EA040561B1 - METHOD OF APPLICATION OF ANTIBODY TO OX40 (VERSIONS) - Google Patents

Description

Link to related applications

This application claims priority under U.S. Provisional Application Nos. 62/333556, 62/327140, 62/264691, 62/239574 and 62/168377, filed May 9, 2016, April 25, 2016, December 8 2015, October 9, 2015 and May 29, 2015, respectively. The content of the above application is incorporated herein by reference.

Sequence listing

This application contains a sequence listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The specified ASCII copy, created on May 26, 2016, is named MXI_543PC_Sequence_Listing.txt and is 417528 bytes in size.

Background of the Invention

OX40 (TNFRSF4), also known as ACT35, IMD16, TXGP1L, and CD134, is a 50 kD type I transmembrane glycoprotein in the TNFSFR family of costimulatory receptors expressed on activated CD4+ T cells. In the context of cancer, OX40-expressing activated T cells are found in tumor-infiltrating lymphocytes. OX40 and its ligand OX40-L play a critical role in inducing and maintaining T cell responses. Recent studies have demonstrated the utility of enhancing antitumor T cell function to fight cancer, with key components of an effective response including activation of CD4+ T cells and promotion of survival signals through memory T cells and effector T cells. Given the continuing need for improved strategies for treating diseases such as cancer, for example by enhancing immune responses such as T cell responses, new agents that modulate T cell responses such as those that target OX40, and treatments (eg, combination therapies) that use such agents would be of therapeutic value.

Brief summary of the invention

Provided herein are antibodies, such as human monoclonal antibodies, that specifically bind to OX40 and have desirable functional properties. These properties include high binding affinity for human OX40 and cynomolgus monkey OX40 and the ability to stimulate antigen-specific T cell responses, for example, in tumor-bearing subjects.

In some embodiments, anti-OX40 antibodies or antigen-binding portions thereof, as described herein, stimulate an antitumor immune response, such as an antigen-specific T cell response. In some embodiments, the antibodies, or antigen-binding portions thereof, increase the production of cytokines (eg, IL-2 and/or IFN-γ) in OX40-expressing T cells and/or increase T cell proliferation. In some embodiments, the antibodies bind to the C1q component of the human complement. In some embodiments, the antibodies induce NK-mediated lysis of activated CD4+ T cells. In some embodiments, the antibody promotes macrophage-mediated phagocytosis of OX40-expressing cells. In some embodiments, the antibody inhibits regulatory T-cell mediated suppression of CD4+ T cell proliferation.

In some embodiments, the anti-OX40 antibodies, or antigen-binding portions thereof, bind to Fc receptors, such as one or more activating FcyRs. In some embodiments, the antibodies, or antigen-binding portions thereof, induce or enhance T cell activation via multivalent cross-linking.

Provided herein are isolated monoclonal antibodies, or antigen-binding portions thereof, that specifically bind to OX40 and contain three variable heavy chain CDRs and three variable light chain CDRs that are in variable heavy chain and variable light chain pairs selected from SEQ ID NO. : 318 and 94. Also provided herein are antibodies or antigen-binding portions thereof that bind to OX40 and contain the heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 87, 317, and 89, respectively, and the sequences of CDR1, CDR2, and CDR3 light chain containing SEQ ID NO: 90-92, respectively.

Provided herein are monoclonal antibodies, or antigen-binding portions thereof, that bind to OX40 and comprise heavy and light chain variable regions, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 318. Provided herein are isolated monoclonal antibodies, or antigen-binding portions thereof, which bind to OX40 and contain the variable regions of the heavy and light chains, and the variable region of the light chain contains the amino acid sequence of SEQ ID NO: 94. Provided herein are isolated monoclonal antibodies or antigen-binding portions thereof that bind to OX40 and contain sequences of the variable region of heavy 1 040561 of the gel and light chains SEQ ID NOs: 318 and 94. Provided herein are isolated monoclonal antibodies, or antigen-binding portions thereof, that bind to OX40 and contain the heavy chain and light chain sequences of SEQ ID NO: 124 and 116. Provided herein is a method for stimulating an antigen-specific T cell response, comprising contacting a T cell with an anti-OX40 antibody, or antigen-binding portion thereof, such that an antigen-specific T cell response is stimulated.

Provided herein is a method for activating or co-stimulating a T cell, such as an effector T cell, comprising contacting the cell, such as an effector T cell, with an anti-OX40 antibody or antigen-binding portion thereof and CD3, wherein the effector T cell is activated or co-stimulated.

Provided herein is a method of reducing or reducing the number of regulatory T cells in a tumor of a subject in need thereof, comprising administering an effective amount of an anti-OX40 antibody or antigen-binding portion, bispecific molecule or conjugate, wherein the antibody or antigen-binding portion is characterized by an effector or enhanced effector function, to reduce the number of regulatory T cells in the tumor.

Provided herein is a method of stimulating an immune response in a subject, comprising administering an effective amount of an anti-OX40 antibody or antigen-binding portion, bispecific molecule, or conjugate to the subject such that an immune response is stimulated in the subject. In some embodiments, the subject is characterized by the presence of a tumor and is stimulated to have an immune response against the tumor.

Provided herein is a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject an anti-OX40 antibody, or an antigen-binding portion thereof, a bispecific molecule, or a conjugate, such that tumor growth is inhibited in the subject.

In one embodiment, the method comprises further administering an anti-PD-1 antibody comprising the CDR1, CDR2 and CDR3 domains of a heavy chain variable region characterized by the sequence shown in SEQ ID NO: 301 and the CDR1, CDR2 and CDR3 domains of a light chain variable region characterized by the sequence shown in SEQ ID NO: 302, and the method involves at least one cycle of administration, and the cycle is a period of two, three or four weeks, and for each at least one cycle one dose of antibody to OX40 is administered at a dose of 1 mg/kg body weight; fixed dose of 20, 40, 80, 160 or 320 mg; dose of about 1 mg/kg body weight or a fixed dose of about 20, 40, 80, 160 or 320 mg, and a single dose of anti-PD-1 antibody is administered at a dose of 240, 360 or 480 mg or a dose of about 240, 360 or 480 mg.

In another embodiment, the method comprises further administering an anti-CTLA-4 antibody comprising the CDR1, CDR2 and CDR3 domains of a heavy chain variable region characterized by the sequence shown in SEQ ID NO: 309 and the CDR1, CDR2 and CDR3 domains of a light chain variable region characterized by the sequence shown in SEQ ID NO: 310, and the method involves at least one cycle of administration, and the cycle is a period of three weeks, and for each of at least one cycle one dose of antibodies to OX40 is administered at a dose of 1 mg/kg body weight; fixed dose of 20, 40, 80, 160 or 320 mg; dose of approximately 1 mg/kg body weight or a fixed dose of approximately 20, 40, 80, 160 or 320 mg, and a single dose of anti-CTLA-4 antibody is administered at a dose of 1 mg/kg or a dose of approximately 1 mg/kg, and the antibody to OX40 co-administered with anti-CTLA-4 antibody for at least one cycle followed by monotherapy with anti-OX40 antibody for at least one cycle. In some embodiments, the treatment consists of 8 cycles. In one embodiment, the anti-OX40 antibody is co-administered with the anti-CTLA-4 antibody for the first 4 cycles followed by anti-OX40 antibody monotherapy for the last 4 cycles.

In some embodiments, the anti-OX40 antibody comprises the heavy and light chain sequences shown in SEQ ID NOs: 124 and 116, respectively.

In some embodiments, the anti-OX40 antibody or anti-OX40 antibody and the anti-PD-1 or anti-CTLA-4 antibody are formulated for intravenous administration. In some embodiments, anti-OX40 and anti-PD-1 or anti-CTLA-4 antibodies are formulated together. In other embodiments, anti-OX40 and anti-PD-1 or anti-CTLA-4 antibodies are formulated separately.

In some embodiments, the treatment consists of 8 cycles. In one embodiment, an anti-OX40 antibody or an anti-OX40 antibody and an anti-PD-1 antibody or an anti-CTLA-4 antibody are administered on the first day of each cycle.

In some embodiments, the anti-OX40 antibody is administered prior to the administration of the anti-PD-1 or anti-CTLA-4 antibody. In one embodiment, the anti-OX40 antibody is administered approximately 30 minutes prior to the administration of the anti-PD-1 antibody or anti-CTLA-4 antibody. In other embodiments, the anti-OX40 antibody is administered following the administration of the anti-PD-1 or anti-CTLA-4 antibody. In other embodiments, the anti-OX40 antibody is administered simultaneously with the anti-PD-1 or anti-CTLA-4 antibody.

Other features and advantages of the present disclosure will be apparent from the following.

- 2 040561 detailed description and examples, which should not be considered as limiting.

Brief disclosure of graphic materials

In FIG. 1A shows the nucleotide sequence (SEQ ID NO: 126) and amino acid sequence (SEQ ID NO: 17) of the heavy chain variable region of the human monoclonal antibody 3F4. The CDR1 (SEQ ID NO: 11), CDR2 (SEQ ID NO: 12) and CDR3 (SEQ ID NO: 13) regions are labeled and germline derivatives V, D and J are indicated;

in fig. 1B - Nucleotide sequence (SEQ ID NO: 127) and amino acid sequence (SEQ ID NO: 18) of the light chain variable region of the human monoclonal antibody 3F4. The CDR1 (SEQ ID NO: 14), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 16) regions are labeled and germline derivatives V and J are indicated;

in fig. 2A shows the nucleotide sequence (SEQ ID NO: 128) and amino acid sequence (SEQ ID NO: 28) of the heavy chain variable region of the human monoclonal antibody 14B6 (14B6-1 and 14B6-2). The CDR1 (SEQ ID NO: 19), CDR2 (SEQ ID NO: 20) and CDR3 (SEQ ID NO: 21) regions are labeled and germline derivatives V, D and J are indicated;

in fig. 2B shows the nucleotide sequence (SEQ ID NO: 129) and amino acid sequence (SEQ ID NO: 29) of the light chain variable region to the human monoclonal antibody 14B6-1. The CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 23), and CDR3 (SEQ ID NO: 24) regions are labeled and germline derivatives V and J are indicated;

in fig. 2C - Nucleotide sequence (SEQ ID NO: 130) and amino acid sequence (SEQ ID NO: 30) of the light chain variable region of the human monoclonal antibody 14B6-2. The CDR1 (SEQ ID NO: 26), CDR2 (SEQ ID NO: 27), and CDR3 (SEQ ID NO: 28) regions are labeled and germline derivatives V and J are indicated;

in fig. 3A shows the nucleotide sequence (SEQ ID NO: 131) and amino acid sequence (SEQ ID NO: 37) of the heavy chain variable region of the human monoclonal antibody 23H3. The CDR1 (SEQ ID NO: 31), CDR2 (SEQ ID NO: 32) and CDR3 (SEQ ID NO: 33) regions are labeled, and germline derivatives V, D and J;

in fig. 3B - Nucleotide sequence (SEQ ID NO: 132) and amino acid sequence (SEQ ID NO: 38) of the light chain variable region of the human monoclonal antibody 23H3. The CDR1 (SEQ ID NO: 34), CDR2 (SEQ ID NO: 35), and CDR3 (SEQ ID NO: 36) regions are labeled and germline derivatives V and J are indicated;

in fig. 4A shows the nucleotide sequence (SEQ ID NO: 133) and amino acid sequence (SEQ ID NO: 48) of the heavy chain variable region of the human monoclonal antibody 6E1 (6E1-1 and 6E12). The CDR1 (SEQ ID NO: 39), CDR2 (SEQ ID NO: 40) and CDR3 (SEQ ID NO: 41) regions are labeled and germline derivatives V, D and J are indicated;

in fig. 4B shows the nucleotide sequence (SEQ ID NO: 134) and amino acid sequence (SEQ ID NO: 49) of the light chain variable region of the human monoclonal antibody 6E1-1. The CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 43), and CDR3 (SEQ ID NO: 44) regions are labeled, and germline derivatives V and J;

in fig. 4C - Nucleotide sequence (SEQ ID NO: 135) and amino acid sequence (SEQ ID NO: 50) of the light chain variable region of the human monoclonal antibody 6E1-2. The CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 46), and CDR3 (SEQ ID NO: 47) regions are labeled and germline derivatives V and J are indicated;

in fig. 5A shows the nucleotide sequence (SEQ ID NO: 136) and amino acid sequence (SEQ ID NO: 57) of the heavy chain variable region of the human monoclonal antibody 18E9. The CDR1 (SEQ ID NO: 51), CDR2 (SEQ ID NO: 52) and CDR3 (SEQ ID NO: 53) regions are labeled and germline derivatives V, D and J are indicated;

in fig. 5B shows the nucleotide sequence (SEQ ID NO: 137) and amino acid sequence (SEQ ID NO: 58) of the light chain variable region of the human monoclonal antibody 18E9. The CDR1 (SEQ ID NO: 54), CDR2 (SEQ ID NO: 55) and CDR3 (SEQ ID NO: 56) regions are labeled and germline derivatives V and J are indicated;

in fig. 6A shows the nucleotide sequence (SEQ ID NO: 138) and amino acid sequence (SEQ ID NO: 65) of the heavy chain variable region of the human monoclonal antibody 8B11. The CDR1 (SEQ ID NO: 59), CDR2 (SEQ ID NO: 60) and CDR3 (SEQ ID NO: 61) regions are labeled and germline derivatives V, D and J are indicated;

in fig. 6B shows the nucleotide sequence (SEQ ID NO: 139) and amino acid sequence (SEQ ID NO: 66) of the light chain variable region of the human monoclonal antibody 8B11. The CDR1 (SEQ ID NO: 62), CDR2 (SEQ ID NO: 63), and CDR3 (SEQ ID NO: 64) regions are labeled and germline derivatives V and J are indicated;

in fig. 7A shows the nucleotide sequence (SEQ ID NO: 140) and amino acid sequence (SEQ ID NO: 73) of the heavy chain variable region of the human monoclonal antibody 20B3. The CDR1 (SEQ ID NO: 67), CDR2 (SEQ ID NO: 68), and CDR3 (SEQ ID NO: 69) regions are designated and indicated

- 3 040561 germline derivatives V, D and J;

in fig. 7B shows the nucleotide sequence (SEQ ID NO: 141) and amino acid sequence (SEQ ID NO: 74) of the light chain variable region of the human monoclonal antibody 20B3.

The CDR1 (SEQ ID NO: 70), CDR2 (SEQ ID NO: 71), and CDR3 (SEQ ID NO: 72) regions are labeled and germline derivatives V and J are indicated;

in fig. 8A shows the nucleotide sequence (SEQ ID NO: 142) and amino acid sequence (SEQ ID NO: 84) of the heavy chain variable region of the human monoclonal antibody 14A2 (14A2-1 and 14A2-2). The CDR1 (SEQ ID NO: 75), CDR2 (SEQ ID NO: 76) and CDR3 (SEQ ID NO: 77) regions are labeled and germline derivatives V, D and J are indicated;

in fig. 8B shows the nucleotide sequence (SEQ ID NO: 143) and amino acid sequence (SEQ ID NO: 85) of the light chain variable region of the human monoclonal antibody 14A2-1. The CDR1 (SEQ ID NO: 78), CDR2 (SEQ ID NO: 79), and CDR3 (SEQ ID NO: 80) regions are labeled and germline derivatives V and J are indicated;

in fig. 8C - Nucleotide sequence (SEQ ID NO: 144) and amino acid sequence (SEQ ID NO: 86) of the light chain variable region to human monoclonal antibody 14A2-2. The CDR1 (SEQ ID NO: 81), CDR2 (SEQ ID NO: 82), and CDR3 (SEQ ID NO: 83) regions are labeled and germline derivatives V and J are indicated;

in fig. 9A shows the nucleotide sequence (SEQ ID NO: 145) and amino acid sequence (SEQ ID NO: 93) of the heavy chain variable region of the human monoclonal antibody 20C1. The CDR1 (SEQ ID NO: 87), CDR2 (SEQ ID NO: 88) and CDR3 (SEQ ID NO: 89) regions are labeled and germline derivatives V, D and J are indicated;

in fig. 9B shows the nucleotide sequence (SEQ ID NO: 146) and amino acid sequence (SEQ ID NO: 94) of the light chain variable region of the human monoclonal antibody 20C1. The CDR1 (SEQ ID NO: 90), CDR2 (SEQ ID NO: 91), and CDR3 (SEQ ID NO: 92) regions are labeled and germline derivatives V and J are indicated;

in fig. 10A shows the nucleotide sequence (SEQ ID NO: 176) and amino acid sequence (SEQ ID NO: 124) of the human monoclonal antibody OX40.21 heavy chain. The nucleotide sequence (SEQ ID NO: 168) and amino acid sequence (SEQ ID NO: 116) of the light chain are shown in FIG. 10B;

in fig. 11A-11D are FACS binding and EC ₅₀ curves (in nM) of various anti-OX40 antibodies for activated human T cells with hIG1 and secondary antibodies as controls;

in fig. 12A-12C: Binding and EC ₅₀ curves (in nM) of various anti-OX40 antibodies for activated cynomolgus monkey T cells with hIG1 and secondary antibodies as controls, as measured by FACS;

in fig. 13A-13C are binding and K _D curves of anti-OX40 antibody, OX40.21, for activated human T cells, HEK293 cells overexpressing human OX40, and CHO cells overexpressing cynomolgus monkey OX40 as assessed by Scatchard;

in fig. 14A Immunohistological staining of various acetone-fixed frozen tissue sections with antibodies to OX40, OX40.8, OX40.6 and OX40.16. Images show characteristic staining at 1 µg/mL for the hyperplastic tonsil and 5 µg/mL for other tissues except for OX40.16 at 10 µg/mL in the endocardium and valves. While all three antibodies positively stained a small number of lymphocytes in the amygdala, OX40.8 also stained myofilament-like structures in the heart, and OX40.6 stained cardiac muscle, endothelial/subendothelial matrix in small arteries in the tonsils and endocardium, and valves in the heart. GC, germinal center; MZ, mantle zone;

in fig. 14B - Immunostaining of various acetone-fixed frozen human tissue sections with an anti-OX40 antibody, OX40.21 (variant OX40.16). The images show typical staining at an antibody concentration of 5 µg/mL. The antibody positively stained a small subset of lymphocytes in the tonsil and thymus. Positive cells in the amygdala were distributed in the germinal center, mantle zone, and interfollicular region, while thymic positive cells were mainly localized in the medulla. No specific staining was observed in the heart, liver, kidneys and lungs. GC, germinal center; Me, medulla; MZ, mantle zone.

in fig. 15A Distribution of tumor-infiltrating OX40+ lymphocytes in hepatocellular carcinoma (HCC), colorectal carcinoma (CRC), head and neck squamous cell carcinoma (HNSCC), and melanoma (Mel). Manual assessment of 12 to 20 cases for each type of tumor was performed by assessing the number of positive cells under a microscope at 20x magnification. Minimum, <1 cell per 20x objective field; moderate, 1 ~ <10 cells per 20x objective field; average, 10 ~ <50 cells per 20x objective field; noticeable, 50 ~ < 200 cells per 20x objective field; intensive > 200 cells per 20x objective field;

in fig. 15B and 15C - Immunohistological staining for CD3, FoxP3 and OX40 on adjacent

- 4 040561

FFPE sections from colorectal carcinoma (CRC) and head and neck squamous cell carcinoma (HNSCC) specimens. In FIG. 15B is a low magnification view showing that both OX40+ and

FoxP3 + TIL constitute a small fraction of CD3+ TIL and are mainly distributed throughout the tumor stroma.

