Leukemia (ALL), or mantle cell lymphoma. In some embodiments, the B cell proliferative disorder is NHL, such as indolent NHL and/or aggressive NHL. In some embodiments, the B cell proliferative disorder is indolent follicular lymphoma or diffuse large B cell lymphoma.

In some embodiments of any of the methods of the invention, administration of an anti-OX 40 antibody or fragment thereof having agonist activity of the invention is combined with administration of a tumor antigen. In some embodiments, the tumor antigen comprises a protein. In some embodiments, the tumor antigen comprises a nucleic acid. In some embodiments, the tumor antigen is a tumor cell.

In some embodiments of any of the methods of the invention, the cancer is indicative of (e.g., is infiltrated by) human effector cells. Methods for detecting human effector cells are well known in the art and include, for example, by IHC. In some embodiments, the cancer exhibits high levels of human effector cells. In some embodiments, the human effector cell is one or more of an NK cell, a macrophage, a monocyte. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.

In some embodiments of any of the methods of the invention, the cancer displays FcR expressing cells (e.g., is infiltrated by FcR expressing cells). Methods for detecting FcR are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits high levels of FcR expressing cells. In some embodiments, the FcR is an Fc γ R. In some embodiments, the FcR is an activating Fc γ R. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.

In certain embodiments, the methods and uses described herein further comprise administering to the individual an effective amount of at least one additional therapeutic agent. The antibodies of the invention may be used alone or in combination with other therapeutic agents in therapy. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent.

Such combination therapies encompass combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case administration of the antibody of the invention can occur prior to, concurrently with, and/or after administration of the other therapeutic agent and/or agents. In one embodiment, the administration of the anti-OX 40 antibody and the administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about 1, 2, 3,4, 5, or 6 days of each other. The antibodies of the invention may also be used in combination with radiotherapy.

In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with a chemotherapeutic or chemotherapeutic agent. In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with a radiation or radiation therapy agent. In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with a targeted therapy or targeted therapeutic. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with an immunotherapy or immunotherapeutic, such as a monoclonal antibody.

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention may be administered in combination with PARP inhibitors (e.g., olaparib, Rucaparib, Niraparib, Cediranib, BMN673, Veliparib), Trabectedin, nab-paclitaxel (albumin-binding Palitazone, ABRAXANE), Trebananib, Pazopandanib, Cediranib, Palbociclib, everolimus, fluorouracil (e.g., FOLFOX, FOLFIRI), IFL, regorafenib, Reolysin, alima, Zykadia, Sutent, Touris (Touris) (neomycin), Inlyta (axitinib, Pfiver), Aeitor (everolimus, Novatis), Neraflavab (bovine CSF), Baysin (Baysin/Bayside), Vibrionid, Vezocine, Velcrotinib, Victore, Velcroide, or Abiramide, Ab, Abiramide, Ab, Abiramide, Ab, Abiramide, Ab, Abiramide, Ab.

In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with a PD-1 axis binding antagonist. PD-1 axis binding antagonists include, but are not limited to, PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. Alternative names for "PD-1" include CD279 and SLEB 2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD 273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PD-L1 and/or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding its binding partner. In a particular aspect, the PD-L1 binding partner is PD-1 and/or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits PD-L2 from binding its binding partner. In a particular aspect, the PD-L2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of: MDX-1106(nivolumab, OPDIVO), Merck3475(MK-3475, pembrolizumab, KEYTRUDA) and CT-011 (Pidilizumab). In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 binding antagonist is selected from the group consisting of: YW243.55.S70, MPDL3280A, MEDI4736 and MDX-1105. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO 2007/005874. Antibody YW243.55.S70 (heavy and light chain variable region sequences shown in SEQ ID Nos. 20 and 21, respectively) is anti-PD-L1 as described in WO 2010/077634A 1. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558 or nivolumab, is an anti-PD-1 antibody described in WO 2006/121168. Merck3475, also known as MK-3475, SCH-900475 or pembrolizumab, is an anti-PD-1 antibody described in WO 2009/114335. CT-011, also known as hBAT, hBAT-1 or pidilizumab, is an anti-PD-1 antibody described in WO 2009/101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO 201I/066342. In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternative names for "MDX-1106" include MDX-1106-04, ONO-4538, BMS-936558, or nivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registry number 946414-94-4).

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with an agonist against an activating costimulatory molecule. In some embodiments, the activating costimulatory molecule can include CD40, CD226, CD28, GITR, CD137, CD27, HVEM, or CD 127. In some embodiments, the agonist to the activating co-stimulatory molecule is an agonistic antibody that binds CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD 127. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with an antagonist against an inhibitory co-stimulatory molecule. In some embodiments, the inhibitory co-stimulatory molecule may comprise CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase. In some embodiments, the antagonist against an inhibitory co-stimulatory molecule is an antagonistic antibody that binds CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3 (e.g., LAG-3-IgG fusion protein (IMP321)), B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with a therapy comprising adoptive transfer of T cells (e.g., cytotoxic T cells or CTLs) expressing a Chimeric Antigen Receptor (CAR).

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can inhibit angiogenesisThe formulations are administered in combination. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with an antibody directed against VEGF, e.g., VEGF-a. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be conjugated to bevacizumab (also known as bevacizumab)

Genentech). In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with an antibody against angiopoietin 2 (also known as Ang 2). In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with MEDI 3617. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with an antibody directed against VEGFR 2. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with ramucirumab. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with a VEGF receptor fusion protein. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with an aflibercept. In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be conjugated to ziv-aflibercept (also known as VEGF trap or VEGF trap)

) The administration is combined. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with a bispecific antibody against VEGF and Ang 2. In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with RG7221 (also known as vanucizumab).

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with an angiogenesis inhibitor and in combination with a PD-1 axis binding antagonist (e.g., a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonist such as an anti-PD-L1 antibody, and a PD-L2 binding antagonist such as an anti-PD-L2 antibody). In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with bevacizumab and a PD-1 axis binding antagonist (e.g., a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonist such as an anti-PD-L1 antibody, and a PD-L2 binding antagonist such as an anti-PD-L2 antibody). In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with bevacizumab and MDX-1106(nivolumab, OPDIVO). In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with bevacizumab and Merck3475(MK-3475, pembrolizumab, KEYTRUDA). In some embodiments, the anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with bevacizumab and CT-011 (Pidilizumab). In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with bevacizumab and yw243.55.s 70. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with bevacizumab and MPDL 3280A. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with bevacizumab and MEDI 4736. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with bevacizumab and MDX-1105.

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with an anti-neoplastic agent. In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with an inhibitor of Bruton's Tyrosine Kinase (BTK).

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with obinutuzumab and a PD-1 axis binding antagonist (e.g., a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonist such as an anti-PD-L1 antibody, and a PD-L2 binding antagonist such as an anti-PD-L2 antibody).

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with a cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the peptide Cancer vaccine is a multivalent long peptide, a multiple peptide, a mixture of peptides, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al, Cancer Sci, 104: 14-21, 2013).

In some embodiments, the anti-OX 40 antibodies of the inventionOr a fragment thereof, may be administered in combination with an adjuvant. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be conjugated to a composition comprising a TLR agonist, e.g., Poly-ICLC (also known as

) Treatment with LPS, MPL, or CpG ODN is administered in combination.

In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with Tumor Necrosis Factor (TNF) alpha. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with an interleukin (e.g., IL-1, IL-10, IL-4, IL-13, IL-17, etc.). In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with a therapy targeting CXCL 10. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with a therapy targeting CCL 5. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with an LFA-1 or ICAMl agonist. In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with a selectin agonist.

In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with an inhibitor of B-Raf.

In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with an inhibitor of B-Raf (e.g., vemurafenib or dabrafenib) and an inhibitor of MEK (e.g., MEK1 and/or MEK2) (e.g., cobimetinib or trametinb).

In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with an inhibitor of c-Met.

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with an agent that selectively degrades estrogen receptors.

In some embodiments, an anti-OX 40 antibody or fragment thereof of the invention can be administered in combination with radiation therapy.

In some embodiments, the anti-OX 40 antibodies or fragments thereof of the invention can be administered in combination with an oncolytic virus.

Further therapies or drugs or active ingredients that can be combined with the anti-OX 40 antibodies or fragments thereof of the invention are disclosed in WO 2015/153513.

Such combination therapies noted above encompass both combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case administration of the antibody of the invention can occur prior to, concurrently with, and/or after administration of the other therapeutic agent and/or adjuvant. The antibodies of the invention may also be used in combination with radiotherapy.

The antibodies of the invention (and any additional therapeutic agent) may be administered by any suitable method, including parenteral, intrapulmonary and intranasal administration, and, if required for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, for example intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, a single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (either alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-OX 40 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of OX40 in a biological sample. The term "detecting" as used herein includes quantitative or qualitative detection. In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample comprises a cell or tissue.

In one embodiment, anti-OX 40 antibodies are provided for use in a diagnostic or detection method. In another aspect, methods of detecting the presence of OX40 in a biological sample are provided. In certain embodiments, the methods comprise detecting the presence of an OX40 protein in the biological sample. In certain embodiments, OX40 is human OX 40. In certain embodiments, the methods comprise contacting the biological sample with an anti-OX 40 antibody as described herein under conditions that allow binding of the anti-OX 40 antibody to OX40, and detecting whether a complex is formed between the anti-OX 40 antibody and OX 40. The method may be an in vitro or in vivo method. In one embodiment, the anti-OX 40 antibody is used to select subjects eligible for treatment with the anti-OX 40 antibody, e.g., where OX40 is a biomarker for selecting patients.

In one embodiment, the antibodies of the invention can be used to diagnose cancer or tumors.

In certain embodiments, labeled anti-OX 40 antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties that are detected indirectly, such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H and 131I, fluorophores such as rare earth chelates or luciferin and derivatives thereof, rhodamine and derivatives thereof, dansyl (dansyl), umbelliferone (umbelliferone), luciferase (luceriferase), e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2, 3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, beta-galactosidase, glucoamylase, lytic enzymes, carbohydrate oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes utilizing a hydrogen peroxide oxidation dye precursor such as HR, lactoperoxidase, or microperoxidase, biotin/avidin, spin labeling, phage labeling, stable free radicals, and the like.

In one aspect, the invention provides diagnostic methods, e.g., for identifying cancer patients likely to respond to treatment with an anti-OX 40 antibody.

In some embodiments, methods are provided for identifying a patient likely to respond to anti-OX 40 antibody treatment or for diagnosing cancer, the method comprising (i) determining the presence or absence or amount (e.g., number per given sample size) of FcR expressing cells in a cancer sample from the patient, (ii) identifying the patient as likely to respond if the sample comprises FcR expressing cells (e.g., a high number of FcR expressing cells), or diagnosing the patient as having an FcR (e.g., a high FcR) containing cancer. Methods for detecting FcR expressing cells are well known in the art and include, for example, IHC. In some embodiments, the FcR is an Fc γ R. In some embodiments, the FcR is an activating Fc γ R. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma. In some embodiments, the method is an in vitro method.

In some embodiments, methods are provided for identifying a patient likely to respond to treatment with an anti-OX 40 antibody or for diagnosing cancer, the method comprising (i) determining the presence or absence or amount (e.g., number per given sample size) of human effector cells (e.g., infiltrating effector cells) in a cancer sample from the patient, and (ii) identifying the patient as likely to respond or diagnosing the patient as having cancer comprising human effector cells if the sample comprises effector cells (e.g., a high number of effector cells). Methods for detecting infiltrating human effector cells are well known in the art and include, for example, IHC. In some embodiments, the human effector cell is one or more of an NK cell, a macrophage, a monocyte. In some embodiments, the effector cell expresses an activating Fc γ R. In some embodiments, the method is an in vitro method. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.

In some embodiments, methods of recommending a treatment for a cancer patient are provided that include the steps of the above-described methods for identifying a patient likely to respond to treatment with an anti-OX 40 antibody or methods for diagnosing cancer, and (iii) recommending a treatment with an anti-OX 40 antibody (e.g., an anti-OX 40 antibody of the invention or fragment thereof) when the sample has FcR expressing cells or has human effector cells.

In some embodiments, methods of treating a cancer patient are provided that include the steps of the above-described methods for identifying a patient likely to respond to treatment with an anti-OX 40 antibody or a method for diagnosing cancer, and (iii) treating the patient with an anti-OX 40 antibody (e.g., an anti-OX 40 antibody of the invention or a fragment thereof) when the sample has FcR-expressing cells or has human effector cells.

In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with an anti-OX 40 antibody. In some embodiments, the sample is obtained prior to treatment with the cancer drug. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin fixed, paraffin coated (FFPE). In some embodiments, the sample is a biopsy (e.g., core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.