In FIG. 15C is a higher magnification view showing a potential partial co-localization of TIL OX40+ and FoxP3+ (Treg) in CRC;

in fig. 16 - The ability of various anti-OX40 antibodies to inhibit OX40 ligand (OX40-L) binding to OX40-transfected 293 cells (hOX40-293 cells) with hIG1 as a control;

in fig. 17 summarizes OX40-L blocking bonds between various anti-OX40 antibodies;

in fig. 18 shows the ability of various anti-OX40 antibodies to inhibit the binding of anti-OX40 antibody clone L106 to hOX40-293 cells as assessed by FACS with PE L106 labeled, mIgG1 PE labeled and unstained cells as control only;

in fig. 19A-19C - The ability of various anti-OX40 antibodies to inhibit the binding of allophycocyanin (APC) conjugated OX40.1 antibody to hOX40-293 (FIGS. 19A and 19B) or hOX40-HT1080 (FIGS. 19C) cells as assessed by FACS. hIG1 and/or hIgG4 were used as controls;

in fig. 19D-19G - The ability of various anti-OX40 antibodies to inhibit the binding of biotin-conjugated OX40.4 or OX40.5 antibody to hOX40-293 cells. hIG1 and streptavidin-APC were used as controls;

in fig. 19H epitope groups are summarized with respect to OX40.1 based on the results shown in FIG. 19A-19C;

in fig. 19I summarize epitope groups with respect to OX40.5 or OX40.4 based on the results shown in FIG. 19D-19G;

in fig. 20A shows that OX40.21 only binds unreduced human OX40, regardless of the presence of N-linked sugars;

in fig. 20B and 20C two N-glycopeptides identified by peptide mapping after deglycosylation: 60% occupancy for both AspN118 (FIG. 20B) and AspN12 (FIG. 20C);

in fig. 20D - regions in OX40 associated with OX40.16, OX40.21 and OX40.8;

in fig. 20E - Identification of peptides recognized by OX40.16, OX40.21 and OX40.8 by LC-MS;

in fig. 21A-21D - Effect of various anti-OX40 antibodies on proliferation of primary human CD4+ T cells when co-cultured with CHO-CD3-CD32A cells. CHO cells only, T cells only, CHO cells cultured with T cells only, and hIG1 were used as controls;

in fig. 22A-22D - Effect of various anti-OX40 antibodies on interferon gamma (IFN-γ) secretion from primary human CD4+ T cells co-cultured with CHO-CD3-CD32A cells. CHO cells only, T cells only, CHO cells cultured with T cells only, and hIG1 were used as controls;

in fig. 23A-23F show the effect of various anti-OX40 antibodies on stimulation of IL-2 secretion from primary T cells in cultures of enterotoxin B-activated staphylococcus (SEB) human peripheral blood mononuclear cells (PBMC) isolated from different donors;

in fig. 24 - Effect of various anti-OX40 antibodies on NK92-induced lysis of activated CD4+ cells;

in fig. 25A and 25B show the effect of various anti-OX40 antibodies on primary NK cell-mediated lysis of activated CD4+ T cells isolated from two donors by NK:target cell ratios;

in fig. 26 - Effect of various anti-OX40 antibodies on phagocytosis of hOX40-293 cells by primary human macrophages;

in fig. 27 - the level of binding of the human C1q complement component to OX40.21. IgG1 and IgG1.1 (having no effector functions) were used as controls;

in fig. 28A-28C show that OX40 is expressed on tumor infiltrating lymphocytes, with the profile generally limited to CD4+ cells with minimal expression on CD8+ cells.

in fig. 28D shows that OX40 is expressed by CD4+ T cells and regulatory T cells in Sa1N mouse tumors;

in fig. 28E shows that OX40 is expressed by CD4+ T cells, CD8+ T cells, and regulatory T cells in MC38 mouse tumors;

in fig. 29 shows the ability of various anti-OX40 antibodies to reverse regulatory T cell (Treg)-mediated suppression of human CD4+ T cells. In both the presence and absence of Treg cells, anti-OX40 antibodies increased the proliferation of immunoreactive T cells (Tresp) compared to the IgG1 isotype control;

in fig. 30A and 30B - recovery of intravenous OX40.6 antibody from monkeys. Two of the monkeys showed accelerated clearance that correlated with drug antibody production;

- 5 040561 fig. 31 is a graph showing T cell proliferation results for percent antigenicity for various anti-OX40 antibodies, and quality control samples QC-1, QC-2, and

QC-3;

in fig. 32A-32C - Effect of various isotypes of the chimeric OX86 antibody (an antibody containing rat OX86 variable regions and a mouse constant region that does not block the interaction between OX40 and OX40-L, i.e., a non-blocking antibody) on antitumor activity as measured by changes in tumor volumes in individual mice exposed to these isotypes (mIgG1 and mIgG2 isotypes) in the MC38 colon adenocarcinoma model: (Fig. 32A) control mouse IgG1 antibody (control), (Fig. 32B) mIgG1 antibody OX86 ('OX-40 mIgG1), ( Fig. 32C) mIgG2 OX86 antibody (OX-40 mIgG2a);

in fig. 33A-33C - Effect of different isotypes (mIgGI and mIgG2a) of the OX86 antibody on the number of CD4+ regulatory T cells in tumors and the periphery and on the number of cells in the spleen;

in fig. 34A-34C - Effect of chimeric OX86 antibodies with human IgG1 on antitumor activity measured by changes in tumor volumes in individual mice exposed to these antibodies in the MC38 colon adenocarcinoma model: (Fig. 34A) human IgG1 isotype control (isotype), (Fig. 34B) OX86-hIgG1 chimeric antibody (OX86-hG1), (FIG. 34C) OX86-hIgG1-S267E antibody (OX86-hG1-S267E). The substitution of S267E in hIG1 increases its effector function by increasing binding to FcRs (CD32A and CD32B). OX86-hIgG1 showed strong antitumor activity;

in fig. 35A-35D - Effect of chimeric hIG1 antibody OX86 on depletion of regulatory T cells;

in fig. 36A-36E Effects of blocking (i.e., blocking interaction between OX40 and OX40 ligand) hamster anti-mouse OX40 antibody (8E5) at different doses on antitumor activity by altering tumor volumes in individual mice exposed to said antibodies in a subcutaneous CT tumor model. -26 mice. (Fig. 36A) Hamster Ig control, (Fig. 36B) 8E5 at 10 mg/kg, (Fig. 36C) 8E5 at 3 mg/kg, (Fig. 36D) 8E5 at 1 mg/kg, (Fig. 36E) 8E5 at 0.3 mg/kg;

in fig. 37A-37D - Effect of combination therapy with OX86-rG1 antibody and anti-PD1 antibody on antitumor activity measured by changes in tumor volumes in individual mice exposed to these antibodies and the combination in the MC38 colon adenocarcinoma model: (Fig. 37A) isotype control, (Fig. 37B) anti-PD1 antibody, (Fig. 37C) OX86rG1 (anti-OX40), (Fig. 36D) OX86-rG1 + anti-PD1 antibody (anti-OX40 + anti-PDl);

in fig. 38A and 38B show the ex vivo secondary immune response to KLH, CD69+ expressing CD4+CD4-T cells at 22 and 41 days, respectively. Animals were immunized with KLH on day 1 of the study. Data refer to individual animal results (male and female) as percentages of CD4+CD8- T cells. The horizontal bars represent group averages;

in fig. 39 - antibody binding to Fab to his captured by FcyR-his proteins. Binding responses are shown as a percentage of theoretical Rmax assuming a 1:1 mAb:FcyR binding stoichiometry. The columns for each antibody are shown in the order indicated by the colored legends at the bottom of the slide;

in fig. 40 - antibody binding to Fab to his captured by FcgR-his proteins. Binding responses are shown as a percentage of theoretical Rmax assuming a 1:1 mAb:FcyR binding stoichiometry. The columns for each antibody are in the order indicated by the colored legends at the bottom of the slide;

in fig. 41A-41D - Effect of combination therapy with anti-OX40 agonist antibody (OX86rG1) and anti-CTLA-4 antibody (9D9-mG2b) on antitumor activity measured by changes in tumor volumes in individual mice exposed to these antibodies and the combination in a model of colon adenocarcinoma CT26: (Fig. 41A) isotype control, (Fig. 41B) anti-CTLA-4 antibody, (Fig. 41C) OX86-rG1, (Fig. 41D) OX86-rG1 + anti-CTLA-4 antibody;

fig. 42 is a diagram of the phase 1/2a clinical trial protocol;

fig. 43 is a diagram of the study visit schedule for the phase 1/2a clinical trial.

Detailed description of the invention

The document describes isolated antibodies, in particular monoclonal antibodies, eg human monoclonal antibodies, which bind specifically to OX40 and thus activate the OX40 downstream signaling pathway (OX40 agonist antibodies). In some embodiments, the antibodies described herein are characterized by certain functional features and/or contain certain structural features, such as CDR regions containing specific amino acid sequences. Also provided herein are methods of using antibodies to enhance an immune response, alone or in combination with other immunostimulatory agents (eg, antibodies), and/or for cancer therapy.

Definitions.

To make the description clearer, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

As used herein, the term OX40 refers to a receptor that is a member of the superfamily of TNF receptors that bind to the OX40 ligand (OX40-L). OX40 is also referred to as the tumor necrosis factor receptor superfamily member 4 (TNFRSF4), ACT35, IMD16, TXGP1L and CD134. The term OX40 includes any variants or isoforms of OX40 that are naturally expressed by cells. Accordingly, the antibodies described herein can cross-react with OX40 from non-human species (eg, cynomolgus monkey OX40). Alternatively, the antibodies may be specific for human OX40 and may not show cross-reactivity with other species. OX40 or any of its variants and isoforms can be isolated from cells or tissues that naturally express them or recombinantly obtained using well-known methods in the present field of technology and/or described herein.

The amino acid sequence of human OX40 precursor (accession number NP_003318.1) is shown in SEQ ID NO: 1. The amino acid sequence of the extracellular domain of mature human OX40 is shown in SEQ ID NO: 2. The amino acid sequence of cynomolgus monkey OX40 is shown in SEQ ID NO: 3. Amino acid sequence Human OX40-L is shown in SEQ ID NO: 4.

As used herein, the terms Programmed Death 1, Programmed Cell Death 1, PD-1 protein, PD-1, PD1, PDCD1, hPD-1, and hPD-I are used interchangeably and include variants, isoforms, species homologues of human PD-1 and analogs containing at least one common epitope with PD-1. The complete PD-1 sequence can be found under GenBank Accession U64863.

The terms cytotoxic T-lymphocyte-associated antigen-4, CTLA-4, CTLA4, CTLA-4 antigen, and CD152 (see, e.g., Murata (1999) Am. J. Pathol. 155: 453-460) are used interchangeably and are included in variants, isoforms, species homologues of human CTLA-4, and analogues containing at least one common epitope with CTLA-4 (see, for example, Balzano (1992) Int. J. Cancer Suppl. 7: 28-32). The complete human CTLA-4 sequence is available from GenBank under accession number L1 5006.

As used herein, the term antibody may include whole antibodies and any antigen-binding fragments (ie, antigen-binding portions) or individual chains thereof. In one embodiment, an antibody refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In some naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region consists of three domains: CH1, CH2 and CH3. In some naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a single CL domain. The V _H and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) that alternate with more conserved regions called framework regions (FRs). Each V _H and VL consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

Antibodies typically bind specifically to their cognate antigen with high affinity, as reflected by a dissociation constant (KD) of 10 ^-7 to 10 ^-11 M or less. Generally, a KD greater than about 10 ^-6 M is considered to indicate non-specific binding. As used herein, an antibody that specifically binds to an antigen refers to an antibody that binds to an antigen and substantially identical antigens with high affinity, which means a KD of 10 ^-7 M or less, preferably 10 ^-8 M or less, yet more preferably 5 x 10 ^-9 M or less and most preferably 10 ^-8 to 10 ^-10 M or less, but does not bind with high affinity to unrelated antigens. An antigen is substantially identical to a given antigen if it exhibits a high degree of sequence identity with respect to the given antigen, for example, if it has a sequence identity of at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably at least 99%, relative to the sequence of the given antigen. As an example, an antibody that specifically binds to human OX40 may cross-react with OX40 from some non-human primate species (e.g., cynomolgus monkey) but cannot cross-react with OX40 from other species (e.g., mouse OX40) or with an antigen other than from OX40.

- 7 040561

The immunoglobulin may be of any of the widely known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. The IgG isotype is subclassified in some species: IgG1, IgG2, IgG3 and IgG4 in humans and IgG1, IgG2a, IgG2b and IgG3 in mice. In some embodiments, anti-OX40 antibodies described herein are of the IgG1 or IgG2 subtype. Immunoglobulins, such as IgG1, exist in several allotypes that differ from each other by no more than a few amino acids. An antibody may include, for example, both natural and non-natural antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthetic antibodies and single chain antibodies.

As used herein, the term antigen-binding portion of an antibody refers to one or more antibody fragments that retain the ability to specifically bind to an antigen (eg, human OX40). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Examples of binding fragments included in the term antigen-binding portion of an antibody, such as an anti-OX40 antibody described herein, include (i) a Fab fragment, a monovalent fragment consisting of the V _L , VH, _CL and CH1 domains; (ii) an F(ab') ₂ fragment, a divalent fragment containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the V _L and VH domains of one antibody arm, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546) which consists of a V _H domain, and (vi) a dedicated complementarity determining region (CDR); or (vii) a combination of two or more dedicated CDRs, which may optionally be connected by a synthetic linker. In addition, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, they can be combined, using recombinant methods, with a synthetic linker that allows them to be a single protein chain in which pairs of V _L and V H regions form monovalent molecules. (known as single chain Fvs (scFvs), see e.g. Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) . Such single chain antibodies are also intended to be included in the term antigen-binding portion of an antibody. These and other potential constructs are described in Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are prepared by conventional methods known to those skilled in the art and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be obtained by recombinant DNA methods or by enzymatic or chemical cleavage of intact immunoglobulins.

A bispecific or bifunctional antibody is an artificial hybrid antibody containing two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be made by a variety of methods, including hybridoma fusion or Fab' linkage. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

As used herein, the term monoclonal antibody refers to an antibody that exhibits a single binding specificity and binding affinity for a particular epitope or antibody composition, wherein all antibodies exhibit a single binding specificity and binding affinity for a particular epitope. Typically, such monoclonal antibodies will be derived from a single cell or nucleic acid encoding the antibody and will increase in number without intentionally introducing any sequence changes. Accordingly, the term human monoclonal antibody refers to a monoclonal antibody that contains variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are generated by a hybridoma, e.g., obtained by fusing a B cell derived from transgenic or transchromosomal non-human animals (e.g., a transgenic mouse having a genome containing a human heavy chain transgene and a light chain), with immortalized cell.

As used herein, the term recombinant human antibody includes all human antibodies that are produced, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., mouse) that is transgenic or transchromosomal for human genes. immunoglobulin or a hybridoma derived therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a library of recombinant combinatorial human antibodies, and produced or isolated by any other means that involve splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain variable and constant regions that use specific human germline immunoglobulin sequences, are encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during

- 8 040561 maturation of antibodies. As known in the art (see, for example, Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable region contains an antigen-binding domain that is encoded by various genes that rearrange to form an antibody specific for a foreign antigen. . In addition to the rearrangement, the variable region can be further modified with several single amino acid changes (called a somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant region will change with further antigen response (i.e., isotype switching). Therefore, rearranged and somatically mutated nucleic acid sequences that encode immunoglobulin light chain and heavy chain polypeptides in response to an antigen may not be identical to the original germline sequences, but will instead be substantially identical or similar (i.e., share the same identity, component of at least 80%).

A human antibody (HuMAb) refers to an antibody containing variable regions in which both the framework and the CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation). However, the term human antibody, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mice, have been grafted onto human scaffold sequences. The terms human antibodies and fully human antibodies are used interchangeably.

A humanized antibody refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment, the humanized form of an antibody is an antibody in which some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, while some, most, or all of the amino acids within one or more of the CDR regions are unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are allowed, as long as they do not alter the ability of the antibody to bind to a specific antigen. The humanized antibody retains antigenic specificity similar to that of the original antibody.

A chimeric antibody refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

As used herein, the term isotype refers to a class of antibodies (eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE) that are encoded by heavy chain constant region genes.

An allotype refers to naturally occurring variants within a defined group of isotypes whose variants differ by a few amino acids (see, for example, Jefferis et al. (2009) mAbs 1:1). Antibodies described herein can be of any allotype. As used herein, antibodies designated as IgG1f or IgG1.1f"isotype are IgG1 and effector-free IgG1.1 antibodies, respectively, of allotype G, i.e. characterized by 214R, 356E and 358M according to the EC index as in Kabat, as shown, for example, in SEQ ID NO: 5 (see underlined residues in SEQ ID NO: 5 in Table 23).

The phrases antibody that recognizes an antigen and antibody specific for an antigen are used interchangeably herein with the term antibody that specifically binds to an antigen.

As used herein, the term isolated antibody is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to OX40 is substantially free of antibodies that specifically bind to antigens other than OX40). An isolated antibody that specifically binds to an OX40 epitope may, however, be cross-reactive with other OX40 proteins from different species.

As used herein, an antibody that inhibits the binding of OX40-L to OX40 is intended to refer to an antibody that inhibits the binding of OX40-L to OX40, for example, in binding assays using hOX40-293 cells with an EC50 of about 1 μg/mL or less, about 0.9 µg/mL or less, about 0.85 µg/mL or less, about 0.8 µg/mL or less, about 0.75 µg/mL or less, about 0.7 µg/mL or less, approximately 0.65 µg/mL or less, about 0.6 µg/mL or less, about 0.55 µg/mL or less, about 0.5 µg/mL or less, about 0.45 µg/mL or less, approximately 0.4 µg/mL or less, about 0.35 µg/mL or less, about 0.3 µg/mL or less, about 0.25 µg/mL or less, about 0.2 µg/mL or less, about 0.15

- 9 040561 μg/ml or less, or about 0.1 μg/ml or less, in methods recognized in the art, for example, the FACS-based binding assays described herein.

Effector function refers to the interaction of the Fc region of an antibody with an Fc receptor or ligand, or the biochemical event that results from this. Exemplary effector functions include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcyR mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of a cell surface receptor (e.g., B-cell receptor, BCR). Such effector functions typically require the Fc region to be associated with a binding domain (eg, an antibody variable domain).

An Fc receptor or FcR is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to an IgG antibody contain receptors of the FcyR family, including allelic variants and alternatively spliced forms of these receptors. The FcyR family consists of three activating (FcyRI, FcyRIII and FcyRIV in mice, FcyRIA, FcyRIIA and FcyRIIIA in humans) and one inhibitory (FcyRIIB) receptor. Various properties of human FcyR are presented in table. 1. Most innate effector cell types co-express one or more activating FcyR and inhibitory FcyRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans) but not inhibitory FcyRIIB in mice and humans. . Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a in terms of the types of activating Fc receptors to which it binds.

Table 1. Properties of human FcyR

fcy Allelic variants Affinity for IgG human Isotype preference Cell distribution FcyRI Nothing is described High (Kd ~ 10 nM) IgGl=3>4»2 Monocytes, macrophages, activated neutrophils, dendritic cells? FcyRIIA H131 Low to Medium IgGl>3>2>4 Neutrophils, monocytes, macrophages, eosinophils, dendritic cells, platelets R131 Low IgGl>3>4>2 FcyRIIIA V158 Medium IgGl=3»4>2 NK cells, monocytes, macrophages, mast cells, eosinophils, dendritic cells? F158 Low IgGl=3»4>2 FcyRIIB 1232 Low IgGl=3=4>2 B cells, monocytes, macrophages, dendritic cells, mast cells T232 Low IgGl=3=4>2

The Fc region (crystallizable fragment of an immunoglobulin) or Fc or Fc domain refers to the C-terminal region of an antibody heavy chain that mediates binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or on the first component (C1q) of the classical complement system. Thus, the Fc region contains an antibody constant region that excludes the first immunoglobulin constant region (eg, CH1 or CL). In the IgG, IgA, and IgD isotypes of an antibody, the Fc region contains C _H2 and C _H3 constant domains in each of the two antibody heavy chains; the IgM and IgE Fc regions contain three heavy chain constant domains ( _CH domains 2-4) in each polypeptide chain. For IgG, the Fc region contains the immunoglobulin domains Cy2 and Cy3 and a hinge between Ογ1 and Cy2. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position C226 or P230 (or the amino acid between those two amino acids) to the carboxyl terminus of the heavy chain, with the numbering corresponding to the EC index , as in Kabat. Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD; see also FIG. 3C-3F in US Patent Application Publication No. 2008/0248028. The C _H2 domain of the human Fc region of IgG extends from about amino acid 231 to about amino acid 340, while the C _H3 domain is located on the C-terminal side of the C _H2 domain in the Fc region, i. it extends from about amino acid 341 to about amino acid 447 IgG. As used herein, an Fc region may be a native Fc sequence including any allotype or Fc variant (eg, a non-naturally occurring Fc). The Fc may also refer to this region by itself or in the context of an Fc containing protein polypeptide, such as a binding protein containing an Fc region, also referred to as an Fc fusion protein (eg, an antibody or an immunoadhesin).

The hinge, hinge domain or hinge region or hinge region of an antibody refers to a heavy chain constant region domain that connects the CH1 domain to the CH2 domain and includes the upper, middle, and lower hinge portions (Roux et al., J. Immunol., 1998, 161:4083). The hinge provides various levels of flexibility between the binding and effector regions of the antibody, and also provides sites for intermolecular disulfide binding between the two heavy chain constant regions. As used herein, the hinge starts at Glu216 and ends at Gly237 for all IgG isotypes (Roux et al., 1998 J. Immunol. 161: 4083). IgG1, IgG2, IgG3 and IgG4 wild-type hinge sequences are shown in Table 1. 2 and 23.

Table 2. Amino acids of the hinge region

C-terminal Type 1g C _n 1* Upper hinge Middle hinge Lower hinge IgGl VDKRV EPKSCDKTHT CPPCP APELLGG (SEQID (SEQ ID NO: 188) (SEQ ID NO: 192) (SEQID NO: NO: 186) 200) IgG2 VDKTV ERK CCVECPPCP APPVAG (SEQ ID NO: 193) (SEQID (SEQID NO: NO:187) 201) IgG3 (17-VDKRV ELKTPLGDTTHT CPRCP APELLGG 15-15-15) (SEQ ID (SEQ ID NO: 189) (EPKSCDTPPPCPRCP) ₃ (SEQID NO: NO: 186) (SEQ ID NO: 194) 200) IgG3 (17-VDKRV ELKTPLGDTTHT CPRCP APELLGG 15-15) (SEQ ID (SEQ ID NO: 189) (EPKSCDTPPPCPRCP) ₂ (SEQID NO: NO: 186) (SEQ ID NO: 195) 200) IgG3 (17-VDKRV ELKTPLGDTTHT CPRCP APELLGG 15) (SEQ ID (SEQ ID NO: 189) (EPKSCDTPPPCPRCP)i (SEQID NO: NO: 186) (SEQ ID NO: 196) 200) IgG3 (15-VDKRV EPKS CDTPPPPCPRCP APELLGG 15-15) (SEQ ID (SEQ ID NO: 190) (EPKSCDTPPPCPRCP) ₂ (SEQID NO: NO: 186) (SEQ ID NO: 197) 200) IgG3 (15) VDKRV EPKS CDTPPPPCPRCP APELLGG (SEQID (SEQ ID NO: 190) (SEQ ID NO: 198) (SEQID NO: NO: 186) 200) IgG4 VDKRV ESKYGPP CPSCP APEFLGG (SEQ ID (SEQ ID NO: 191) (SEQ ID NO: 199) (SEQID NO: NO: 186) 200)

* C-terminal amino acid sequences of CH1 domains.

The term hinge includes wild-type hinges (such as those listed in Table 23) as well as variants thereof (eg, non-naturally occurring or modified hinges). For example, the term IgG2 hinge includes wild-type IgG2 hinge, as shown in Table 1. 23, and variants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and/or no more than 5, 4, 3, 2, or 1 mutations, such as substitutions, deletions, or additions. Exemplary IgG2 hinge variants include IgG2 hinges in which 1, 2, 3, or all 4 cysteines (C219, C220, C226, and C229) are replaced with a different amino acid. In a specific embodiment, the IgG2 contains a C219S substitution. In some embodiments, the hinge is a hybrid hinge that contains sequences from at least two isotypes. For example, a hinge may comprise an upper, middle, or lower hinge from one isotype and the remainder of the hinge from one or more other isotypes. For example, the hinge may be an IgG2/IgG1 hinge and may comprise, for example, an IgG2 upper and middle hinge and an IgG1 lower hinge. The hinge may have an effector function or be devoid of an effector function. For example, wild-type IgG1 lower hinge provides effector function.

The term CH1 domain refers to a heavy chain constant region linking a variable domain to a hinge in the heavy chain constant region. As used herein, the CH1 domain starts at A118 and ends at V215. The term CH1 domain includes CH1 domains

- 11 040561 wild type (such as SEQ ID NO: 202 for IgGl and SEQ ID NO: 203 for IgG2, Table 23), as well as variants thereof (eg, non-naturally occurring CH1 domains or modified CH1 domains). For example, the term CH1 domain includes wild-type CH1 domains and variants characterized by having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and/or no more than 5, 4, 3, 2 or 1 mutation, such as substitutions, deletions or additions. Illustrative CH1 domains include CH1 domains with mutations that modify the biological activity of the antibody, such as ADCC, CDC, or half-life. Modifications to the CH1 domain that affect the biological activity of an antibody are provided herein.

The term CH2 domain refers to the heavy chain constant region linking the hinge to the CH3 domain in the heavy chain constant domain. As used herein, the CH2 domain starts at P238 and ends at K340. The term CH2 domain includes wild-type CH2 domains (such as SEQ ID NO: 204 for IgG1 and SEQ ID NO: 205 for IgG2, Table 23) as well as variants thereof (for example, non-naturally occurring CH2 domains or modified CH2 ). For example, the term CH2 domain includes wild-type CH2 domains and variants characterized by having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and/or no more than 5, 4, 3, 2 or 1 mutation, such as substitutions, deletions or additions. Exemplary CH2 domains include CH2 domains with mutations that modify the biological activity of the antibody, such as ADCC, CDC, or half-life. In some embodiments, the CH2 domain contains A330S/P331S substitutions that reduce effector function. Other modifications to the CH2 domain that affect the biological activity of the antibody are provided herein.

The term CH3 domain refers to a heavy chain constant region that is C-terminal to the CH2 domain in the heavy chain constant region. As used herein, the CH3 domain starts at G341 and ends at K447. The term CH3 domain includes wild-type CH3 domains (such as SEQ ID NO: 206 for IgG1 and SEQ ID NO: 207 for IgG2, Table 23) as well as variants thereof (for example, non-naturally occurring CH3 domains or modified CH3 ). For example, the term CH3 domain includes wild-type CH3 domains and variants characterized by having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and/or no more than 5, 4, 3, 2 or 1 mutation, such as substitutions, deletions or additions. Exemplary CH3 domains include CH3 domains with mutations that modify the biological activity of the antibody, such as ADCC, CDC, or half-life. Modifications to the CH3 domain that affect the biological activity of an antibody are provided herein.

The native sequence of the Fc region or the native sequence of Fc contains an amino acid sequence that is identical to the amino acid sequence of the Fc region found in nature. Native sequences of human Fc regions include a native sequence of a human IgG1 Fc region; a native sequence of the human IgG2 Fc region; a native sequence of a human IgG3 Fc region and a native sequence of a human IgG4 Fc region, as well as naturally occurring variants thereof. The native Fc sequence includes various Fc allotypes (see, for example, Jefferis et al. (2009) mAbs 1:1).

The term epitope or antigenic determinant refers to a site on an antigen (eg, OX40) to which an immunoglobulin or antibody specifically binds. Epitopes in protein antigens can be formed from both contiguous amino acids (usually a linear epitope) and from non-contiguous amino acids aligned by the protein's tertiary fold (usually a conformational epitope). Epitopes formed from contiguous amino acids are generally, but not always, retained upon exposure to denaturing solvents, while epitopes formed from tertiary folds are generally lost upon exposure to denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (e.g., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, where overlapping or contiguous peptides (e.g., from OX40) are tested for reactivity with a given an antibody (eg, an anti-OX40 antibody). Methods for determining the spatial conformation of epitopes include methods in the art and described herein, such as X-ray crystallography, 2D nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

The term epitope mapping refers to the process of identifying the molecular determinants of an antigen involved in antibody-antigen recognition.

The term binds to the same epitope in relation to two or more antibodies means that the antibodies bind to the same group of amino acid residues, as determined by this method. Techniques for determining whether antibodies with the same epitope on OX40 bind to the antibodies described herein include epitope mapping techniques known in the art, such as X-ray analysis of crystals of antigen:antibody complexes, providing atomic resolution of epitope and mass -hydrogen spectrometry

- 12 040561 deuterium exchange (HDX-MS). Other methods control antibody binding to fragments of the antigen (eg, proteolytic fragments) or to mutated variations of the antigen, where loss of binding due to modification of an amino acid residue in the antigenic sequence is often considered to be indicative of an epitope component (eg, alanine-scanning mutagenesis - Cunningham & Wells (1985) Science 244:1081). In addition, computational combinatorial methods can also be used to map epitopes. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. It is expected that antibodies containing the same or close VH and VL or the same CDR1, 2 and 3 sequences will bind to the same epitope.

Antibodies that compete with another antibody for binding to a target refer to antibodies that (partially or completely) inhibit the binding of another antibody to a target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody will inhibit the binding of another antibody to a target, can be determined using known competition experiments. In some embodiments, the antibody competes with another antibody and inhibits the binding of the other antibody to the target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition may be different depending on which antibody is the blocking antibody (ie, a cold antibody that is first incubated with the target). Competitive assays can be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101/pdb.prot4277 or Chapter 11 Using Antibodies by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, an overlapping epitope, or a neighboring epitope (eg, as evidenced by steric hindrance).

Other competitive binding assays known in the art include: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), competitive sandwich assay (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeled RIA using label I-125 (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)) and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, the terms specific binding, selective binding, selectively binds, and specifically binds refer to the binding of an antibody to an epitope on a predetermined antigen, but not to other antigens. Typically, antibody (i) binds to an equilibrium dissociation constant (KD) of less than about 10 ^-7 M, such as less than about 10 ^-8 , 10 ^-9 , or 10 ^-10 M, or even lower, as defined, for example, by surface plasmon resonance (SPR) technology in the BIACORE 2000 Surface Plasmon Resonance (SPR) instrument using a predetermined antigen, such as recombinant human OX40, as the analyte and an antibody as the ligand, and (ii) binds to the target antigen with an affinity that at least twice its binding affinity for a non-specific antigen (eg BSA, casein) other than a predetermined antigen or a closely related antigen. Accordingly, an antibody that specifically binds to human OX40 refers to an antibody that binds to soluble or cell-bound human OX40 with a KD of 10 ^-7 M or less, such as less than about 10 ^-8 , 10 ^{-9 ,} or 10 ^-10 M or even lower. An antibody that cross-reacts with cynomolgus monkey OX40 refers to an antibody that binds to cynomolgus monkey OX40 with a K _D of 10 ^-7 M or less, such as less than about 10 ^-8 , 10 ^-9 or 10 ^-10 M or even lower. In some embodiments, antibodies that do not cross-react with OX40 from non-human species (eg, mouse OX40) exhibit virtually undetectable binding to these proteins in standard binding assays.

As used herein, the term _kassoc or _ka is intended to refer to the association rate constant for a particular antibody-antigen interaction, while as used herein, the term kd _is or kd is intended to refer to the dissociation rate constant of a particular antibody-antigen interaction. As used herein, the term KD is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of kd to _ka (ie kd/ka) and is expressed as a molar concentration (M). Antibody KD values can be determined using methods well known in the art. The preferred method for determining the KD of an antibody is using surface plasmon resonance, preferably using a biosensor system such as the Biacore® SPR system or flow cytometry and Scatchard assay.

As used herein, the term high affinity for an IgG antibody refers to an antibody having a K _D of 10 ^-8 M or less, more preferably 10 -9 M or less, and even

- 13 040561 more preferably 10-10 M or less for the target antigen. However, high affinity binding may vary for other antibody isotypes. For example, high affinity binding for an IgM isotype refers to an antibody having a K _D of 10 ^-7 M or less, more preferably ^10'8 M or less.

The term EC50 in the context of an in vitro or in vivo assay using an antibody or antigen-binding fragment thereof refers to the concentration of the antibody, or antigen-binding portion thereof, that induces a response that is 50% of the maximum response, i.e. is in the middle between the maximum response and the baseline.

The term binds to immobilized OX40 refers to the ability of an antibody described herein to bind to OX40, eg expressed on a cell surface or attached to a solid support.

As used herein, the term cross-reactivity refers to the ability of an antibody described herein to bind to OX40 from another species. For example, an antibody described herein that binds to human OX40 can also bind to OX40 from another species (eg, cynomolgus monkey OX40). As used herein, cross-reactivity can be measured by detecting specific reactivity with purified antigen in binding assays (eg, SPR, ELISA) or binding to, or otherwise functionally interacting with, cells that physiologically express OX40. Methods for determining cross-reactivity include standard binding assays as described herein, for example, using a BIACORE® surface plasmon resonance (SPR) assay using a BIACORE® 2000 SPR instrument (Biacore AB, Uppsala, Sweden) or flow cytometric technique .

The term naturally occurring, as used herein to apply to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses), that can be isolated from a source in nature, and that has not been intentionally modified by humans in a laboratory, is a naturally occurring one.

A polypeptide refers to a chain containing at least two consecutively linked amino acid residues with no upper chain length limit. One or more amino acid residues in a protein may contain a modification such as, but not limited to, glycosylation, phosphorylation, or a disulfide bond. A protein may contain one or more polypeptides.

As used herein, the term nucleic acid molecule is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single or double stranded and may be cDNA.

Conservative modifications of the sequences presented herein are also contemplated, for example, in Table. 23, that is, modifications to the nucleotide and amino acid sequences that do not abolish the binding of an antibody encoded by a nucleotide sequence or containing an amino acid sequence to an antigen. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as additions and deletions of nucleotides and amino acids. For example, modifications can be introduced to the sequence in Table. 23 by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions in which an amino acid residue is replaced with an amino acid residue containing a similar side chain. Families of amino acid residues containing similar side chains have been defined in the present technical field. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine , phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in an anti-OX40 antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999) and Burks et al Proc Natl Acad Sci USA 94:412-417 (1997)). Alternatively, in another embodiment, mutations can be introduced randomly across all or part of the coding sequence of an anti-OX40 antibody, eg, by saturation mutagenesis, and the resulting modified anti-OX40 antibodies can be screened for improved binding activity.

For nucleic acids, the term significant homology indicates that two nucleic acids

- 14 040561 acids or their designated sequences, when optimally aligned and compared, are identical with the corresponding nucleotide insertions or deletions by at least about 80% of nucleotides, typically at least 90 to 95% and more preferably at least 98 to 99 .5% nucleotides. Alternatively, significant homology exists when the segments will hybridize under selective hybridization conditions with a complementary strand.