Sequences of exemplary anti-OX 40 antibodies of the invention

These and other aspects and embodiments of the invention are described in the accompanying drawings (brief description of the drawings follows) and in the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention, however, it is to be understood that the examples are described by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

Examples

Example 1 production and purification of anti-OX 40 antibodies and control antibodies

anti-OX 40 antibodies were identified by Adimab by interrogating the Adimab antibody platform. Libraries and their use in screening processes are described, for example, in Xu et al, 2013; WO 2009036379; WO 2010105256; w02012009568.

The following anti-OX 40 antibodies of the invention were expressed and purified in yeast or CHO-S cells or 293HEK cells:

name of antibody
	ADI-20057
ADI-23504
	ADI-23507
ADI-23509

The following control antibodies used in the examples were expressed and purified in 293HEK cells:

as used herein, pogallizumab is a human IgG1 OX40 antibody transiently expressed in 293HEK cells, using antibodies from the proposed INN: list 114 (see http:// www.who.int/media/publications/pharmaceuticals/drugs/information/inputs/PL114. pdf) of heavy and light chain sequences. As used herein, Hu106-222 is a humanized IgG1 OX40 antibody transiently expressed in 293HEK cells that utilizes heavy and light chain sequences from US 9006399. As used herein, 11D4 is a humanized IgG1 OX40 antibody transiently expressed in 293HEK cells, which utilizes heavy and light chain sequences from US 8236930. As used herein, tavolixizumab is a humanized IgG1 OX40 antibody transiently expressed in 293HEK cells that utilizes light from a proposed INN: list 115 (see http:// www.who.int/media/publications/pharmaceuticals/drugs/information/inputs/PL115. pdf) of heavy and light chain sequences.

For the treatment of yeast material:

yeast clones were grown to saturation and then induced at 30 ℃ for 48 hours with shaking. After induction, yeast cells were pelleted and supernatants harvested for purification. IgG was purified using a protein A column and eluted with acetic acid (pH 2.0). Fab fragments were generated by papain digestion and purified by KappaSelect (GE healthcare Life sciences).

For CHO-S cells treatment:

used according to the manufacturer's instructions

The kit (Invitrogen) generated an expression CHO-S cell line. For mAb expression, the DNA sequences of the heavy and light chains were inserted into the pcho1.0 plasmid, with the heavy chain upstream of the light chain. The full length human OX40CDS sequence (purchased from nano Biological) was inserted into pcho1.0 vector for the generation of stable over-expressed cell lines.

Treatment of 293HEK cells

For transient expression of proteins in 293HEK cells, the vector pV120 was used, in which the heavy and light chains of the antibody were cloned into separate vectors. Transfection into 293HEK cells was performed with PEI using standard procedures; supernatants were collected after 7 days of culture and purified on AKTA system (GE).

Example 2: binding kinetics and affinity assays for anti-OX 40 antibodies

The kinetics and equilibrium dissociation constant (KD) for binding of the antibodies of the invention to human OX40 were determined using MSD-SET and a biophotonic interferometry (ForteBio) assay.

ForteBio KD assay (Bio light interferometry)

ForteBio affinity assays were performed according to the current protocol (Estep, P et al, High throughput solution Based measurement of antibody-antibody affinity and affinity binding. MAbs, 2013.5 (2): p.270-8). Briefly, the sensor was equilibrated in assay buffer for 30 minutes under the line, then the baseline was established by on-line detection for 60 seconds, and ForteBio affinity measurements were performed on an AHQ sensor (ForteBio) on-line loading of purified antibody obtained as described above. The sensor with the loaded antibody was exposed to 100nM of OX40 antigen for an additional 5 minutes before the sensor was transferred to assay buffer for dissociation for 5 minutes for dissociation rate measurements. Kinetic analysis was performed using a 1: 1 binding model.

In experiments performed as described in the assays above, ADI-20057, ADI-23504, ADI-23507 and ADI-23509 (Fab of anti-OX 40 antibody in the form of IgG1 expressed in yeast) were tested in the two-digit nanomolar to single-digit nanomolar range with monovalent K_DBinding to human OX40_ Fc (human OX40 bound to the Fc part of an antibody, purchased from R)&D Systems). ADI-20057, ADI-23504, ADI-23507, and ADI-23509 (anti-OX 40 antibody in IgG1 form and expressed in yeast) bind K in the single-digit nanomolar to sub-nanomolar range in bivalency when the antibody is on the sensor tip_DValues bind to human OX40_ Fc (from R)&D Systems); ADI-20057, ADI-23504, ADI-23507 and ADI-23509 (in IgG1 form and expressed in yeast) bound to cynomolgus monkey OX40_ Fc (purchased from Acro Biosystems) at single digit nanomolar levels. ADI-20057, ADI-23504, ADI-23507, and ADI-23509 (anti-OX 40 antibody expressed in yeast in IgG1 form) did not bind mouse OX40_ Fc (purchased from Acro Biosystems) when the antibody was on the sensor tip (Table 7).

Table 7: measurement of binding of an antibody of the invention in the form of IgG1 by Bio-optical interferometry

MSD-SET kinetic detection

Equilibrium affinity assays are described in Estep, p. et al, MAbs, 2013.5 (2): p.270-8. Solution Equilibrium Titration (SET) was performed in PBS + 0.1% IgG-free BSA (PBSF), where the antigen (biotin-OX-40 monomer (biotinylated OX-40, from Acro Biosystems) was kept constant at 10-100pM and incubated with 3 to 5-fold serial dilutions of Fab or mAb starting at 5-100nM (experimental conditions were sample dependent). antibody diluted at 20nM in PBS was coated at 4 ℃ or overnight at room temperature in standard PBSBound to MSD-ECL plates (Multi-array 96-well plates, cat # L15XA-3, https:// www.mesoscale.com/en/products/115xa-3/) for 30 min. The plates were blocked with BSA for 30 min while shaking at 700 rpm. The plate was then washed 3 times with wash buffer (PBSF + 0.05% Tween 20). SET samples were applied and shaken at 700rpm, incubated on the plate for 150s, and then washed once. Antigen captured on the plate was detected by incubation on the plate with 250ng/mL sulfo-tagged streptavidin in PBSF for 3 minutes. Plates were washed three times with wash Buffer and then Read on an MSD Sector Imager 2400 instrument using a1 × Read Buffer T with surfactant (cat # R92TC-1 https:// www.mesoscale.com/en/products/R92tc-1 /). The percentage of free antigen was plotted in Prism as a function of titrated antibody and a quadratic equation was fitted to extract K_D. To improve throughput, a liquid handling robot was used throughout the MSD-SET experiment, including SET sample preparation.

In experiments performed as described for the above assays, the Fab forms of ADI-23504 and ADI-23507, in IgG1 form and expressed in yeast, were at detectable sub-nanomolar monovalent K_DValues bound to human OX40 (table 8).

Table 8: MSD binding of an antibody of the invention in the form of IgG1

Example 3: binding of anti-OX 40 antibodies of the invention to human OX40

Binding of the antibodies of the invention to human OX40 can be measured in a flow cytometry-based assay.

1. Binding to human OX40 on CHO cells

CHO cells overexpressing human OX40 (CHO-hOX40 cells) were generated by transfection of pCHO1.0 vector (Invitrogen) with human OX40cDNA (Sino Biological, HG10481-G) cloned into MCS. CHO-hOX40 cells (0.2X 10)⁶Individual cells) were incubated with 100nM of the experimental antibody in PBS 0.1% BSA on ice for 30 minutes. The cells were then washed at least twice and with secondary antibody (PE-labeled, SouthemBiotech, final concentration)5 μ g/ml) were incubated in PBS 0.1% BSA on ice (protected from light) for 30 min. Cells were washed at least twice and analyzed by flow cytometry. Flow cytometry was performed on the Canto II system (BD Biosciences) and MFI calculated accordingly.

In experiments performed as described in the assay above, ADI-20057, ADI-23504, ADI-23507, and ADI-23509(IgGl form, expressed in yeast) bind to OX40 overexpressed on CHO cells with MFI values > 1000-fold difference compared to stained wild-type CHO cells (Table 9).

Table 9: flow cytometry assay of binding of IgGl forms produced in Yeast antibodies of the invention to CHO cells expressing human OX40

In experiments performed as described in the test methods above, ADI-20057 in the form of IgG1 expressed in 293HEK cells bound to OX40 overexpressed on CHO cells with EC50 values of 2.873nM (table 10).

Table 10: flow cytometry binding of antibodies of the invention in the form of IgG1 produced by 293HEK cells to human OX40 expressing CHO cells

IgG1 comprises SEQ ID NO: 41 and the heavy chain of SEQ ID NO: 43, as for the IgG1 control in the following.

In experiments performed as described in the assay above, ADI-20057 in the form of IgG2 expressed in 293HEK cells bound to OX40 overexpressed on CHO cells with EC50 values of 5.187nM (table 11).

Table 11: flow cytometry binding of antibodies of the invention in the form of IgG2 produced by 293HEK cells to human OX40 expressing CHO cells

IgG2 comprises SEQ ID NO: 44 and the heavy chain shown in SEQ ID NO: 45, as was the case for the IgG2 control in the following.

2. Binding to human OX40 on 293HEK cells

Binding of the antibodies of the invention to human OX40 can be measured in a flow cytometry assay. 293HEK cells overexpressing human OX40 (0.2X 10)⁶Prepared in a similar manner as described above for CHO cells) were incubated with 100nM of the experimental antibody in PBS 0.1% BSA on ice for 30 minutes. The cells were then washed at least twice and incubated with a secondary antibody (PE-labeled, southern biotech, final concentration 5 μ g/ml) in PBS 0.1% BSA on ice (protected from light) for 30 minutes. Cells were washed at least twice and analyzed by flow cytometry. Flow cytometry was performed on the Accuri C6 system (BD Biosciences) and MFI was calculated accordingly.

In experiments performed as described in the assay above, ADI-20057(IgG1 format, expressed in yeast) binds OX40 strongly at concentrations of 200nM, 40nM, 8nM and 1.6nM, compared to the weak or non-binding of the negative control IgG1 control (Table 12).

Table 12: antibodies of the invention in the form of IgG1 produced in yeast cells by flow cytometry bind to 293HEK cells expressing human OX40

3. Binding to human OX40 on activated primary CD4+ T cells

Binding of the antibodies of the invention to human OX40 on primary T cells can be measured in a flow cytometry assay.

Primary CD4+ T cells from healthy donors were activated with anti-CD 3/CD28 DynaBeads (Invitrogen) for 48 hours and 0.2X 10 cells were plated⁶Individual cells were incubated with 100nM of the experimental antibody in PBS 0.1% BSA on ice for 30 minutes. The cells were then washed at least twice and incubated with a secondary antibody (PE-labeled, southern Biotech, final concentration 5. mu.g/ml) in PBS 0.1% BSA on ice (protected from light) for 30 minA clock. Cells were washed at least twice and analyzed by flow cytometry. Flow cytometry was performed on the Accuri C6 system (BD Biosciences) and MFI was calculated accordingly.

In experiments performed as described in the assay above, ADI-20057(IgG1 format, expressed in yeast) binds OX40 with a high MFI signal compared to the negative control IgG1 control (table 13).

Table 13: binding of antibodies of the invention in the form of IgG1 produced in yeast cells to activated CD4+ T cells (MFI) as determined by flow cytometry

Example 4 blocking of human OX40L interaction with OX40 by antibodies of the invention

The ability of an antibody of the invention to block the binding of human OX40 to OX40L can be measured by flow cytometry.

The method comprises the following steps: human OX 40-expressing CHO cells prepared as described in example 3 above were 0.2X 10⁶One was incubated with the experimental antibody (100nM) in PBS 0.1% BSA on ice for 30 min. The cells were then washed 3 times and incubated with OX40L-hFc (obtained from Acro biosystems) (. about.10. mu.g/ml) linked to NHS-fluorescein (Promega) for 30 min in PBS 0.1% BSA on ice (protected from light). Cells were washed 3 times. Flow cytometry was performed on an Accuri C6 system (BD Biosciences) and MFI was calculated on C6 software.

In experiments performed as described in the assay above, ADI-20057 (a form of IgG1 expressed in yeast) did not block human OX40L-FITC from binding to CHO cells expressing human OX40 (prepared as described in example 3), as the MFI values were similar to those of the control IgG1 control (table 14).

Table 14: flow cytometry-determined blockade of binding of OX40L to OX40 expressed on CHO cells by antibodies of the invention in the form of IgG1 produced in yeast cells

The method 2 comprises the following steps: OX40L fused to murine Fc (OX40L-mFc was obtained from Acro biosystems) was also used, followed by an anti-murine FC-FITC secondary antibody (Biolegend) using a staining procedure as described in method 1. Flow cytometry was performed on the Accuri C6 system (BD Biosciences) and MFI was calculated on the C6 software.