For polypeptides, the term significant homology indicates that two polypeptides or their designated sequences, when optimally aligned and compared, are identical with the corresponding amino acid insertions or deletions in at least about 80% amino acids, typically at least about 90 to 95%, and more preferably at least 98 to 99.5% amino acids.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions/total positions x 100), with the optimal alignment determined by the number of gaps and the length of each spaces that must be entered to optimally align the two sequences. Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm as described in the non-limiting examples below.

Percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com) using the NWSgapdna.CMP matrix and a gap opening penalty of 40, 50, 60, 70 or 80 and a gap lengthening penalty of 1, 2, 3, 4, 5, or 6. Percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989 )) that was included in the ALIGN program (version 2.0) using the PAM120 weight substitution table of residues, gap extension penalty of 12, and sequence gap penalty of 4. In addition, percent identity between two amino acid sequences can be determined using algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)), which was included in the GAP program in the GCG software package (available at http://www.gcg.co m) using either a Blossum 62 die or a PAM250 die and a gap open penalty of 16, 14, 12, 10, 8, 6, or 4 and a gap extension penalty of 1, 2, 3, 4.5, or 6.

The nucleic acid and protein sequences described herein can also be used as a query sequence to perform open database searches, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST (version 2.0) programs of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. Protein BLAST searches can be performed using the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules described herein. The Gapped BLAST program can be used to generate gapped alignments for comparison purposes as described in Altschul et al., (1997) Nucleic. Acids. Res. 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default settings for the respective programs (eg XBLAST and NBLAST) may apply. See www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or in partially purified or substantially pure form. Nucleic acid is isolated or rendered substantially pure when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other parts of the chromosome) or proteins, by standard techniques including alkaline/SDS exposure, CsCl banding, column chromatography , agarose gel electrophoresis, and others well known in the art. See F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

Nucleic acids, such as cDNA, can be mutated according to standard techniques to provide gene sequences. For coding sequences, these mutations may, if desired, affect the amino acid sequence. In particular, DNA sequences are contemplated that are substantially homologous to or derived from native V, D, J, constants, switches, and other similar sequences described herein.

As used herein, the term vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a plasmid, which refers to a cyclic double-stranded loop of DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell and thus replicate along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors). In general, expression vectors in recombinant DNA methods are often in the form of plasmids. In the present description, the plasmid and vector can be used interchangeably, since the plasmid is the most commonly used form of the vector. However, other forms of expression vectors are also included, such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions.

As used herein, the term recombinant host cell (or simply host cell) is intended to refer to a cell that contains a nucleic acid that is not naturally present in the cell, and optionally a cell into which a recombinant expression vector has been inserted. It should be understood that such terms are intended to refer not only to a particular cell of a subject, but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not be essentially identical to the original cell, but are still included within the scope of the term host cell as used herein.

As used herein, the term antigen refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. The antigen may be OX40 or a fragment thereof. The antigen may also be a tumor antigen against which protective or therapeutic immune responses are desired, eg antigens expressed by the tumor cell (eg for use as a tumor vaccine to be administered in combination with an anti-OX40 antibody). Antigens include tumor-associated antigens for the prevention or treatment of cancers. Examples of tumor-associated antigens include, without limitation, sequences containing all or part of the sequences ehCG, gp100 or Pmel17, HER2/neu, WT1, mesothelin, CEA, gp100, MARTI, TRP-2, Melan-A, NY-ESO -1, NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MAGE-3, MAGE-A3, tyrosinase, telomerase, SSX2 and MUC-1 and germline-derived tumor antigens . Tumor-associated antigens also include blood group antigens, eg Lea, Le ^b , LeX, LeY, H-2, B-1, B-2 antigens. Alternatively, more than one antigen may be included in the construct. For example, the MAGE antigen can be combined with other antigens such as melanin A, tyrosinase and gp100 along with adjuvants such as GM-CSF or IL-12 and linked to an anti-APC antibody.

The sequences of the above antigens are well known in the art. For example, an exemplary MAGE-3 cDNA sequence is provided in US Pat. No. 6,235,525 (Ludwig Cancer Research Institute); exemplary nucleic acid and NY-ESO-1 protein sequences are provided in US Pat. No. 5,804,381 and US Pat. No. 6,069,233 (Ludwig Institute for Cancer Research); examples of nucleic acid and Melan-A protein sequences are provided in US Pat. Nos. 5,620,886 and 5,854,203 (Ludwig Cancer Research Institute); examples of NY-BR-1 nucleic acid and protein sequences are given in US Pat. Ludwig Institute for Cancer Research); an example amino acid sequence of the HER-2/neu protein is available from GENBANK® with accession number AAA58637; and the nucleotide sequence (mRNA) for human carcinoembryonic antigen-like protein 1 (CEA-1) is available from GENBANK® with accession number NM020219.

The immune response refers to the biological response within a vertebrate against foreign agents that protects the body from those agents and the diseases they cause. The immune response is mediated by the action of a cell of the immune system (eg, T-lymphocyte, B-lymphocyte, natural killer cell, macrophage, eosinophil, mast cell, dendritic cell, or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines and complement), resulting in the selective targeting, binding, injury, destruction and/or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, malignant or other abnormal cells or, in the case of autoimmunity or pathological inflammation, normal human cells or fabrics. The immune response includes, for example, activation or inhibition of a T cell, such as an effector T cell or Th cell, such as CD4+ or CD8+ T cells, or inhibition of a Treg cell.

An immunomodulator or immunoregulator refers to an agent, such as a component of a signal

- 16 040561 pathway that can be involved in the modulation, regulation or modification of the immune response. Modulation, regulation, or modification of an immune response refers to any change in a cell of the immune system or in the activity of such a cell (eg, an effector T cell). Such modulation includes stimulation or suppression of the immune system, which may manifest itself in an increase or decrease in the number of different types of cells, an increase or decrease in the activity of these cells, or any other changes that may occur in the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have improved function in the tumor microenvironment. In preferred embodiments, the immunomodulator is located on the surface of the T cell. An immunomodulatory target or immunoregulatory target is an immunomodulator that targets binding and whose activity is altered by the binding of a substance, agent, fragment, compound, or molecule. Immunomodulatory targets include, for example, cell surface receptors (immunomodulatory receptors) and receptor ligands (immunomodulatory ligands).

Immunotherapy refers to the treatment of a subject suffering from infection or at risk of infection or suffering from a relapse of the disease, by a method involving the induction, enhancement, suppression or otherwise change the immune response.

Immunostimulatory therapy refers to therapy that results in an increase (induction or enhancement) of an immune response in a subject, such as in the treatment of cancer.

Potentiating an endogenous immune response means increasing the effectiveness or potency of an existing immune response in a subject. This increase in efficiency and power can be achieved, for example, by overcoming mechanisms that suppress the host's endogenous immune response, or by stimulating mechanisms that enhance the host's endogenous immune response.

Effector T cells (T _eff ) refer to T cells (eg, CD4+ and CD8+ T cells) with cytolytic activity, as well as T helper (Th) cells, which secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (Treg cells). Anti-OX40 antibodies described herein activate T _eff cells, eg CD4+ and CD8+ T _eff cells.

The increased ability to stimulate an immune response or the immune system may result from increased T-cell costimulatory receptor agonistic activity and/or increased inhibitory receptor antagonistic activity. An increased ability to stimulate an immune response or the immune system can be reflected in a multiple fold increase in EC ₅₀ or maximum activity level in an assay that measures the immune response, e.g., an assay that measures changes in cytokine or chemokine release, cytolytic activity (determined directly on cells -targets or indirectly through the detection of CD107a or granzymes) and proliferation. The ability to stimulate the immune response or the activity of the immune system can be increased by at least 10, 30, 50, 75%, 2, 3, 5 times or more.

As used herein, the term bound refers to the association of two or more molecules. The bond may be covalent or non-covalent. The connection may also be genetic (ie, recombinantly fused). Such linkages can be achieved using a wide variety of methods recognized in the art, such as chemical conjugation and recombinant protein production.

As used herein, the term administration refers to the physical administration of a composition containing a therapeutic agent to a subject using any of the various delivery methods and systems known to those skilled in the art. Preferred routes of administration for the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral route of administration, such as by injection or infusion. As used herein, the phrase parenteral administration means methods of administration other than enteral and topical administration, generally by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intracranial, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intraarticular injection and infusion, as well as in vivo electroporation. Alternatively, an antibody described herein can be administered via a non-parenteral route, such as a topical, epidermal, or mucosal route, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. The introduction can also be performed, for example, once, repeatedly and/or for one or more long periods.

As used herein, the term T cell mediated response refers to a T cell mediated response, including effector T cells (eg, CD8+ cells) and helper T cells (eg, CD4+ cells). T cell mediated responses include, for example, cytotoxicity and T cell proliferation.

As used herein, the term cytotoxic T-lymphocyte (CTL) response refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8+ T cells.

As used herein, the terms inhibit or block (eg, referring to inhibition/blocking of OX40-L binding to OX40 on cells) are used interchangeably and encompass both partial and complete inhibition/blocking. In some embodiments, the anti-OX40 antibody inhibits OX40-L binding to OX40 by at least about 50%, such as about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as defined, for example, as described above. In some embodiments, the anti-OX40 antibody inhibits OX40-L binding to OX40 by no more than 50%, such as about 40%, 30%, 20%, 10%, 5%, or 1%, as determined, for example, as further described above.

As used herein, the term inhibits tumor growth includes any measurable reduction in tumor growth, e.g., inhibition of tumor growth by at least 10%, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99 or 100%.

As used herein, the term cancer refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade neighboring tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

As used herein, the terms treat, treatment, and effect refer to any type of intervention or process performed on a subject or administering an active agent to a subject for the purpose of reversing, alleviating, arresting, inhibiting, or slowing or preventing the progression, development, severity, or recurrence of a symptom, complication, conditions or biochemical signs associated with the disease. Prophylaxis refers to the administration to a subject who does not have a disease in order to prevent the onset of the disease or to minimize its consequences if it occurs.

Hematologic malignancies include lymphoma, leukemia, myeloma or lymphoid malignancy, and malignancy of the spleen and lymph nodes. Illustrative lymphomas include both B-cell lymphomas (B-cell hematologic malignancy) and T-cell lymphomas. B-cell lymphomas include both Hodgkin's lymphomas and most non-Hodgkin's lymphomas. Non-limiting examples of B cell lymphomas include diffuse large B cell lymphoma, follicular lymphoma, mucosal lymphatic tissue lymphoma, small lymphocytic lymphoma (overlapping with chronic lymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B cell lymphoma, Waldenström macroglobulinemia, marginal zone nodular B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis. Non-limiting examples of T-cell lymphomas include extranodal T-cell lymphoma, cutaneous T-cell lymphomas, anaplastic large cell lymphoma, and angioimmunoblastic T-cell lymphoma. Hematologic malignancies also include leukemia, such as, without limitation, secondary leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, and acute lymphoblastic leukemia. Hematologic malignancies further include myelomas such as, but not limited to, multiple myeloma and smoldering multiple myeloma. Other hematologic and/or B-cell or T-cell associated malignancies are encompassed by the term hematologic malignancy.

The term effective dose or effective dosage is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A therapeutically effective amount or therapeutically effective dose of a drug or therapeutic agent is any amount of a drug that, when used alone or in combination with another therapeutic agent, promotes regression of the disease, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of periods without symptoms of the disease or preventing deterioration or disability due to disease.

For solid tumors, an effective amount comprises an amount sufficient to cause shrinkage of the tumor and/or decrease in tumor growth rate (eg, to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to slow tumor growth. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount may be administered in one or more administrations. An effective amount of the drug or composition may: (i) reduce the number of malignant cells; (ii) reduce tumor size; (iii) inhibit, restrain, slow down to some extent and can stop the penetration of malignant cells into peripheral organs; (iv) inhibit, i.e. slow down to some extent and stop tumor metastases; (v) inhibit tumor growth; (vi) prevent or delay the onset and/or recurrence of the tumor; and/or (vii) alleviate to some extent one or more of the symptoms associated with the cancer. In one example, the effective amount is an amount of an anti-OX40 antibody and an amount of an anti-PD-1 antibody (eg, nivolumab) or an anti-CTLA-4 antibody (eg, ipilimumab), in combination, that is clinically proven to significantly reduce cancer. or slowing the progression of a malignant tumor, such as an advanced solid tumor. As used herein, the terms fixed dose, fixed dose, and fixed fixed dose are used interchangeably and refer to a dose that is administered to a patient without regard to the weight or body surface area (BSA) of the patient. Therefore, a fixed or constant dose is not provided as a mg/kg dose, but rather as an absolute amount of the agent.

As used herein, the term body surface area (BSA) dose refers to a dose that is adjusted to the body surface area (BSA) of an individual patient. The BSA-based dose may be provided as mg/kg body weight. Various calculations have been published for obtaining BSA without direct measurement, the most widely used of which is the Dubois formula (see Du Bois D., Du Bois E.F. (Jun 1916) Archives of Internal Medicine 17 (6): 863-71 and Verbraecken, J et al (Apr 2006) Metabolism - Clinical and Experimental 55 (4): 515-24. Other exemplary BSA formulas include Mosteller's formula (Mosteller R.D.N. Engl. J. Med., 1987; 317:1098), Haycock's formula (Haycock G.B. et al., J. Pediatr. 1978, 93:62-66), Gehan's formula and George (Gehan E.A., George S.L., Cancer Chemother. Rep. 1970, 54:225235), Boyd's formula (Current J.D. (1998), The Internet Journal of Anesthesiology 2 (2) and Boyd, Edith (1935), University of Minnesota The Institute of Child Welfare, Monograph Series, No. x. London: Oxford University Press), Fujimoto's formula (Fujimoto S. et al., Nippon Eiseigaku Zasshi 1968;5:443-50), Takahira's formula (Fujimoto S. et al., Nippon Eiseigaku Zasshi 1968;5:443-50) and the Schlich formula (Schlich E. et al., Ernahrungs Umschau 2010;57:178-183).

A prophylactically effective amount or prophylactically effective dosage of a drug is an amount of a drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from a relapse of the disease, prevents the development or recurrence of the disease. The ability of a therapeutic or prophylactic agent to stimulate regression of a disease or to inhibit the development or recurrence of a disease can be assessed using various methods known to the practitioner, such as in humans during clinical trials, in animal model systems that predict efficacy in humans, or by analyzing the activity of the agent in in vitro analyses.

By way of example, an antineoplastic agent is a drug that slows down the progression of a cancer or promotes regression of a cancer in a subject. In preferred embodiments, the therapeutically effective amount of the drug promotes regression of the cancer to the extent that the cancer is eliminated. Promoting regression of a malignant tumor means that administration of an effective amount of a drug, alone or in combination with an anticancer agent, results in a decrease in tumor growth or size, tumor necrosis, a decrease in the severity of at least one symptom of the disease, an increase in the frequency and duration of periods without symptoms of the disease, preventing deterioration or disability due to the disease or worsening of the patient's symptoms of the disease. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to an acceptably low level of toxicity or other adverse physiological effects at the cellular, organ and/or organism level (side effects) resulting from the administration of a drug.

As an example for the treatment of tumors, a therapeutically effective amount or dosage of a drug preferably inhibits cell growth or tumor growth by at least 20%, more preferably at least 40%, even more preferably at least 60%, and even more preferably at least about 80% relative to untreated subjects. In the most preferred embodiments, the therapeutically effective amount or dosage of the drug completely inhibits cell growth or tumor growth, i.e., preferably inhibits cell growth or tumor growth by 100%. Way

- 19 040561 The ability of a compound to inhibit tumor growth can be assessed using the assays described below. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit cell growth, such inhibition can be measured in vitro using assays known to the skilled practitioner. In other preferred embodiments described herein, tumor regression can be observed and can continue for a period of at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days.

The terms patient and subject refer to any human or non-human animal receiving either prophylactic or therapeutic treatment. For example, the methods and compositions described herein may be used to treat a subject or patient with a cancer, such as an advanced solid tumor. The term non-human animal includes all vertebrates, such as mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

The various aspects described herein are described in more detail in the following subsections.

I. Antibodies to OX40.

This document describes antibodies, such as fully human antibodies, which are characterized by specific functional features or properties. For example, antibodies specifically bind to human OX40. In addition, antibodies can cross-react with OX40 from one or more non-human primates, such as cynomolgus monkey OX40. Such antibodies are useful in the treatment of cancer when used as a monotherapy or when used in combination with an immuno-oncology agent such as an anti-PD-1 antibody (eg, nivolumab) or an anti-CTLA-4 antibody (eg, ipilimumab).

Anti-OX40 antibodies described herein exhibit one or more or all of the following functional properties:

(1) binding to soluble human OX40, eg, a KD of 10 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by the BIACORE® SPR assay;

(2) binding to membrane-bound human OX40, eg EC ₅₀ 1 nM or less (eg 0.01 nM to 1 nM), eg as measured by FACS;

(3) binding to cynomolgus monkey OX40, eg, binding to membrane-bound cynomolgus monkey OX40, eg, with an EC ₅₀ of 10 nM or less (eg, 0.01 to 10 nM), eg, as measured by FACS;

(4) inducing or enhancing T cell activation as evidenced by (i) increased production of IL-2 and/or IFN-γ in OX40 expressing T cells and/or (ii) increased T cell proliferation;

(5) inhibition of OX40 ligand binding to OX40, eg with an EC ₅₀ of 1 nM or less as measured by FACS, eg in an assay with hOX40-293 cells;

(6) binding to an epitope on the extracellular portion of mature human OX40 (SEQ ID NO: 2), such as an epitope in the DVVSSKPCKPCTWCNLR region (SEQ ID NO: 178), or

DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);

(7) competition for human OX40 binding to 3F4, 14B6-1, 14B6-2, 23H3, 18E9, 8B11, 20B3 and 20C1;

(8) competition for human OX40 binding to 6E1-1, 6E1-2, 14A2-1 and 14A2-2.