In experiments performed as described in the assay above, ADI-20057-g2 (antibody in IgG2 form and expressed in CHO cells) also did not block binding of human OX40L (fused to mouse Fc (60 μ g/ml)) to OX40 on CHO cells, compared to control pogalizumab and OX40L (figure 1). As can be seen from the figure, the ADI-20057-g2 antibody had a similar effect as control IgG4 on the binding of OX40 to OX 40L. Wherein the control IgG4 comprises SEQ ID NO: 42 and the heavy chain of SEQ ID NO: 43, the same as above.

Example 5 agonist Activity of anti-OX 40 antibodies of the invention

1. Plate-bound antibody T cell activation assay

T cell isolation was performed according to the manufacturer's instructions in the unouched CD4+ T cell isolation kit (Invitrogen). A magnet fitted with a 1.5ml tube rack was used to remove unwanted magnetic beads (QIAGEN).

Agonist activity of the anti-OX 40 antibodies of the invention can be assessed by measuring inflammatory cytokines released by T cells following T cell activation. The test antibody was coated on a 96-well flat-bottom plate (Corning) with suboptimal anti-CD 3 (0.25. mu.g/ml) antibody (Biolegend) and anti-OX 40 (6. mu.g/ml) at 37 ℃ for 2 hours or 4 ℃ overnight. After washing, 100,000 CD4+ primary T cells were added to each well of a total of 200. mu.l of medium containing 2. mu.g/ml anti-CD 28 antibody (Biolegend) in solution. After 5 days, the level of IL-2 secretion was tested by ELISA (Ready-SET-Go!; eBioscience).

In experiments performed as described in the assay above, ADI-20057 in the form of IgG1, produced in yeast cells, increased IL-2 and IFNg secretion over IgG4 control, with IL-2 levels up to 100 fold higher and IFNg levels up to 3 fold different in T cells from two different healthy donors (table 15).

Table 15: t cell activated secretion of IL-2 and IFNg with suboptimal anti-CD 3 activation plus the IgG1 form of the antibody ADI-20057 of the invention produced in yeast cells

2. Soluble antibody T cell activation assay

Test 1:

agonist activity of the anti-OX 40 antibodies of the invention can be assessed by measuring inflammatory cytokines released by T cells following T cell activation. IL-2 secretion was tested by ELISA (Ready-SET-Go!; eBioscience) by stimulating 100,000 CD4+ T cells with PHA (10. mu.g/ml, Sigma) and 200nM of a candidate anti-OX 40 antibody for 5 days.

In experiments performed as described in the assay above, ADI-20057 in the form of IgG1 produced in yeast cells increased IL-2 secretion over IgG4 control, with IL-2 levels increased up to 4-fold in T cells from six different healthy donors (Table 16).

Table 16: IL-2 secretion upon PHA activation plus T cell activation of the antibody of the invention in the form of IgG1 produced in yeast cells

And (3) testing 2:

agonist activity of the anti-OX 40 antibodies of the invention can be assessed by measuring inflammatory cytokines released by T cells upon T cell activation. IL-2 secretion was tested by ELISA (Ready-SET-Go!; eBioscience) by stimulating 100,000 CD4+ T cells for 5 days with anti-CD 3 antibody (Biolegend) (concentration 1. mu.g/ml), anti-CD 28 antibody (Biolegend) (2. mu.g/ml) and anti-OX 40 antibody at 10. mu.g/ml, 20. mu.g/ml or 40. mu.g/ml.

In experiments performed as described in the test methods above, concentrations of 10ug/ml, 20ug/ml and 40ug/ml of HEK293 cells expressed anti-OX 40 antibody ADI-20057 in the form of IgG1 increased IL-2 secretion over the IgG1 control (Table 17), as well as all concentrations of HEK293 cells expressed anti-OX 40 antibody ADI20057 in the form of IgG2 (Table 18).

Table 17: IL-2 secretion upon T cell activation with anti-CD 3, anti-CD 28 antibodies in solution plus antibodies of the invention in the form of IgG1 produced by HEK293 cells, ADI-20057

Table 18: IL-2 secretion upon T cell activation with anti-CD 3, anti-CD 28 antibodies in solution plus antibodies of the invention in the form of IgG2 produced by HEK293 cells, ADI-20057

And (3) testing:

agonist activity of the anti-OX 40 antibodies of the invention can be assessed by measuring inflammatory cytokines released by T cells following T cell activation. Prior to testing for IL-2 secretion by ELISA (Ready-SET-Go!; eBioscience), 100,000 PBMCs were stimulated with PHA (5. mu.g/ml; Sigma) and 5. mu.g/ml, 10. mu.g/ml or 20. mu.g/ml anti-OX 40 antibody for 5 days.

In experiments performed as described in the test methods above, ADI-20057-g1(IgG1 format), ADI-20057-g2(IgG2 format) expressed in CHO cells increased IL-2 secretion at 5. mu.g/ml, 10. mu.g/ml and 20. mu.g/ml over IgG1 control and IgG2 control, above the control antibodies 11D4, Hu106-222 and pogalizumab benchmarks (FIG. 2).

3. Luciferase reporter T cell activation assay

Agonist activity of the anti-OX 40 antibodies of the invention can be assessed by measuring the promotion of NFkB-mediated transcriptional activation in a luciferase reporter assay. Jurkat cells (ATCC USA) overexpressing human OX40 (purchased from Sino) and NFkB-luciferase construct (NFkB promoter-luc, Promega) were activated with PHA5 (5. mu.g/ml; Sigma) or anti-CD 3 (2. mu.g/ml; Biolegend) plus anti-CD 28 (2. mu.g/ml; Biolegend) and anti-OX 40 antibody in solution (100nM) for 18 hours and then tested on a detection device (Molecular Devices) after cell lysis and addition of substrate and bioluminescence measurements, indicating relative luciferase expression induction.

In experiments performed as described in the assay above, ADI-20057 in the form of IgG1 produced in yeast cells increased luciferase expression greater than the IgG4 control, with signals up to about a 3.79-fold increase and about a 4.27-fold increase in activation using PHA (Sigma) or anti-CD 3(Biolegend), respectively (Table 19).