Preferably, the antibodies bind to OX40 with high affinity, e.g. K _D 10 ^-7 M or less, 10 ^-8 M or less, 10 -9 M or less, 10 ^-10 M or less, 10 ^-11 M or less, 10 ^-12 M or less, 10 ^-12 to 10 ^-7 M, 10 ^-11 to 10 ^-7 M, 10 ^-10 to 10 ^-7 M, or 10 ^-9 to 10 ^-7 M In some embodiments, the antibody to OX40 binds to soluble human OX40, for example, as determined by the BIACORE® SPR assay, with a KD of 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M (1 nM) or less, 10 ¹⁰ M or less, from 10 ^-12 to 10 ^-7 M, from 10 ^-11 to 10 ^-7 M, from 10 ^-10 to 10 ^-7 M, from 10 ^-9 to 10 ^-7 M or from 10 ^-8 to 10 ^-7 M. In some embodiments, the anti-OX40 antibody binds to bound (e.g., cell membrane bound) human OX40, e.g., on activated human T cells, e.g., as determined by flow cytometry, with a KD of 10 ^-7 M or less, 10 ^{- 8} M or less, 10 ^-9 M (1 nM) or less, 10 ^-10 M or less, 10 ^-12 to 10 ^-7 M, from 10 ^-11 to 10 ^-8 M, from 10 ^-10 to 10 ^-8 M, from 10 ^-9 to 10 ^-8 M, from 10 ^-11 to 10 ^-9 M or from 10 ^-10 to 10 ^-9 M. In some embodiments, anti-OX40 antibodies bind to bound (e.g., cell membrane bound) human OX40, such as activated human T cells, e.g., as determined by FACS, with an EC50 of 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M (1 nM) or less, 10 ^-10 M or less, 10 ^-12 to 10 ^-7 M, 10 ^-11 to 10 ^-8 M, 10 ^-10 to 10 ^-8 M, 10 ^-9 to 10 ^-8 M, 10 ^-11 to 10 ^-9 M, or 10 ^-10 to 10 ^-9 M. In some embodiments, the anti-OX40 antibody binds to soluble human OX40 with a KD of 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M (1 nM) or less, 10 ^-10 M or less, from 10 ^-12 to 10 ^-7 M, from 10 ^-11 to 10 ^-7 M, from 10 ^-10 to 10 ^-7 M, 10 ^-9 to 10 ^-7 M, or 10 ^-8 to 10 ^-7 M, and with human cell membrane-bound OX40 with a K _D or EC ₅₀ of 10 ^-7 M or less, 10 ^-8 M or less 10 ^-9 M (1 nM) or less, 10 ^-10 M or less, from 10 ^-12 to 10 ^-7 M,

- 20 040561 10 ^-11 to 10' ⁸ M, 10 ^-10 to 10 ^-8 M, 10 ^-9 to 10 ^-8 M, 10 ^-11 to 10' ⁹ M or 10 ^-10 to 10 ^-9 M.

Anti-OX40 antibodies described herein can bind to cynomolgus monkey OX40, e.g., bind to membrane-bound cynomolgus monkey OX40, e.g., EC ₅₀ 100 nM or less, 10 nM or less, 100 to 0.01 nM, 100 to 0.1 nM, 100 to 1 nM, or 10 to 1 nM, eg, as measured by FACS (eg, as described in the examples).

Anti-OX40 antibodies described herein can stimulate or enhance an immune response, for example, by activating T _eff cells, limiting suppression of T effector cells by Treg cells, depleting and/or inhibiting Treg tumor cells, and/or activating NK cells, for example, in a tumor . For example, antibodies to OX40 can activate or co-stimulate T _eff cells, as evidenced by, for example, increased secretion of cytokines (eg, IL-2 and IFN-γ) and/or increased proliferation. In some embodiments, CD3 stimulation is also provided. In some embodiments, the OX40 antibody increases IL-2 secretion by 50, 100% (i.e., 2-fold), 3-fold, 4-fold, 5-fold, or more, optionally up to a maximum of 10, 30, 100-fold, as measured, for example, on primary human T cells or T cells expressing human OX40 (eg, as described in the examples below). In some embodiments, the OX40 antibody increases IFN-γ secretion by 50, 100% (i.e., 2-fold), 3-fold, 4-fold, 5-fold or more, optionally up to a maximum of 10-fold, 30-fold, 100-fold, as measured, for example, on primary human T cells or T cells expressing human OX40 (eg, as described further in the examples).

The anti-OX40 antibodies described herein can inhibit the binding of human OX40L to human OX40 on cells, e.g., 293 cells expressing human OX40 (i.e., hOX40-293 cells), e.g., with an EC ₅₀ of 10 nM or less, 1 nM or less , 0.01 to 10 nM, 0.1 to 10 nM, or 0.1 to 1 nM (see example 6).

The anti-OX40 antibodies described herein bind to an epitope on OX40 as determined, for example, by binding to fragments of human OX40. For example, in some embodiments, the antibody binds to all or part of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) of human OX40 (SEQ ID NO: 2) as determined, for example, by HDX or by binding antibodies to human OX40 fragments followed by enzymatic cleavage (see example 11). In other embodiments, the antibody binds to all or part of the sequence DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179) of human OX40 (SEQ ID NO: 2).

In some embodiments, the anti-OX40 antibodies described herein bind to all or part of the sequence SQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR (SEQ ID NO: 182).

In other embodiments, anti-OX40 antibodies described herein bind to all or part of the sequence of PCKPCTWCNLR (SEQ ID NO: 183).

In other embodiments, anti-OX40 antibodies that bind to all or part of the DVVSSKPCKPCTWCNLR sequence (SEQ ID NO: 178) further bind to all or part of the QLCTATQDTVCR sequence (SEQ ID NO: 184).

In additional embodiments, anti-OX40 antibodies described herein bind all or part of the SQNTVCRPCGPGFYN sequence (SEQ ID NO: 185).

Anti-OX40 antibodies described herein can compete for binding to OX40 (or inhibit binding) with anti-OX40 antibodies containing the CDRs or variable regions described herein, e.g., 3F4, 14B6-1, 14B6-2, 23H3, 6E1- 1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and/or 20C1.

In some embodiments, anti-OX40 antibodies inhibit the binding of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and/or 20C1 to human OX40 at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1 inhibit the binding of anti-OX40 antibodies to human OX40 at least by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%.

In some embodiments, the antibodies induce or enhance the activation of multivalently crosslinked T cells through, for example, FcR binding. In some embodiments, the antibodies are multivalent, eg, divalent. In some embodiments, the antibodies are not monovalent.

In some embodiments, antibodies are characterized by 1, 2, 3, 4, 5, or 6 of the following:

(1) binding to soluble human OX40, eg, a KD of 10 nM or less (eg, 0.01 to 10 nM), eg, as measured by the BIACORE® SPR assay;

(2) binding to membrane-bound human OX40, eg EC50 1 nM or less (eg 0.01 to 1 nM), eg as measured by FACS;

(3) binding to cynomolgus monkey OX40, e.g. binding to membrane-bound cynomolgus OX40

- 21 040561 monkey, for example with an EC ₅₀ of 10 nM or less (for example, from 0.01 to 10 nM), for example, as measured by FACS;

(4) inducing or enhancing T cell activation as evidenced by (i) increased production of IL-2 and/or IFN-γ in OX40 expressing T cells and/or (ii) increased T cell proliferation;

(5) inhibition of OX40 ligand binding to OX40, eg, with an EC ₅₀ of 1 nM or less, as measured by FACS, eg, in an assay with hOX40-293 cells;

(6) binding to an epitope on the extracellular portion of mature human OX40 (SEQ ID NO: 2), such as an epitope in the DVVSSKPCKPCTWCNLR region (SEQ ID NO: 178) or DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);

(7) competition for human OX40 binding to 3F4, 14B6-1, 14B6-2, 23H3, 18E9, 8B11, 20B3 and 20C1;

(8) competition for human OX40 binding to 6E1-1, 6E1-2, 14A2-1 and 14A2-2.

Accordingly, it will be understood that an antibody that exhibits one or more of these functional properties (e.g., biochemical, immunochemical, cellular, physiological, or other biological activity, or the like), as determined in accordance with techniques known in the art and described in herein, refers to a statistically significant difference in specific activity compared to no antibody (eg, or when a control antibody of irrelevant specificity is present). Preferably, the anti-OX40 antibody increases the measured parameter (e.g., T cell proliferation, cytokine production) by at least 10% of the measured parameter, more preferably by at least 20, 30, 40, 50, 60, 70, 80, 90 , 95, 100% (i.e. 2 times), 3, 5 or 10 times. Conversely, an antibody can reduce a measured parameter (e.g., tumor volume, OX40-L binding to OX40, number of regulatory T cells in tumors) by at least 10% of the measured parameter, more preferably by at least 20, 30, 40 , 50, 60, 70, 80 or 90, 95 or 99%.

Standard assays for evaluating the binding capacity of antibodies to OX40 of various species are known in the art, including, for example, ELISA, Western blots, and RIA. Appropriate analyzes are described in detail in the examples. The binding kinetics (eg, binding affinity) of antibodies can also be assessed by standard assays known in the art, such as the BIACORE® SPR assay. Assays to evaluate the effect of antibodies on OX40 functional properties (eg, ligand binding, T cell proliferation, cytokine production) are described in more detail below and in the examples.

In some embodiments, anti-OX40 antibodies are not native antibodies or are not naturally occurring antibodies, e.g., anti-OX40 antibodies with post-translational modifications that differ from antibodies that occur naturally, such as less or a different type of post-translational modification.

II. Illustrative antibodies to OX40.

Specific antibodies described herein are antibodies, e.g., monoclonal antibodies, containing the CDR and/or variable region sequences of antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and 20C1 isolated and structurally characterized as described in Example 1, as well as antibodies characterized by at least 80% identity (for example, at least 85% identity, at least 90% identity , at least 95% or at least 99%) with respect to the sequences of the variable region or CDRs of antibodies 3F4, 14B6-1, 14B6-2, 23h3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A21, 14A2-2 and 20C1. The V _H amino acid sequences of 3F4, 14B6 (14B6-1 and 14B6-2), 23H3, 6E1 (6E11 and 6E1-2), 18E9, 8B11, 20B3, 14A2 (14A2-1 and 14A2-2) and 20C1 are shown in SEQ ID NO: 17, 28, 37, 48, 57, 65, 73, 84 and 93, respectively. The V _L amino acid sequences of 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and 20C1 are shown in SEQ ID NOs: 18, 29, 30 , 38, 49, 50, 58, 66, 74, 85, 86 and 94 respectively.

Accordingly, provided herein are antibodies, or an antigen-binding portion thereof, comprising heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 28, 37, 48, 57, 65 , 73, 84 and 93.

Also provided are antibodies or antigen-binding portions thereof comprising heavy and light chain variable regions, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 29, 30, 38, 49, 50, 58, 66 , 74, 85, 86 and 94.

Provided herein are antibodies, or an antigen-binding portion thereof, comprising heavy and light chain variable region sequences comprising SEQ ID NOs: 17 and 18; 28 and 29; 28 and 30; 37 and 38; 48 and 49; 48 and 50; 57 and 58; 65 and 66; 73 and 74; 84 and 85; 84 and 86; 93 and 94.

Anti-OX40 antibodies described herein may contain CDR1, CDR2, and CDR3 of the heavy and light chains 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2- 1, 14A2-2 and 20C1 or combinations thereof. Amino acid sequences of CDR1 V _H in 3F4, 14B6 (14B6-1 and 14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11, 20B3, 14A2 (14A2-1 and 14A2-2) and 20C1 are shown in SEQ ID NOs: 11, 19, 31, 39, 51, 59, 67, 75 and 87, respectively. Amino acid sequences of CDR2 V _H in 3F4, 14B6 (14B6-1 and 14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11, 20B3, 14A2 (14A2-1 and 14A2-2) and 20C1 are shown in SEQ ID NOs: 12, 20, 32, 40, 52, 60, 68, 76 and 88, respectively. The amino acid sequences of CDR3 VH in 3F4, 14B6 (14B6-1 and 14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11, 20B3, 14A2 (14A2-1 and 14A2-2) and 20C1 are in SEQ ID NOs: 13, 21, 33, 41, 53, 61, 69, 77 and 89. CDR1 V _L amino acid sequences in 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9 , 8B11, 20B3, 14A21, 14A2-2 and 20C1 are shown in SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62, 70, 78, 81 and 90, respectively. The amino acid sequences of CDR2 VL in 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and 20C1 are shown in SEQ ID NOs: 15, 23, 26, 35, 43, 46, 55, 63, 71, 79, 82 and 91, respectively. The amino acid sequences of the VL CDR3s in 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and 20C1 are shown in SEQ ID NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83 and 92, respectively. CDR regions are delineated using the Kabat system (Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

Given that each of these antibodies binds to OX40 and that antigen-binding specificity is provided primarily by the CDR1, 2 and 3 regions, the CDR1, 2 and 3 VH sequences and the CDR1, 2 and 3 V _L sequences can be mixed and matched (i.e. CDRs from different antibodies can be mixed and matched, although each antibody must contain CDR1, 2 and 3 VH and CDR1, 2 and 3 V _L ) to create other binding antibodies to OX40. OX40 binding of such mixed and matched antibodies can be examined using the binding assays described above and in the examples (eg, ELISA). Preferably, when VH CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from the particular VH sequence is replaced with structurally similar CDR sequence(s). Similarly, when V _L CDR sequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequence from a particular V _L sequence is preferably replaced with structurally similar CDR sequence(s). One skilled in the art will appreciate that new V _H and V _L sequences can be generated by replacing one or more of the V _H and/or V _L CDR region sequences with structurally similar sequences from the CDR sequences disclosed herein for the 3F4 monoclonal antibodies, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and 20C1. Mixed and matched antibodies having binding affinity, biological activity, and/or other properties equivalent to or superior to the specific antibodies disclosed herein may be selected for use in the methods of the present invention.

Provided herein are isolated antibodies, or antigen-binding portions thereof, comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 19, 31, 39, 51, 59, 67, 75, and 87;

(b) a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 20, 32, 40, 52, 60, 68, 76 and 88;

(c) a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 21, 33, 41, 53, 61, 69, 77, and 89;

(d) a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62, 70, 78, 81, and 90;

(e) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 23, 26, 35, 43, 46, 55, 63, 71, 79, 82, and 91; and (f) a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83, and 92, wherein the antibody specifically binds to human OX40.

In one embodiment, the antibody comprises heavy and light chain variable regions, wherein the heavy chain variable region CDR1, CDR2 and CDR3 comprise: SEQ ID NOs: 11-13; 19-21; 31-33; 39-41; 51-53; 59-61; 67-69; 74-77; 87-89 and 87, 317 and 89, respectively;

wherein the antibody specifically binds to human OX40.

In another embodiment, the antibody comprises heavy and light chain variable regions, wherein the light chain variable region CDR1, CDR2 and CDR3 comprise: SEQ ID NOs: 14-16; 22-24; 25-27; 34-36; 42-44; 45-47; 54-56; 62-64; 70-72; 78-80; 81-83 and 90-92, respectively;

wherein the antibody specifically binds to human OX40.

In a specific embodiment, the antibody comprises heavy and light chain variable regions, wherein the antibody comprises:

(a) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOS: 87, 317 and 89,

- 23 040561, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO:

90-92, respectively;

(b) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 11-13, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 1416, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 19-21, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 2224, respectively;

(d) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 19-21, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 2527, respectively;

(e) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 31-33, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 3436, respectively;

(f) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 39-41, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 4244, respectively;

(g) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 39-41, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 4547, respectively;

(h) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 51-53, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 5456, respectively;

(i) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 59-61, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 6264, respectively;

(j) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 67-69, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 7072, respectively;

(k) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 75-77, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 7880, respectively;

(l) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 75-77, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 8183, respectively; or (m) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO: 87-89, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO: 9092, respectively, wherein the antibody specifically binds to OX40 human.

In another embodiment, the antibody comprises heavy and light chain variable regions, wherein the antibody comprises:

(a) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 87, 317 and 89, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 90-92, respectively;

(b) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 11-13, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 1416, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 19-21, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 2224, respectively;

(d) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 19-21, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 2527, respectively;

(e) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 31-33, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 3436, respectively;

(f) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 39-41, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 4244, respectively;

(g) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 39-41, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 4547, respectively;

- 24 040561 (h) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 51-53, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 5456, respectively;

(i) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 59-61, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 6264, respectively;

(j) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 67-69, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 7072, respectively;

(k) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 75-77, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 7880, respectively;

(l) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 75-77, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 8183, respectively; or (m) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 87-89, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 9092, respectively.

The VH domain, or one or more of its CDRs described herein, may be linked to a constant domain to form a heavy chain, eg, a full length heavy chain. Similarly, a VL domain or one or more of the CDRs described herein can be linked to a constant domain to form a light chain, such as a full length light chain. The full-length heavy chain (excluding the C-terminal lysine (K) or excluding the C-terminal glycine and lysine (GK), which may be absent) and the full-length light chain combine to form a full-length antibody. N-terminal glutamine and glutamate residues can also be converted to pyroglutamate residues in both light and heavy chains.

The VH domain described herein can be fused to a human IgG constant domain, eg, IgG1, IgG2, IgG3, or IgG4, which is either naturally occurring or modified, eg, as described below. For example, the heavy chain may comprise the amino acid sequence of any VH domain described herein fused to the human IgG1 amino acid sequence shown in SEQ ID NO: 5.

The constant domain of human IgG1 can also be an allotypic variant. For example, an IgG1 allotype variant contains R107K, E189D and M191L (underlined above, numbered as in SEQ ID NO: 6). In the full length region of the heavy chain, these amino acid substitutions are numbered R214K, E356D and M358L.

The VL domain described herein can be fused to a human k or λ light chain constant domain. For example, the light chain may comprise the amino acid sequence of any of the VL domains described herein fused to the anti-human IgG1 light chain amino acid sequence shown in SEQ ID NO: 7.

In some embodiments, the heavy chain constant region contains a lysine or other amino acid at the C-terminus, for example, it contains the following last amino acids: LSPGK (SEQ ID NO: 8) for the heavy chain. In some embodiments, the heavy chain constant region lacks one or more amino acids at the C-terminus and is characterized by, for example, the C-terminal sequence of LSPG (SEQ ID NO: 9) or LSP.

Amino acid sequences of illustrative heavy and light chains are shown in table. 23 and correspond to SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 124 and 125 for heavy chains and SEQ ID NO: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 and 122 for light chains.

Heavy and light chains containing an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, or 70% identical to any of the heavy or light chains shown in Table 1. 23 (or their variable regions), for example, SEQ ID NOS: 95 and 96; 97 and 98; 99 and 100; 101 and 102; 103 and 104; 105 and 106; 107 and 108; 109 and 110; 111 and 112; 113 and 114; 115 and 116; 117 and 118; 119 and 120; 121 and 122; 123 and 116; 124 and 116; and 125 and 116 can be used to generate anti-human OX40 antibodies having the desired characteristics, such as those further described herein. Illustrative variants are those containing an allotypic variation, for example in the constant region, and/or a mutation in the variable or constant regions, such as the mutations described herein. Heavy and light chains containing an amino acid sequence that differs by no more than 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acid (by substitution, addition or deletion) from any of the heavy or light chains shown in Table. 23 (or their variable regions) can be used to generate anti-human OX40 antibodies having the desired characteristics, such as those described later herein.

In various embodiments, the antibodies described above exhibit one or more, two or more, three or more, four or more, five or more, six or all of the following functional properties:

(1) binding to soluble human OX40, eg, a KD of 10 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by Biacore;

(2) binding to membrane-bound human OX40, eg, EC50 1 nM or less (eg, 0.01 nM to 1 nM), eg, as measured by FACS;

(3) binding to cynomolgus monkey OX40, eg, binding to membrane-bound cynomolgus monkey OX40, eg, with an EC50 of 10 nM or less (eg, 0.01 to 10 nM), eg, as measured by FACS;

(4) inducing or enhancing T cell activation as evidenced by (i) increased production of IL-2 and/or IFN-γ in OX40 expressing T cells and/or (ii) increased T cell proliferation;

(5) inhibition of OX40 ligand binding to OX40, eg, with an EC50 of 1 nM or less, as measured by FACS, eg, in an assay with hOX40-293 cells;

(6) binding to an epitope on the extracellular portion of mature human OX40 (SEQ ID NO: 2), such as an epitope in the DVVSSKPCKPCTWCNLR region (SEQ ID NO: 178), or

DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);

(7) competition for human OX40 binding to 3F4, 14B6-1, 14B6-2, 23H3, 18E9, 8B11, 20B3 and 20C1;

(8) competition for human OX40 binding to 6E1-1, 6E1-2, 14A2-1 and 14A2-2.

Such antibodies include, for example, human antibodies, humanized antibodies, or chimeric antibodies.

In some embodiments, anti-OX40 antibodies described herein bind to amino acid residues in the following region of mature human OX40 (SEQ ID NO: 2):

DVVSSKPCKPCTWCNLR (SEQ III NO: 178) corresponding to amino acid residues 46-62 of mature human OX40 (SEQ ID NO: 2).

In some embodiments, anti-OX40 antibodies described herein bind to amino acid residues in the following region of mature human OX40 (SEQ ID NO: 2)

DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179) corresponding to amino acid residues 89-124 of mature human OX40 (SEQ ID NO: 2).

In some embodiments, anti-OX40 antibodies described herein that bind to all or part of the sequence of DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) bind to all or part of the sequence of SQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR (SEQ ID NO: 182).

In other embodiments, anti-OX40 antibodies described herein that bind to all or part of the sequence of DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) bind to all or part of the sequence of PCKPCTWCNLR (SEQ ID NO: 183).

In other embodiments, anti-OX40 antibodies that bind to all or part of the DVVSSKPCKPCTWCNLR sequence (SEQ ID NO: 178) further bind to all or part of the QLCTATQDTVCR sequence (SEQ ID NO: 184).

In additional embodiments, anti-OX40 antibodies described herein that bind to all or part of the sequence of DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) further bind to all or part of the sequence of SQNTVCRPCGPGFYN (SEQ ID NO: 185).

Modified heavy chain constant domains.

The heavy chain constant region of the anti-OX40 antibodies described herein can be of any isotype, eg, IgG1, IgG2, IgG3, and IgG4, or combinations and/or modifications thereof. In some embodiments, anti-OX40 antibodies comprise a modified heavy chain constant region that alters the properties of the antibody.

As discussed further herein and in the examples, cross-linking of anti-OX40 antibodies to unmodified hIG1 constant regions (hIG1 isotype antibodies) induces OX40 signaling and promotes T cell activation and, in particular, promotes T cell proliferation, IFN-γ secretion and secretion of IL-2. Cross-linking can occur by, for example, binding to human CD32A Fcy receptors (FcyR) expressed on the surface of transfected CHO cells in an assay using co-cultures of CHO-CD3-CD32A cells and primary human CD4 T cells. Cross-linking can also occur, for example, by adding a soluble polyclonal human Fcy antibody to cultures of staphylococcus enterotoxin B (SEB) activated human peripheral blood mononuclear cells (PBMC).

- 26 040561

Anti-OX40 antibodies with modified heavy chain constant regions (e.g., IgG1 constant region with CH1/hinge replaced with CH1/hIgG2 hinge region) may have the ability to alter antibody activities relative to antibodies with a full IgG1 heavy chain constant region. Exemplary activities that may be altered include, without limitation: (1) T cell activation with or without crosslinking, (2) T cell proliferation with or without crosslinking, and/or (3) cytokine secretion ( eg IFN-γ, IL-2) with or without crosslinking. The methods described in the examples can be used to determine if anti-OX40 antibodies with modified heavy chain constant regions have these altered activities (see, for example, example 27). In preferred embodiments, these altered activities do not significantly affect the antigen-binding properties of antibodies, which can be assessed using, for example, FACS, SPR).

Accordingly, provided herein are methods for altering the activity of anti-OX40 antibodies, comprising an anti-OX40 antibody that contains a non-IgG2 hinge and replacing the non-IgG2 hinge with an IgG2 hinge. In some embodiments, the modified heavy chain constant region comprises an IgG2 isotype hinge (IgG2 hinge) and a CH1, CH2, and CH3 domain. In some embodiments, the modified heavy chain constant region comprises an IgG2 hinge and a CH1, CH2, and CH3 domain, wherein at least one of the CH1, CH2, and CH3 domains is not of the IgG2 isotype. The IgG2 hinge can be a wild-type IgG2 hinge, such as a wild-type human IgG2 hinge (e.g., ERKCCVECPPCPAPPVAG, SEQ ID NO: 208) or a variant thereof, provided that the IgG2 hinge retains the ability to confer an altered activity on the antibody relative to the same antibody that contains non-IgG2 hinge. In some embodiments, the IgG2 hinge variant retains the same stiffness or hardness as the wild-type IgG2 hinge. Hinge stiffness can be determined, for example, by computer simulation, electron microscopy, spectroscopy such as nuclear magnetic resonance (NMR), X-ray crystallography (B-factors), or ultracentrifugation to analytically study the sedimentation rate (AUC) to measure or compare the gyration radius of antibodies containing a hinge. A hinge may have similar or higher stiffness to another hinge if an antibody containing a hinge has a value obtained in one of the tests described in the previous sentence that differs from the value of the same antibody with another hinge, for example, an IgG1 hinge, by less than 5, 10, 25, 50, 75 or 100%. One skilled in the art will be able to determine from the tests whether a hinge has at least the same rigidity as another hinge by interpreting the results of these tests. An exemplary human IgG2 hinge is an IgG2 hinge that contains a substitution of one or more of four cysteine residues (ie C219, C220, C226 and C229). Cysteine may be replaced by serine. An exemplary IgG2 hinge is a human IgG2 hinge containing the C219S mutation (eg, ERKSCVECPPCPAPPVAG, SEQ ID NO: 209). Other IgG2 hinge variants that may be used include human IgG2 hinges containing a C220, C226 and/or C229 substitution, such as a C220S, C226S or C229S mutation (which may be combined with a C219S mutation). The IgG2 loop can also be an IgG2 hinge in which part of the hinge is part of a different isotype (i.e. it is a chimeric hinge), provided that the rigidity of the chimeric hinge is at least the same as that of the wild-type IgG2 hinge. For example, the IgG2 hinge may be an IgG2 hinge, wherein the lower hinge (as defined in Table 2) has an IgG1 isotype and is, for example, a wild-type IgG1 lower hinge. Additional IgG2 mutations that can be used in the IgG2 hinge include the SE (S267E), SELF (S267E/L328F), SDIE (S239D/I332E), SEFF and GASDALIE (G236A/S239D/A330L/I332E) mutations.

A hybrid or chimeric hinge is assigned to a particular isotype if more than half of the consecutive amino acids of the hinge belong to that isotype. For example, a hinge containing the upper and middle IgG2 hinges and the lower IgG1 hinge is considered an IgG2 hinge.

In some embodiments, the anti-OX40 antibody comprises a modified heavy chain constant region that contains an IgG2 hinge containing one of the following sequences:

ERKCCVECPPCPAPPVAG (SEQ ID NO: 208);

- 27 040561

ERKSCVECPPCPAPPVAG (SEQ ID NO: 209);

ERKCSVECPPCPAPPVAG (SEQ ID NO: 210);

ERKXCVECPPCPAPPVAG (SEQ ID NO: 211);

ERKCXVECPPCPAPPVAG (SEQ ID NO: 212);

ERKCCVECPPCPAPPVAGX (SEQ ID NO: 213);

ERKSCVECPPCPAPPVAGX (SEQ ID NO: 214);

ERKCSVECPPCPAPPVAGX (SEQ ID NO: 215);

ERKXCVECPPCPAPPVAGX (SEQ ID NO: 216);

ERKCXVECPPCPAPPVAGX (SEQ ID NO: 217);

ERKCCVECPPCPAPELLGG (SEQ ID NO: 218);

ERKSCVECPPCPAPELLGG (SEQ ID NO: 219);

ERKCCSVECPPCPAPELLGG (SEQ ID NO: 220);

ERKXCVECPPCPAPELLGG (SEQ ID NO: 221);

ERKCXVECPPCPAPELLGG (SEQ ID NO: 222);

ERKCCVECPPCPAPELLG (SEQ ID NO: 223);

ERKSCVECPPCPAPELLG (SEQ ID NO: 224);

ERKCCSVECPPCPAPELLG (SEQ ID NO: 225);

ERKXCVECPPCPAPELLG (SEQ ID NO: 226);

ERKCXVECPPCPAPELLG (SEQ ID NO: 227);

ERKCCVECPPCPAP (SEQ ID NO: 228);

ERKSCVECPPCPAP (SEQ ID NO: 229);

ERKCSVECPPCPAP (SEQ ID NO: 230);

ERKXCVECPPCPAP (SEQ ID NO: 231); or

ERKCXVECPPCPAP (SEQ ID NO: 232), where X is any amino acid except cysteine, or any of the above sequences in which 1-5, 1-3, 1-2 or 1 amino acid is inserted between the amino acid residues of CVE and CPP. In some embodiments, THT or GGG is administered. In some embodiments, 1, 1-2, or 1-3 amino acids are inserted between the hinge and the CH2 domain. For example, glycine can be inserted between the hinge and the CH2 domain.

In some embodiments, the hinge comprises SEQ ID NOs 208, 209, 210, 211, or 212, where 1, 2, 3, or all 4 amino acids P233, V234, A235, and G237 (corresponding to C-terminal amino acid 4 of PVAG (SEQ ID NO: 233 ) are deleted or replaced with another amino acid, for example, the C-terminal amino acids of the IgG1 hinge (ELLG (SEQ ID NO: 234) or ELLGG (SEQ ID NO: 235) In some embodiments, the hinge comprises SEQ ID NOs: 208, 209, 210, 211 or 212, wherein V234, A235, and G237 are removed or replaced by another amino acid.In some embodiments, the hinge comprises SEQ ID NO: 208, 209, 210, 211, or 212, wherein A235 and G237 are deleted or replaced by another amino acid.In some embodiments, the hinge is contains SEQ ID NO: 208, 209, 210, 211, or 212, with G237 removed or replaced with another amino acid In some embodiments, the hinge contains SEQ ID NO: 447, 448, 449, 450, or 451, with V234 and A235 removed or replaced other goy amino acid. Replacement of PVAG (SEQ ID NO: 233) in IgG2 with the corresponding IgG1 hinge amino acids, ie. (ELLG (SEQ ID NO: 234) or ELLGG (SEQ ID NO: 235)) to obtain a hybrid hinge, such as shown above, which provides a hinge with the benefits of an IgG2 hinge and the effector function of IgG1 hinges.

In some embodiments, the modified heavy chain constant region comprises a hinge that consists essentially of one of the sequences shown above, such as any of SEQ ID NOs: 208-232, and in some embodiments, does not contain additional hinge amino acid residues.

In some embodiments, the modified heavy chain constant region comprises a CH1 domain that is a wild-type CH1 domain of an IgG1 or IgG2 isotype (an IgG1 CH1 domain or an IgG2 CH1 domain, respectively). The CH1 domains of the IgG3 and IgG4 isotypes (IgG3 CH1 domain and IgG2 CH1 domain, respectively) can also be used. The CH1 domain can also be a wild-type CH1 domain variant, e.g. Ig1, IgG2, IgG3 domain variant

- 28 040561 or wild-type IgG4. Exemplary CH1 domain variants include A114C and T173C and/or

C131, e.g. C131S.

In some embodiments, the modified heavy chain constant region comprises a CH2 domain that is a wild-type CH2 domain of an IgG1, IgG2, IgG3, or IgG4 isotype (IgG1 CH2 domain, IgG2 CH2 domain, IgG3 CH2 domain, or IgG4 CH2 domain, respectively). The CH2 domain can also be a wild-type CH2 domain variant, for example, a wild-type IgG1, IgG2, IgG3 or IgG4 domain variant. Exemplary CH2 domain variants include those that modulate the biological activity of an antibody Fc region, such as ADCC or CDC, or modulate an antibody's half-life or stability. In one embodiment, the CH2 domain is a human IgG1 CH2 domain with A330S and P331S mutations, wherein the CH2 domain reduces effector function compared to the same CH2 domain without the mutation. Other mutations are further set forth elsewhere in this document.

In some embodiments, the modified heavy chain constant region comprises a CH3 domain that is a wild-type CH3 domain of an IgG1, IgG2, IgG3, or IgG4 isotype (IgG1 CH3 domain, IgG2 CH3 domain, IgG3 CH3 domain, or IgG4 CH3 domain, respectively). The CH3 domain can also be a wild-type CH3 domain variant, for example, a wild-type IgG1, IgG2, IgG3 or IgG4 CH3 domain variant. Exemplary CH3 domain variants include those that modulate the biological activity of an antibody Fc region, such as ADCC or CDC, or modulate an antibody's half-life or stability.

Typically, CH1, hinge, CH2 or CH3 domain variants may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations and/or no more than 10, 9, 8, 7 6, 5, 4, 3, 2, or 1 mutation, or 1-10 or 1-5 mutations, or contain an amino acid sequence that is at least about 75, 80, 85, 90, 95 96, 97, 98, or 99 % is identical to the corresponding wild-type domain (CH1 domain, hinge, CH2 or CH3, respectively) provided that the heavy chain constant region containing the particular variant retains the desired biological activity.

In table. 3 depicts exemplary human heavy chain constant regions containing human CH1, hinge, CH2 and/or CH3 domains, each domain being either a wild-type domain or a variant thereof that confers the desired biological activity on the heavy chain constant region. Empty cell in the table. 3 indicates whether the domain is present or not, and if present, may be of any isotype, such as IgG1, IgG2, IgG3, or IgG4. For example, an antibody containing constant region 1 of the heavy chain in table. 3 is an antibody that contains a heavy chain constant region containing at least an IgG2 hinge, and which may also contain a CH1, CH2 and/or CH3 domain, and if present, the CH1, CH2 and/or CH3 domain is isotype IgG1, IgG2, IgG3 or IgG4. As another example to understand Table. 3, an antibody containing heavy chain constant region 8 is an antibody containing a heavy chain constant region containing an IgG1 CH1 domain and an IgG2 hinge, an IgG1 CH2 domain, and which may or may not contain CH3, which, if present, may be an IgG1 isotype, IgG2, IgG3 or IgG4.

- 29 040561

Table 3 Exemplary Human Heavy Chain Constant Region Configurations

* Modified heavy chain constant region.

In some embodiments, the anti-OX40 antibody comprises the heavy chain constant region shown in Table 1. 3 and may have altered activity relative to the same heavy chain constant region containing antibody that does not contain that particular heavy chain constant region. In some embodiments, an antibody comprising the heavy chain constant region shown in Table 3 or 4 may have altered activity against the same antibody containing a heavy chain constant region that does not contain an IgG2 hinge or the same IgG2 hinge. In some embodiments, an antibody comprising the heavy chain constant region shown in Table 3 or 4 may have altered activity against the same antibody containing a heavy chain constant region that contains a non-IgG2 hinge and contains, for example, an IgG1, IgG3 or IgG4 hinge. In some embodiments, an antibody comprising the heavy chain constant region shown in Table 3 or 4 may have altered activity relative to the same antibody containing a heavy chain constant region that does not contain one or more of the same CH1, hinge, CH2, or CH3 domains. For example, in some embodiments, an antibody comprising the heavy chain constant region shown in Table 3 or 4 may have altered activity relative to the same antibody containing a heavy chain constant region that does not contain an IgG2 hinge and CH1, CH2 and/or CH3 domain of a particular isotype. For example, an antibody containing the constant region 22 of the heavy chain shown in table. 3 may have altered activity relative to (i) the same antibody containing a heavy chain constant region that does not contain an IgG2 hinge, but contains, for example, a non-IgG2 hinge (eg, an IgG1, IgG3, or IgG4 hinge); (ii) the same antibody containing a heavy chain constant region that does not contain an IgG2 hinge and an IgG1 CH1, but contains, for example, a non-IgG2 hinge and/or a non-IgG1 CH1; (iii) the same antibody containing a constant

- 30 040561 a heavy chain region that does not contain an IgG2 and IgG2 CH2 hinge, but contains, for example, a non-IgG2 and/or non-IgG2 CH2 hinge; (iv) the same antibody containing a heavy chain constant region that does not contain an IgG2 and CH3 IgG1 hinge, but contains, for example, a non-IgG2 and/or CH3 non-IgG1 hinge; (v) the same antibody containing a heavy chain constant region that does not contain an IgG2 hinge, IgG1 CH1 and IgG2 CH2, but contains, for example, a non-IgG2 hinge and/or non-IgG1 CH1 and/or non-IgG2 CH2 ; (vi) the same antibody containing a heavy chain constant region that does not contain an IgG2 hinge, an IgG1 CH1 and an IgG1 CH3, but contains, for example, a non-IgG2 hinge and/or a non-IgG1 CH1 and/or a non-IgG1 CH3 ; (vii) the same antibody containing a heavy chain constant region that does not contain an IgG2 hinge, IgG2 CH2 and IgG1 CH3, but contains, for example, a non-IgG2 and/or CH2 non-IgG2 and/or CH3 non-IgG1 hinge; (viii) or the same antibody containing a heavy chain constant region that does not contain an IgG2, CH1 IgG1, CH2 IgG2 and CH3 IgG1 hinge, but contains, for example, a non-IgG2 and/or CH1 non-IgG1 hinge, and/or CH2 non-IgG2, and/or CH3 non-IgG1.