Table 19: luciferase reporter gene assay in Jurkat stably transfected with human OX40 and NFkB promoter-luc by stimulation with PHA or anti-CD 3 plus anti-CD 28 plus antibodies of the IgG1 format of the invention produced in yeast cells

In experiments performed as described in the assay above, Raji cells (ATCC) were further added to provide co-stimulatory signals and FcgRIIB for IgG cross-linking, ADI-20057-g1 is human IgG1 OX40 antibody, ADI-20057-g2 is human IgG2OX40 antibody, both expressed in CHO cells, whose luciferase expression increased by OX40 activation was greater than IgG controls (IgG1 control and IgG2 control), with EC50 values of 0.09635nM and 0.5674nM, respectively (FIG. 3).

In experiments performed essentially as described in this assay, human IgG1 OX40 antibody, ADI-23504-g1, produced in CHO cells, was an affinity matured form of the human IgG1 OX40 antibody, ADI-20057-g1, produced in CHO cells, which exhibited better agonist activity compared to IgG1 controls, with EC50 values of 1.265nM and 0.8231nM, respectively (FIG. 4).

4. Mixed Lymphocyte Reaction (MLR)/DC T cell co-culture assay

Agonist activity of the antibodies of the invention against OX40 signaling can be assessed by measuring the release of IL-2 during a mixed lymphocyte reaction or a DC-T cell co-culture assay. 2 x 10 of⁶PBMC were plated in each well of 6-well tissue culture plates or T25 tissue cultureAnd (5) culturing in bottles. Cells were incubated for 2-3 hours to allow attachment of monocytes. Monocytes (1X 10) from PBMC were cultured in X-VIVO 15 medium containing 1% AB serum, 10mM HEPES, 50. mu.M beta-Me, IL-4(1000U/ml) and GM-CSF (1000U/ml), or 25-50ng/ml of each of the above components⁶Individual cells/ml) to produce immature bone marrow modcs. After 2 days, fresh medium supplemented with IL-4 and GM-CSF was added. On day 5, cells were frozen or prepared by addition of a solution containing rTNFa (1000U/ml), IL-1b (5ng/ml), IL-6(10ng/ml) and 1. mu.M PGE₂Stimulation mixture of (2) at 3X 10⁵Cell density of individual cells/ml for 2 days induced maturation. T cell isolation was performed according to the manufacturer's instructions in the unouched CD4+ T cell isolation kit (Invitrogen). A magnet fitted with a 1.5ml tube rack was used to remove unwanted magnetic beads (QIAGEN).

100,000-200,000 isolated T cells (from donors) were mixed with 10,000-20,000 allogeneic MODCs (as above) in a total volume of 200 μ l in 96-round bottom tissue culture plates for 4-5 days at 37 ℃. SEE (1ng/ml) was added to increase T cell activation. Test antibody was added at the beginning of the MLR and incubated throughout the culture. Detection of IL-2 was performed according to the manufacturer's instructions (eBioscience). OD measurements were determined on Multiskan FC system (Thermo).

In experiments performed as described in the assay above, human antibody ADI-20057-g1 in the form of IgG1 expressed in CHO cells and human IgG2 antibody ADI-20057-g2 expressed in CHO cells increased IL-2 secretion above that of pogalizumab, 11D4, Hu106-222 and tavolixizumab when used at concentrations of-1.48 ug/ml, -4.4 ug/ml, -13.3 ug/ml and-40 ug/ml. Pogallizumab had poor agonist activity at all concentrations (fig. 5, where IgG control was IgGl control).

Example 6 detection of OX40L mediated T cell activation of OX40 by antibodies of the invention based on luciferase reporter genes

The blocking activity of the anti-OX 40 antibodies of the invention can be assessed by measuring the ability of the antibodies to block OX 40L-mediated OX40 activation of T cells assessed using NFkB-mediated transcriptional activity (see example 5). anti-CD 3 (2. mu.g/ml; Biolegend), anti-CD 28 (2. mu.g/ml; Biolegend) and recombinant OX40L (60. mu.g/ml; Acro Biosystems) were used, together with increasing concentrations of anti-OX 40 antibody (ADI-20057-G1 expressed in CHO cells of the IgG1 form of the invention, ADI-20057-G2 expressed in CHO cells of the IgG2 form of the invention) and IgG4 controls, Pogalizumab, OX40L (obtained from Acro Biosystems), in solution for 18 hours, to activate Jurkat cells (American ATCC) overexpressing human OX40 and NFkB-luciferase constructs (NFkB promoter-luc, Promega, USA), followed by detection and absorbance measurements after cell lysis and addition of substrate.

Pogalizumab readily blocked OX 40L-based T cell activation at concentrations greater than about 0.08nM or higher, whereas human OX40 antibody ADI-20057-g1 in the form of IgG1 produced in CHO cells and human OX40 antibody ADI-20057-g2 in the form of IgG2 produced in CHO cells did not only not block OX40 binding to OX40L, but also increased OX 40L-based T cell activation and thus enhanced OX 40L-based T cell activation of OX40 (compared to the negative control IgG4) (fig. 6).

Example 7 antibody dependent cell mediated cytotoxicity of antibodies of the invention

Luciferase reporter-based antibody-dependent cell-mediated cytotoxicity assays

Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the OX40 antibody can be measured using the luciferase reporter gene assay (Promega). Target cells (human OX40 expressing CHO cells (prepared as described above)) were plated at 1.2X 10⁶Each cell/ml was inoculated in 25. mu.l RPMI medium (Gibco) containing 10% Ultra-Low IgG FBS (Sigma). A1: 3 serial dilution of 1. mu.g/ml antibody was prepared and 25. mu.l/well was added. 25 ul of 6X 10 inoculum⁶Cells/ml ADCC Effector cells (Promega) and 5% CO at 37 ℃₂Incubate for 6 hours. Add 75. mu.l luciferase assay reagent (Promega) and bring the mixture to room temperature for 20 min. Plates were centrifuged at 300g/min for 2 minutes. 120 μ l of cell supernatant was carefully transferred to Optiplates. The plate was read in a luminometer. Curves were fitted and EC50 of antibody responses were determined using GraphPad Prism 6.0.

In experiments performed as described in the assay above, ADI-20057 in the form of IgG1 expressed in HEK293 cells increased luciferase expression, supporting ADCC activity (figure 7). ADI-20057 of the IgG2 isotype expressed in HEK293 cells did not elicit ADCC activity (FIG. 8).