Exemplary modified heavy chain constant regions that can be associated with anti-OX40 variable regions, such as the variable regions described herein, are shown in Table 1. 4, which sets out the identification of each of the domains.

Table 4 Exemplary Modified Heavy Chain Constant Regions

Modified heavy chain constant regions CHI Hinge CH2 snz SEQ ID NO whole MHCCR IgGl-IgG2-IgGl f wild-type IgGl SEQID NO: 202 IgG2/IgGl SEQ ID NO:240 wild-type IgGl SEQ ID NO:204 wild-type IgGl SEQ ID NO:204 SEQ ID NO:244 IgGl-IgG2-IgGl f2 IgGl wild type SEQ ID NO:202 IgG2 wild type SEQ ID NO:238 wild-type IgGl SEQ ID NO:204 wild-type IgGl SEQ ID NO:206 SEQ ID NO:245 IgGl-IgG2CS- IgGl IgG2C219S/I IgGl wild IgGl wild SEQ ID IgGlf wild type gGl type type NO:246

- 31 040561

SEQ ID SEQ ID NO:241 SEQ ID SEQ ID NO:206 NO:202 NO:204 IgGl-IgG2CS- IgGl IgG2 C219S IgGl wild IgGl wild SEQ ID IgGlf2 wild type SEQ ID NO:202 SEQ ID NO:239 type SEQ ID NO:204 type SEQ ID NO:206 NO:247 IgG2-IgGlf IgG2 IgG2/IgGl IgGl wild IgGl wild SEQ ID wild type SEQ ID NO:203 SEQ ID NO:240 type SEQ ID NO:204 type SEQ ID NO:206 NO:248 IgG2-IgGlf2 IgG2 IgG2 wild IgGl wild IgGl wild SEQ ID wild type SEQ ID NO:203 type SEQ ID NO:238 type SEQ ID NO:204 type SEQ ID NO:206 NO:249 IgG2CS-IgGlf IgG2 IgG2C219S/I IgGl wild IgGl wild SEQ ID wild type SEQ ID NO:203 gGl SEQ ID NO:241 type SEQ ID NO:204 type SEQ ID NO:206 NO:250 IgG2CS-IgGlf2 IgG2 IgG2 C219S IgGl wild IgGl wild SEQ ID wild type SEQ ID NO:203 SEQ ID NO:239 type SEQ ID NO :204 type SEQ ID NO:206 NO:251 IgGl-IgG2- IgGl IgG2 wild IgGl IgGl wild SEQ ID IgGl.lf wild type SEQ ID NO:202 type SEQ ID NO:238 A330S/P331 S SEQ ID NO:243 type SEQ ID NO:206 NO: 252 IgGl-IgG2CS- IgGl IgG2 C219S IgGl IgGl wild SEQ ID IgGl.lf wild type SEQ ID NO:202 SEQ ID NO:239 A330S/P331 S SEQ ID NO:243 type SEQ ID NO:206 NO: 253 IgG2-IgGl.lf IgG2 IgG2 wild IgGl IgGl wild SEQ ID wild type SEQ ID NO:203 type SEQ ID NO:238 A330S/P331 S SEQ ID NO:243 type SEQ ID NO:206 NO:254 IgG2CS-IgGl.lf IgG2 IgG2 C219S IgGl IgGl wild SEQ ID wild type SEQ ID NO:203 SEQ ID NO:239 A330S/P331 S SEQ ID NO:243 type SEQ ID NO:206 NO: 255

Additional illustrative modified heavy chain constant regions are shown in Table 1. 5.

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Table 5

Constructions SEO ID NO Description constant areas IgGlf 256 wild-type IgGlf IgGl. If 257 standard inert IgGl.If IgG2.3 258 form IgG2 A(C219S) IgG2.5 259 form IgG2 B (C131S) IgG2.3Gl-KH 260 CH1, upper hinge and lower hinge/upper CH2 IgG2.3, all other IgGlf IgG2.5Gl-KH 261 CH1, upper hinge and lower hinge/upper CH2 IgG2.5, all other IgGlf IgG2.3Gl-AY 262 CH1 and upper hinge IgG2.3, all other IgGlf IgG2.5Gl-AY 263 CH1 and upper hinge IgG2.5, all others IgGlf IgG2.3Gl.lf-KH 264 CH1, upper hinge and lower hinge/upper CH2 IgG2.3, all others IgGl.If IgG2.5Gl.lf-KH 265 CH1, upper hinge and lower hinge/upper CH2 IgG2.5, all others IgGl.If IgG2.5Gl-V27 266 IgG2-B form variant IgG2.3Gl-V27 297 hHC-IgG2-C219S/hHC-IgGlf-S267E

In some embodiments, the anti-OX40 antibody comprises a modified heavy chain constant region comprising an IgG2 hinge comprising any of SEQ ID NOs: 238, 239, 240, 241, and 208-232, or a variant thereof, such as an IgG2 hinge comprising the amino acid sequence, which (i) differs from any of SEQ ID NOs: 238, 239, 240, 241, and 208-232 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from any of SEQ ID NOs: 238, 239, 240, 241, and 208-232 by no more than 5, 4, 3, 2, or 1 amino acid substitution, addition, or deletion; (iii) differs from any of SEQ ID NOs: 238, 239, 240, 241, and 208-232 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and/ or (iv) contains an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 238, 239, 240, 241, and 208-232 , and in any of (i)-(iv) the amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified heavy chain constant region provides an altered activity of the anti-OX40 antibody relative to another heavy chain constant region, e.g., a heavy chain constant region that contains a non-IgG2 hinge, or relative to the same modified heavy chain constant region that contains a non-IgG2 hinge. .

In some embodiments, the anti-OX40 antibody comprises a modified heavy chain constant region comprising an IgG1 CH1 domain comprising SEQ ID NO: 202, or an IgG2 CH1 domain comprising SEQ ID NO: 203, or a variant of SEQ ID NO: 202 or 203, wherein the variant (i) differs from SEQ ID NO: 202 or 203 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 202 or 203 by no more than 5, 4, 3, 2, or 1 amino acid substitution, addition, or deletion; (iii) differs from SEQ ID NO: 202 or 203 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and/or (iv) contains an amino acid sequence that at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 202 or 203, wherein in any of (i)-(iv) the amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified heavy chain constant region may provide altered activity of the anti-OX40 antibody relative to that of another heavy chain constant region, e.g., a heavy chain constant region that contains a non-IgG2 hinge, or relative to the same modified heavy chain constant region that contains a non-IgG2 hinge. -IgG2.

In some embodiments, the anti-OX40 antibody comprises a modified heavy chain constant region comprising an IgG1 CH2 domain comprising SEQ ID NO: 204 or 298, or a variant of SEQ ID NO: 204 or 298, variant (i) being different from SEQ ID NO: ID NO: 204 or 298 for 1, 2, 3, 4 or 5 amino acid substitutions, additions or deletions; (ii) differs from SEQ ID NO: 204 or 298 by no more than 5, 4, 3, 2, or 1 amino acid substitution, addition, or deletion; (iii) differs from SEQ ID NO: 204 or 298 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and/or (iv) contains an amino acid sequence that at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 204 or 298, wherein in any of (i)-(iv) the amino acid substitution may be a conservative amino acid substitution or

- 33 040561 non-conservative amino acid substitution; and wherein the modified heavy chain constant region may provide altered activity of the anti-OX40 antibody relative to that of another heavy chain constant region, e.g., a heavy chain constant region that contains a non-IgG2 hinge, or relative to the same modified heavy chain constant region that contains a non-IgG2 hinge. -IgG2.

In some embodiments, the anti-OX40 antibody comprises a modified heavy chain constant region comprising an IgG1 CH3 domain comprising SEQ ID NO: 206, or a variant of SEQ ID NO: 206, wherein variant (i) differs from SEQ ID NO: 206 by 1, 2 , 3, 4 or 5 amino acid substitutions, additions or deletions; (ii) differs from SEQ ID NO: 206 by no more than 5, 4, 3, 2, or 1 amino acid substitution, addition, or deletion; (iii) differs from SEQ ID NO: 206 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and/or (iv) contains an amino acid sequence that is at least approximately 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 206, wherein in any of (i)-(iv) the amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution ; and wherein the modified heavy chain constant region may provide altered activity relative to that of another heavy chain constant region, e.g., a heavy chain constant region that contains a non-IgG2 hinge, or relative to the same modified heavy chain constant region that contains a non-IgG2 hinge.

The modified heavy chain constant regions may also contain a combination of the CH1, hinge, CH2, and CH3 domains described above.

In some embodiments, the anti-OX40 antibody comprises a modified heavy chain constant region comprising any of SEQ ID NOs: 244-281 or a variant of any of SEQ ID NOs: 244-281, variant (i) being different from any of SEQ ID NOs: 244 -281 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, additions, or deletions; (ii) differs from any of SEQ ID NOs: 244-281 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution, addition, or deletion; (iii) differs from any of SEQ ID NOs: 244-281 in 1-5, 1-3, 1-2, 2-5, 3-5, 1-10 or 5-10 amino acid substitutions, additions or deletions, and/ or (iv) contains an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 244-281, wherein in any of (i) -(iv) the amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified heavy chain constant region may provide altered activity relative to that of another heavy chain constant region, e.g., a heavy chain constant region that contains a non-IgG2 hinge, or relative to the same modified heavy chain constant region that contains a non-IgG2 hinge.

Modified heavy chain constant regions can have (i) a similar, reduced, or increased effector function (e.g., binding to FcyR) relative to the wild-type heavy chain constant region, and (ii) a similar, reduced, or increased half-life (or binding to the FcRn receptor). ) relative to the wild-type heavy chain constant region.

III. Antibodies containing specific serial germline.

In some embodiments, anti-OX40 antibodies described herein comprise a heavy chain variable region from a specific germline immunoglobulin heavy chain gene and/or a light chain variable region from a specific immunoglobulin light chain gene.

As discussed in the examples of this disclosure, human antibodies specific for OX40 have been generated that contain a heavy chain variable region that is a product of or derived from VH gene 1-08, VH gene 6-6, VH gene 5-51, gene 3 -9 VH, DP44 VH gene, 330.3 VH gene, 3-10 VH gene and/or 3-13 VH gene of the human germline. Accordingly, provided herein are isolated monoclonal antibodies specific for human OX40, or antigen-binding portions thereof, comprising a heavy chain variable region that is a product or derivative of a human VH germline gene selected from the group consisting of: VH gene 1-08 , 6-6 VH gene, 5-51 VH gene, 3-9 VH gene, DP44 VH gene, 3-30.3 VH gene, 3-10 VH gene, and/or 3-13 VH gene.

OX40-specific human antibodies have been generated that contain a light chain variable region that is a product of or derived from the VK L5 gene, VK L6 gene, VK L15 gene, VK A27 gene, and/or human germline O14/O4 VK. Accordingly, provided herein are isolated monoclonal antibodies, or antigen-binding portions thereof, comprising a light chain variable region that is a product or derivative of a human germline VK gene selected from the group consisting of VK L5 gene, VK L6 gene, VK L15 gene, gene A27 VK and/or O14/O4VK.

Preferred antibodies described herein are those containing a heavy chain variable region that is a product of or derived from

- 34 040561 of the above human germline VH genes, and also contain a light chain variable region that is a product or derivative of one of the above human germline VK genes.

As used herein, a human antibody contains heavy or light chain variable regions that are the product of or are derived from a particular germline sequence, if the antibody variable regions are derived from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with an antigen of interest, or screening a human immunoglobulin gene library displayed on phage with an antigen of interest. A human antibody that is the product of or is derived from a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e. with the highest % identity) with respect to the human antibody sequence. A human antibody that is the product of or is derived from a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to natural somatic mutations or the deliberate introduction of a site-directed mutation. However, the selected human antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene and contains amino acid residues that identify the human antibody as human compared to the amino acid sequence of germline immunoglobulin from other species ( e.g. mouse germline sequences). In some instances, the human antibody may be at least 95% or even at least 96%, 97%, 98%, or 99% amino acid sequence identical to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, a human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

IV. homologous antibodies.

Provided herein are heavy and light chain variable region antibodies comprising amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-OX40 antibodies described herein.

For example, an isolated anti-OX40 antibody, or antigen-binding portion thereof, may comprise a heavy chain variable region and a light chain variable region, wherein:

(a) the heavy chain variable region contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 318, 17, 28 , 37, 48, 57, 65, 73, 84 and 93, or contains 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15 , 1-20, 1-25 or 1-50 amino acid changes (i.e. amino acid substitutions, additions or deletions) relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 318, 17, 28, 37, 48, 57, 65, 73, 84 and 93;

(b) the light chain variable region contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 29, 30 , 38, 49, 50, 58, 66, 74, 85, 86 and 94, or contains 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10 , 1-15, 1-20, 1-25 or 1-50 amino acid changes (i.e. amino acid substitutions, additions or deletions) relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 29, 30, 38 , 49, 50, 58, 66, 74, 85, 86 and 94;

(c) the antibody specifically binds to OX40; and (d) the antibody exhibits 1, 2, 3, 4, 5, 6 or all of the following functional properties:

(1) binding to soluble human OX40, eg, a KD of 10 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by Biacore;

(2) binding to membrane-bound human OX40, eg, EC50 1 nM or less (eg, 0.01 nM to 1 nM), eg, as measured by FACS;

(3) binding to cynomolgus monkey OX40, eg, binding to membrane-bound cynomolgus monkey OX40, eg, with an EC ₅₀ of 10 nM or less (eg, 0.01 to 10 nM), eg, as measured by FACS;

(4) induction or enhancement of T cell activation as evidenced by (i) increased production of IL-2 and/or IFN-γ in OX40-expressing T cells and/or (ii) increased proliferation of T-

- 35 040561 cells;

(5) inhibition of OX40 ligand binding to OX40, eg, with an EC ₅₀ of 1 nM or less, as measured by FACS, eg, in an assay with hOX40-293 cells;

(6) binding to an epitope on the extracellular portion of mature human OX40 (SEQ ID NO: 2), such as an epitope in the DVVSSKPCKPCTWCNLR region (SEQ ID NO: 178) or DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);

(7) competition for human OX40 binding to 3F4, 14B6-1, 14B6-2, 23H3, 18E9, 8B11, 20B3 and 20C1;

(8) competition for human OX40 binding to 6E1-1, 6E1-2, 14A2-1 and 14A2-2. The antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

In some embodiments, the anti-OX40 antibody, or antigen-binding portion thereof, may comprise a heavy chain and a light chain, wherein:

(a) the heavy chain contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 124 and 125, or contains 1, 2, 3, 4, 5, 1-2, 1-3, 1- 4, 1-5, 1-10, 1-15, 1-20, 1-25 or 1-50 amino acid changes (i.e. amino acid substitutions, additions or deletions) relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 124 and 125;

(b) the light chain contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 and 122, or contains 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25 or 1-50 amino acid changes (i.e. amino acid substitutions, additions or deletions) relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 96, 98 , 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 and 122;

(c) the antibody specifically binds to OX40; and (d) the antibody exhibits 1, 2, 3, 4, 5, 6 or all of the following functional properties:

(1) binding to soluble human OX40, eg, a KD of 10 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by Biacore;

(2) binding to membrane-bound human OX40, eg, EC50 1 nM or less (eg, 0.01 nM to 1 nM), eg, as measured by FACS;

(3) binding to cynomolgus monkey OX40, eg, binding to membrane-bound cynomolgus monkey OX40, eg, with an EC ₅₀ of 10 nM or less (eg, 0.01 to 10 nM), eg, as measured by FACS;

(4) inducing or enhancing T cell activation as evidenced by (i) increased production of IL-2 and/or IFN-γ in OX40 expressing T cells and/or (ii) increased T cell proliferation;

(5) inhibition of OX40 ligand binding to OX40, eg, with an EC50 of 1 nM or less, as measured by FACS, eg, in an assay with hOX40-293 cells;

(6) binding to an epitope on the extracellular portion of mature human OX40 (SEQ ID NO: 2), such as an epitope in the DVVSSKPCKPCTWCNLR region (SEQ ID NO: 178), or

DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);

(7) competition for human OX40 binding to 3F4, 14B6-1, 14B6-2, 23H3, 18E9, 8B11, 20B3 and 20C1;

(8) competition for human OX40 binding to 6E1-1, 6E1-2, 14A2-1 and 14A2-2. Also provided are anti-OX40 antibodies containing VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and/or VLCDR3 that differ from the corresponding CDR in 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11 , 20B3, 14A2-1, 14A2-2 and/or 20C1, by 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 amino acid changes (i.e. amino acid substitutions , additions or deletions). In some embodiments, the antibody contains 1-5 amino acid changes in each of 1, 2, 3, 4, 5, or 6 CDRs relative to the corresponding sequence in 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and/or 20C1. In some embodiments, the antibody contains a total of 1-5 amino acid changes in all CDRs compared to the CDRs in 3F4, 14B6-1, 14B6-2, 23H3, 6E11, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and/or 20C1.

Antibodies containing sequences with homology to those in 3F4, 14B6 (14B61 and 14B6-2), 23H3, 6E1 (6E1-1 and 6E1-2), 18E9, 8B11, 20B3, 14A2 (14A2-1 and 14A2-2) and 20C1, for example, the V _H and V _L regions in SEQ ID NOs: 17 and 18; 28 and 29; 28 and 30; 37 and 38; 48 and 49; 48 and 50; 57 and 58; 65 and 66; 73 and 74; 84 and 85; 84 and 86; 93 and 94, respectively, or heavy and light chains in SEQ ID NOs: 95 and 96; 97 and 98; 99 and 100; 101 and 102; 103 and 104; 105 and 106; 107 and 108; 109 and 110; 111 and 112; 113 and 114; 115 and 116; 117 and 118; 119 and 120; 121 and 122; 123 and 116; 124 and 116; and 125 and 116, respectively, or CDRs can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ ID nO: 17, 28, 37, 48, 57, 65, 73, 84 and 93 and/or SEQ ID NOs: 18, 29, 30, 38, 49, 50,

- 36 040561

58, 66, 74, 85, 86 and 94, or SEQ ID NOs: 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,

124 and 125 and/or SEQ ID NOs: 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 and 122 followed by testing for an encoded altered antibody to preserve function (i.e. the functions set forth in (1)-(7) above using the functional analyzes described herein.