Example 8 anti-tumor Activity of antibodies of the invention

The anti-tumor efficacy of the OX40 antibodies of the invention can be studied in a humanized mouse model (NOG hu-PBMC LoVo tumor model).

LoVo human colon carcinoma cells (ATCC # CCL-229) were cultured according to ATCC instructions (F-12K). 200 million LoVo cells suspended in 0.2ml PBS containing 66 million individual PBMCs were implanted into the right flank of female NOG (laboratory animal technologies, Inc., Viton, Beijing).

Tumors and body weights were measured twice weekly throughout the study, mice were euthanized when tumors reached the endpoint or when mice had > 20% weight loss. At 3 days post-implantation, mice were randomized into 6-7 groups, and the mean tumor volume was estimated with a digital caliper for each group, and the tumor volume (mm) was calculated by the following formula³): each group had a (width) of 2X a length/2 of about 50mm³。

Mice were dosed Intraperitoneally (IP) with PBS, 10mg/kg related IgG isotype control (equitech-Bio) or anti-OX 40 antibody (human OX40 antibody ADI-20057-g1 in the form of IgG1 expressed in CHO cells or human OX40 antibody ADI-20057-g2 in the form of IgG2 expressed in CHO cells) on days 3, 7, 11 and 14 (or 15) post-implantation.

In experiments performed as described above for the assay, tumor volume growth was reduced in human OX40 antibody ADI-20057-g1 in the form of IgG1 expressed in CHO cells compared to an IgG control (equitech-Bio) (fig. 9), and compared to pogulizumab and 11D4 (fig. 11). In experiments performed as described in the assay above, human OX40 antibody ADI-20057-g2 in the form of IgG2 expressed in CHO cells significantly reduced tumor volume growth compared to IgG controls (equitech-Bio) (fig. 10), pogalizumab and 11D4 (fig. 11). ADI-20057-g1 and ADI-20057-g2 did not significantly affect mouse body weight compared to IgG controls, pogalizumab and 11D4 (FIG. 12).

SEQUENCE LISTING

<110> Xinda biopharmaceuticals (Suzhou) Limited

Zengji Renwei

H. Baruhe

Liu Xiao Lin

<120> anti-OX 40 antibodies and uses thereof

<130> PF 170116CNI

<140> CN 201710185399.9

<141> 2017-03-25

<160> 46

<170> PatentIn version 3.2

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

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Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

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Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Glu Gly Ser Thr Arg Tyr Ala Gln Lys Phe

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Gln Gly 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

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Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

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Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Val Ile Asn Pro Ser Gly Gly Asp Thr Ser Tyr Ala Gln Lys Phe

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Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

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Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Leu Glu Tyr

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Val Ile Asn Pro Ser Gly Gly Ser Thr Ile Tyr Ala Gln Lys Phe

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Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

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Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

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

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Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

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

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Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

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

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Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Asn Tyr Pro Tyr

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Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

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Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu

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

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Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

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Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

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Gln Gly 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

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Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

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Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

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Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

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Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

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Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

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Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

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Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys

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Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

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

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

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Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

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Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

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

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

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

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Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

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Leu Ser Pro Gly Lys

450

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

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Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

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Gln Gly 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 Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys

210 215 220

Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 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 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Thr 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 Met 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

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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 Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Asn Tyr Pro Tyr

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe Pro 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> 35

<211> 453

<212> PRT

<213> Artificial sequence

<400> 35

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 Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Glu Gly Ser Thr Arg Tyr Ala Gln Lys Phe

50 55 60

Gln Gly 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 Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

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

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

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

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

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

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 36

<211> 448

<212> PRT

<213> Artificial sequence

<400> 36

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 Asn Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Glu Gly Ser Thr Arg Tyr Ala Gln Lys Phe

50 55 60

Gln Gly 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 Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys

210 215 220

Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 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 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Thr 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 Met 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> 37

<211> 453

<212> PRT

<213> Artificial sequence

<400> 37

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

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Gly Gly Asp Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly 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 Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

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

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

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

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

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

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 38

<211> 448

<212> PRT

<213> Artificial sequence

<400> 38

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

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Gly Gly Asp Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly 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 Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys

210 215 220

Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 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 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Thr 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 Met 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> 39

<211> 453

<212> PRT

<213> Artificial sequence

<400> 39

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 Leu Glu Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Gly Gly Ser Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Gly 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 Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

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

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

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

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

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

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 40

<211> 448

<212> PRT

<213> Artificial sequence

<400> 40

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 Leu Glu Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Ser Gly Gly Ser Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Gly 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 Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys

210 215 220

Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 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 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Thr 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 Met 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> 41

<211> 468

<212> PRT

<213> Artificial sequence

<400> 41

Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg

1 5 10 15

Val Leu Ser Glu Val Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asn Tyr Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Thr Ser Arg Asp Asp Ser Lys Asn

85 90 95

Ala Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gly Gly Pro Gly Trp Tyr Ala Ala Asp Val Trp

115 120 125

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

195 200 205

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

210 215 220

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser

225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

245 250 255

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

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

305 310 315 320

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

370 375 380

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

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

405 410 415

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

420 425 430

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

435 440 445

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

450 455 460

Ser Pro Gly Lys

465

<210> 42

<211> 465

<212> PRT

<213> Artificial sequence

<400> 42

Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg

1 5 10 15

Val Leu Ser Glu Val Arg Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asn Tyr Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Thr Ser Arg Asp Asp Ser Lys Asn

85 90 95

Ala Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gly Gly Pro Gly Trp Tyr Ala Ala Asp Val Trp

115 120 125

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

195 200 205

Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

210 215 220

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

225 230 235 240

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

245 250 255

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

260 265 270

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

275 280 285

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

290 295 300

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

305 310 315 320

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

325 330 335

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

340 345 350

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

355 360 365

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

370 375 380

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

385 390 395 400

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

405 410 415

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

420 425 430

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

435 440 445

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

450 455 460

Lys

465

<210> 43

<211> 236

<212> PRT

<213> Artificial sequence

<400> 43

Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ala Asp Leu Pro Ala Phe Ala Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 44

<211> 463

<212> PRT

<213> Artificial sequence

<400> 44

Met Gly Trp Ser Leu Ile Leu Leu Phe Leu Val Ala Val Ala Thr Arg

1 5 10 15

Val Leu Ser Gln Leu Gln Leu Gln Lys Ser Gly Pro Gly Leu Val Lys

20 25 30

Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Thr Phe

35 40 45

Ser Asn Tyr Ala Met Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu

50 55 60

Glu Trp Ile Gly Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn

85 90 95

Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gly Gly Pro Gly Trp Tyr Ala Ala Asp Val Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

130 135 140

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

195 200 205

Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp

210 215 220

His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys

225 230 235 240

Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

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

305 310 315 320

Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

<210> 45

<211> 236

<212> PRT

<213> Artificial sequence

<400> 45

Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

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

20 25 30

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

35 40 45

Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln

50 55 60

Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Ile

65 70 75 80

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln

100 105 110

Ala Asp Leu Pro Ala Phe Ala Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 46

<211> 20

<212> PRT

<213> Artificial sequence

<400> 46

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

20

Claims

1. An anti-OX 40 antibody or antigen-binding fragment thereof comprising a heavy chain variable region HCVR and a light chain variable region LCVR, wherein the HCVR comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 and the LCVR comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:8, respectively; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, respectively.

2. An anti-OX 40 antibody or antigen-binding fragment thereof comprising a heavy chain variable region HCVR and a light chain variable region LCVR, wherein the HCVR comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 and the LCVR comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8, respectively; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, respectively.

3. An anti-OX 40 antibody or antigen-binding fragment thereof comprising a heavy chain variable region HCVR and a light chain variable region LCVR, wherein the HCVR comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 and the LCVR comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are SEQ ID NO:1, SEQ ID NO:6 and SEQ ID NO:8, respectively; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, respectively.

4. An anti-OX 40 antibody or antigen-binding fragment thereof comprising a heavy chain variable region HCVR and a light chain variable region LCVR, wherein the HCVR comprises complementarity determining regions HCDR1, HCDR2 and HCDR3 and the LCVR comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:8, respectively; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, respectively.

5. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising a heavy chain variable region HCVR comprising or consisting of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an amino acid sequence selected from SEQ ID NOs 16, 17, 18, and 19.

6. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising or consisting of a Light Chain Variable Region (LCVR) comprising or consisting of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the amino acid sequence set forth in SEQ ID NO 21.

7. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising a heavy chain variable region HCVR and a light chain variable region LCVR, wherein the HCVR comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs 16, 17, 18, or 19 and the LCVR comprises or consists of an amino acid sequence as set forth in SEQ ID NOs 21.

8. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain variable region HCVR comprises or consists of the amino acid sequence set forth in SEQ ID NO 16; the light chain variable region LCVR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21.

9. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain variable region HCVR comprises or consists of the amino acid sequence set forth in SEQ ID NO. 17; the light chain variable region LCVR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21.

10. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain variable region HCVR comprises or consists of the amino acid sequence set forth in SEQ ID NO 18; the light chain variable region LCVR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21.

11. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 7, wherein the heavy chain variable region HCVR comprises or consists of the amino acid sequence set forth in SEQ ID NO 19; the light chain variable region LCVR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21.

12. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-4 or claims 8-11, comprising a heavy chain, wherein the heavy chain comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to an amino acid sequence selected from SEQ ID NOs 32, 33, 35, 36, 37, 38, 39, or 40.

13. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-4 or claims 8-11, comprising a light chain, wherein the light chain comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to the amino acid sequence set forth in SEQ ID No. 34.

14. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-4 or claims 8-11, comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs 32, 33, 35, 36, 37, 38, 39, or 40; the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 34.

15. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 14, wherein the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO 32; the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 34.

16. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 14, wherein the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO. 33; the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 34.

17. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 14, wherein the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO 35; the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 34.

18. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 14, wherein the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO 36; the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 34.

19. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 14, wherein the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO 37; the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 34.

20. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 14, wherein the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO 38; the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 34.

21. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 14, wherein the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO 39; the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 34.

22. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 14, wherein the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO 40; the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO. 34.

23. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-4 or claims 8-11 or claims 15-22, wherein the antibody is an antibody or antigen-binding fragment of the IgG1 format or the IgG2 format or the IgG4 format.

24. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-4 or claims 8-11 or claims 15-22, wherein the antibody is a monoclonal antibody.

25. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-4 or claims 8-11 or claims 15-22, wherein the antibody is a humanized or human antibody.

26. The antibody or antigen-binding fragment thereof of any one of claims 1-4 or claims 8-11 or claims 15-22, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of: fab, Fab '-SH, Fv, scFv or (Fab')₂And (4) fragment.

27. An isolated nucleic acid encoding the anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-26.

28. A vector comprising the nucleic acid of claim 27.

29. The vector of claim 28, wherein the vector is an expression vector.

30. A host cell comprising the nucleic acid of claim 27 or the vector of claim 28 or 29.

31. The host cell of claim 30, wherein the host cell is prokaryotic or eukaryotic.

32. The host cell of claim 31, which is selected from a yeast cell, a mammalian cell, or other cell suitable for use in the production of an antibody or antigen-binding fragment thereof.

33. A method of making an anti-OX 40 antibody or antigen-binding fragment thereof, the method comprising culturing the host cell of any one of claims 30-32 under conditions suitable for expression of a nucleic acid encoding the anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-26.

34. The method of claim 33, wherein the method further comprises isolating the antibody or antigen-binding fragment thereof.

35. The method of claim 33 or 34, wherein the method further comprises recovering the anti-OX 40 antibody or antigen-binding fragment thereof from the host cell.

36. A pharmaceutical composition comprising the anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-26.

37. The pharmaceutical composition of claim 36, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

38. An immunoconjugate comprising the anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1 to 26 and a cytotoxic agent.

39. Use of an effective amount of the anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-26 or the immunoconjugate of claim 38 in the preparation of a pharmaceutical composition for activating T cells or inducing T cell-mediated anti-tumor activity or enhancing an immune response of a body in a subject.

40. Use of an effective amount of an anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-26 or the immunoconjugate of claim 38 in the manufacture of a pharmaceutical composition for treating cancer in a subject.

41. The use of claim 40, wherein the cancer is lung cancer, liver cancer, stomach cancer, or colon cancer.

42. The use of claim 41, wherein the lung cancer is non-small cell lung cancer.

43. The use of any one of claims 39 to 42, wherein the pharmaceutical composition is administered in combination with an effective amount of a second drug or active agent.

44. The use of claim 43, wherein the second drug or active agent is a chemotherapeutic agent.

45. The use of claim 43, wherein the second drug or active agent is selected from a PD-1 axis binding antagonist or an anti-angiogenic agent.

46. The use of claim 43, wherein the second medicament or active agent is selected from an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody or bevacizumab.