V. Antibodies with conservative modifications.

Anti-OX40 antibodies provided herein may comprise a heavy chain variable region comprising the sequences CDR1, CDR2, and CDR3, and a light chain variable region comprising the sequences CDR1, CDR2, and CDR3, wherein one or more of these CDR sequences comprise said amino acid sequences based on the antibodies described herein (e.g., 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1) or conservative modifications thereof, and antibodies retain the desired functional properties of the anti-OX40 antibodies described herein. Accordingly, an anti-OX40 antibody, or antigen-binding portion thereof, may comprise a heavy chain variable region comprising the sequences CDR1, CDR2, and CDR3, and a light chain variable region comprising the sequences CDR1, CDR2, and CDR3, wherein:

(a) the heavy chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 13, 21, 33, 41, 53, 61, 69, 77, and 89 and their conservative modifications, e.g., 1 , 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions;

(b) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83, and 92, and their conservative modifications, for example, 1, 2, 3, 4, 5, 12, 1-3, 1-4 or 1-5 conservative amino acid substitutions;

(c) the antibody specifically binds to OX40; and (d) the antibody exhibits 1, 2, 3, 4, 5, 6 or all of the following functional properties:

(1) binding to soluble human OX40, eg K _D 10 nM or less (eg 0.01 to 10 nM), eg as measured by Biacore;

(2) binding to membrane-bound human OX40, eg, EC ₅₀ 1 nM or less (eg, 0.01 to 1 nM), eg, as measured by FACS;

(3) binding to cynomolgus monkey OX40, eg, binding to membrane-bound cynomolgus monkey OX40, eg, with an EC ₅₀ of 10 nM or less (eg, 0.01 to 10 nM), eg, as measured by FACS;

(4) inducing or enhancing T cell activation as evidenced by (i) increased production of IL-2 and/or IFN-γ in OX40 expressing T cells and/or (ii) increased T cell proliferation;

(5) inhibition of OX40 ligand binding to OX40, eg, with an EC ₅₀ of 1 nM or less, as measured by FACS, eg, in an assay with hOX40-293 cells;

(6) binding to an epitope on the extracellular portion of mature human OX40 (SEQ ID NO: 2), such as an epitope in the DVVSSKPCKPCTWCNLR region (SEQ ID NO: 178), or

DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);

(7) competition for human OX40 binding to 3F4, 14B6-1, 14B6-2, 23H3, 18E9, 8B11, 20B3 and 20C1;

(8) competition for human OX40 binding to 6E1-1, 6E1-2, 14A2-1 and 14A2-2. In a preferred embodiment, the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 12, 20, 32, 40, 52, 60, 68, 76, 88, and 317, and conservative modifications thereof. eg 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions; and the light chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 15, 23, 26, 35, 43, 46, 55, 63, 71, 79, 82, and 91 and conservative modifications thereof. eg 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions. In another preferred embodiment, the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 11, 19, 31, 39, 51, 59, 67, 75, and 87, and conservative modifications thereof, for example, 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions; and the light chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62, 70, 78, 81 and 90 and conservative modifications thereof eg 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, or 1-5 conservative amino acid substitutions.

In various embodiments, the antibodies may be, for example, human antibodies, humanized antibodies, or chimeric antibodies.

Conservative amino acid substitutions can also be made in parts of the antibodies other than or in addition to the CDRs. For example, conservative amino acid modifications can be

- 37 040561 are made in the frame region or in the Fc region. The variable region or heavy or light chain may contain 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25 or 1-50 conservative amino acid substitutions relative to the sequences provided herein for antibodies to

OX40. In some embodiments, an anti-OX40 antibody contains a combination of conservative and non-conservative amino acid modifications.

VI. Competing antibodies and the same epitope-binding antibodies.

Also contemplated herein are antibodies that compete for binding to OX40 with the anti-OX40 antibodies described herein (e.g., antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3 , 14A2-1, 14A2-2 and 20C1). Such competing antibodies can be identified based on their ability to competitively inhibit OX40 binding of one or more monoclonal antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1 , 14A2-2 and 20C1 in standard OX40 binding assays. For example, standard ELISA assays or competitive ELISA assays may be used in which recombinant human OX40 protein is immobilized on a plate, various concentrations of unlabeled first antibody are added, the plate is washed, a second labeled antibody is added, washed, and the amount of bound label is measured. If increasing the concentration of the unlabeled (first) antibody (also called blocking antibody) inhibits the binding of the labeled (second) antibody, the first antibody is said to inhibit the binding of the second antibody to the target on the plate, or is said to compete for binding with the second antibody. Additionally or alternatively, the SPR BIACORE® assay can be used to assess the ability of antibodies to compete. The ability of the test antibody to inhibit the binding of the anti-OX40 antibody described herein to OX40 indicates that the test antibody can compete with the antibody for binding to OX40.

Accordingly, provided herein are anti-OX40 antibodies that inhibit the binding of anti-OX40 antibodies described herein to OX40 on cells, e.g., activated T cells, at least 10, 20, 30, 40, 50 55, 60, 65 , 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% using, for example, FACS as described in the examples.

In other embodiments, provided herein are anti-OX40 antibodies that bind to the same epitope as one or more of the anti-OX40 antibodies described herein (e.g., antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1 , 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1) as determined using art-known epitope mapping methods, as such, which are described below.

Methods known in the art for epitope mapping include, for example, structural methods such as X-ray crystal structure determination (e.g., WO 2005/044853), molecular modeling, and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination. frequencies of hydrogen-deuterium exchange of labile amide hydrogens in OX40 when free and complexed with the antibody of interest (Zinn-Justin et al. Biochemistry 1992;31:11335-47; Zinn-Justin et al. Biochemistry 1993;32,6884 -91).

For X-ray crystallography, crystallization can be carried out using any method known in the art (e.g., Giege et al. Acta Crystallogr 1994; D50:339-50; McPherson, Eur. J. Biochem. 1990; 189:1-23), including microchip (eg, Structure 19976;5:1269-74), hanging drop method by vapor diffusion (eg, McPherson, J. Biol. Chem. 1976;251:6300-3), seeding, and dialysis. It is desirable to use a protein preparation at a concentration of at least about 1 mg/ml, and preferably from about 10 mg/ml to about 20 mg/ml. Crystallization can best be achieved in a precipitant solution containing 1000-20000 polyethylene glycol (PEG, average molecular weight from about 1000 to about 20000 Da), preferably from about 5000 to about 7000 Da, more preferably about 6000 Da, with concentrations ranging from about 10% to about 30% (w/v). It may also be desirable to include a protein stabilizing agent, such as glycerol, at a concentration of from about 0.5% to about 20%. A suitable salt, such as sodium chloride, lithium chloride, or sodium citrate, may also be desirable in the non-solvent solution, preferably at a concentration in the range of from about 1 mM to about 1000 mM. The precipitate is preferably buffered to a pH of about 3.0 to about 5.0, preferably about 4.0. The specific buffers used in the precipitant solution may vary and are well known in the art (Scopes, Protein Purification: Principles and Practice, Third ed., (1994) Springer-Verlag, New York). Examples of such buffers include, without limitation, HEPES, Tris, MES, and acetate. Crystals can grow over a wide range of temperatures including 2, 4, 8 and 26°C. Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc., see e.g. Blundell & Johnson , Meth Enzymol 1985;114 & 115, H.W. Wyckoff et al., eds., Academic Press, U.S. Patent Application Publication No. 2004/0014194) and

- 38 040561

BUSTER (Bricogne, Acta Cryst. 1993; D49:37-60; Bricogne, Meth Enzymol 1997; 276A:361-423; Carter &

Sweet, eds.; Roversi et al., Acta Cryst. 2000;D56:1313-23).

Other epitope mapping methods monitor antibody binding to antigenic fragments or mutated variations of an antigen, where loss of binding due to modification of an amino acid residue in the antigen sequence is often considered indicative of an epitope component. One such method is alanine-scanning mutagenesis as described, for example, in Cunningham and Wells, Science 1989;244:1081-5. Another suitable method is deep mutational scanning (see, for example, Araya et al., Trends in Biotechnology 2011;29:435-42; Forsyth et al., mAbs 2013;5:523-32).

Additionally or alternatively, computational combinatorial epitope mapping techniques can be used, including conformational discontinuous epitope mapping.

Additionally or alternatively, epitope mapping can be achieved by examining antibody binding to peptides containing OX40 fragments, eg undenatured or denatured fragments.

A series of overlapping peptides spanning the OX40 sequence (e.g., human OX40) can be synthesized and screened for binding, e.g., in a direct ELISA, competitive ELISA (where the peptide is evaluated for its ability to prevent antibody from binding to OX40 bound to a well of a microtiter plate) or on a chip. Other methods rely on the ability of the antibody of interest to affinity isolate specific short peptides (either in native three-dimensional form or in denatured form) from combinatorial phage display peptide libraries. The peptides are then considered as leaders to determine the epitope recognized by the antibody used to screen the peptide library.

Epitopes can also be identified using MC-based protein footprinting such as hydrogen/deuterium exchange mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP). HDX-MS can be performed, for example, as described in the examples in this document and Wei et al., Drug Discovery Today 2014; 19:95. FPOP can be performed, for example, as described by Hambley et al. (J American Soc Mass Spectrometry 2005; 16:2057).

Antibodies that compete for binding with the anti-OX40 antibodies described herein can be generated and identified using methods known in the art. For example, mice can be immunized with human OX40 described herein, hybridomas generated, and the resulting monoclonal antibodies screened for the ability to compete with the antibody described herein for binding to OX40 using the methods described above.

Antibodies that bind to the same epitope as anti-OX40 antibodies described herein can be generated by immunizing mice with a smaller fragment of OX40 containing the epitope to which the antibody binds. An epitope or an epitope-containing region can be identified using the methods described above. Alternatively, the method of Jespers et al. (Biotechnology 1994; 12:899) can be used to guide the selection of antibodies that recognize the same epitope and therefore exhibit similar properties to the anti-OX40 antibodies described herein. For example, when using phage display, an anti-OX40 antibody heavy chain is first paired with a repertoire of (preferably human) light chains to select an OX40 binding antibody, and then a new light chain is paired with a repertoire of (preferably human) heavy chains to select (preferably human) a human) OX40-binding antibody recognizing the same epitope or epitope region on OX40 as an anti-OX40 antibody described herein. Alternatively, variants of an antibody described herein can be generated by mutagenesis of cDNA encoding the heavy and light chains of the antibody.

In some embodiments, provided herein are antibodies that bind to all or part of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) corresponding to amino acid residues 46-62 of mature human OX40 (SEQ ID NO: 2) as determined by the methods in the examples.

In some embodiments, anti-OX40 antibodies described herein bind to all or part of the SQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR sequence (SEQ ID NO: 182) as determined by the methods in the examples.

In other embodiments, anti-OX40 antibodies described herein bind to all or part of the sequence of PCKPCTWCNLR (SEQ ID NO: 183) as determined by the methods in the examples.

In other embodiments, anti-OX40 antibodies that bind to all or part of the DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) sequence further bind to all or part of the QLCTATQDTVCR (SEQ ID NO: 184) sequence as determined by the methods in the examples.

In additional embodiments, anti-OX40 antibodies described herein bind to all or part of the sequence SQNTVCRPCGPGFYN (SEQ ID NO: 185),

- 39 040561 as determined by the methods in the examples.

In additional embodiments, the anti-OX40 antibody binds in the region

DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179) corresponding to amino acid residues 89-124 of mature human OX40 (SEQ ID NO: 2) as determined by the methods in the examples,

VII. Engineered and modified antibodies.

Regions VH and VL.

Also provided herein are engineered and modified antibodies that can be produced using an antibody containing one or more of the V _H and/or VL sequences disclosed herein as a starting material for constructing the modified antibody, wherein the modified antibody may be characterized by altered relatively original antibody properties. An antibody can be constructed by modifying one or more residues in one or both of the variable regions (ie, V _H and/or VL), for example, within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, the antibody can be engineered by modifying residues within the constant region(s), eg, to change the effector function(s) of the antibody.

One type of variable region design that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues that are located in the six complementarity determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences within the CDR are more diverse between individual antibodies than those outside the CDR. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific reference antibodies by constructing expression vectors that include CDR sequences from a particular reference antibody grafted onto framework sequences from another antibody with different properties ( see, for example, Riechmann L. et al.(1998) Nature 332:323-327; Jones P. et al. (1986) Nature 321:522525; Queen C. et al. (1989) Proc. Natl. Acad. See U.S.A. 86:10029-10033, U.S. Patent No. 5,225,539 to Winter and U.S. Patents Nos. 5,530,101, 5,558,589, 5,697,662, and 6,180,370 to Queen et al.).

Accordingly, another embodiment relates to a monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising the sequences CDR1, CDR2, and CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID SEQ ID NOs: 11-13; 19-21; 31-33; 39-41; 51-53; 59-61; 67-69; 74-77 and 87-89, respectively, and a light chain variable region containing the sequences CDR1, CDR2 and CDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-16; 22-24; 25-27; 34-36; 42-44; 45-47; 54-56; 62-64; 70-72; 78-80; 81-83 and 90-92, respectively. Thus, such antibodies contain the CDR sequences VH and VL of monoclonal antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and 20C1, but may contain different framework sequences.

Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences for the human heavy and light chain variable region genes can be found in the VBase human germline sequence database (available online at: www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson IM, et al. (1992) The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of V _H Segments with Different Hypervariable Loops J. Mol. Biol. 227:776-798 and Cox JPL et al. (1994) A Directory of Human Germ-line VH Segments Reveals a Strong Bias in their Usage Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference.

Preferred framework sequences for use in the antibodies described herein are those that are structurally similar to the framework sequences used in the antibodies described herein. V _H CDR1 sequences 2 and 3 and V _L CDR1 sequences 2 and 3 may be grafted onto framework regions that contain a sequence identical to that found in the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequences may be grafted onto framework regions. regions that contain up to 20 preferentially conservative amino acid substitutions compared to germline sequences. For example, it has been found useful in some cases to mutate residues in framework regions to maintain or enhance the antigen-binding capacity of an antibody (see, for example, US Pat.

The engineered antibodies described herein include those in which modifications have been made to the framework residues within V _H and/or V _L , for example, to improve

- 40 040561 properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to mutate back to the original form one or more scaffold residues to the corresponding germline sequence. More specifically, an antibody that has undergone a somatic mutation may contain scaffold residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody backbone sequences with the germline sequences from which the antibody is derived. To return the framework sequences to their germline configuration, somatic mutations can be mutated back to their original form in the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such mutated antibodies should also be covered.

Another type of framework modification involves mutating one or more residues in the framework region, or even within one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as deimmunization and is described in more detail in US Patent Publication No. 20030153043 to Carr et al.

Another type of variable region modification is the mutation of amino acid residues in CDR regions to improve one or more binding properties (eg, affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding or other functional property of interest can be assessed in in vitro or in vivo assays as described herein and provided in the examples . Preferably conservative modifications are introduced (as described above). Mutations can be amino acid additions, deletions or, preferably, substitutions. In addition, as a rule, no more than one, two, three, four or five residues in the CDR region change.

Accordingly, anti-OX40 monoclonal antibodies, or antigen-binding portions thereof, are also provided herein, comprising a heavy chain variable region comprising: (a) a CDR1 VH region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 19, 31 , 39, 51, 59, 67, 75 and 87, or an amino acid sequence containing one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 11, 19, 31, 39, 51 , 59, 67, 75 and 87; (b) a CDR2 V _H region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 20, 32, 40, 52, 60, 68, 76, 88 and 317, or an amino acid sequence containing one, two , three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 12, 20, 32, 40, 52, 60, 68, 76 and 88; (c) a CDR3 VH region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 21, 33, 41, 53, 61, 69, 77 and 89, or an amino acid sequence containing one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 13, 21, 33, 41, 53, 61, 69, 77 and 89; (d) a CDR1 V _L region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62, 70, 78, 81 and 90, or an amino acid sequence, containing one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62, 70, 78, 81 and 90; (e) a VL CDR2 region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 23, 26, 35, 43, 46, 55, 63, 71, 79, 82, and 91, or an amino acid sequence containing one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 15, 23, 26, 35, 43, 46, 55, 63, 71, 79, 82, and 91; and (f) a VL CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83, and 92, or an amino acid sequence, containing one, two, three, four or five amino acid substitutions, deletions or additions, compared to SEQ ID NOs: 16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83 and 92.

Methionine residues in the CDRs of antibodies can be oxidized, leading to potential chemical degradation and subsequent loss of antibody efficacy. Accordingly, one or more methionine residues in the CDRs of the heavy and/or light chains of the anti-OX40 antibodies described herein may be replaced with amino acid residues that do not undergo oxidative degradation.

Similarly, deamidation sites can be removed from antibodies, especially in CDRs.

Potential glycosylation sites in the antigen-binding domain are preferably removed to prevent glycosylation that may interfere with antigen binding. See, for example, US Pat. No. 5,714,350.

Targeted antigen binding.

In various embodiments, the antibodies described herein are modified to selectively block antigen binding in tissues and environments where antigen binding would be detrimental, but allow antigen binding where it would be beneficial. According to one

In an embodiment, a blocking peptide mask is formed that specifically binds to the antigen-binding surface of the antibody and prevents binding to the antigen, the mask being linked to each of the binding arms of the antibody by a peptidase-cleavable linker. See, for example, US patent No. 8518404 in the name of CytomX. Such constructs are useful in the treatment of cancers in which protease levels are significantly increased in the tumor microenvironment compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows the masking/blocking peptide to dissociate, allowing the antigen to bind selectively in the tumor rather than in peripheral tissues where antigen binding can cause undesirable side effects.

Alternatively, according to an appropriate embodiment, a bivalent binding compound (masking ligand) containing two antigen-binding domains is prepared that binds both to the antigen-binding surfaces of the (bivalent) antibody and interferes with antigen binding, in which the masks of the two binding domains are linked to each other (but not with an antibody) a cleavable linker, such as a cleavable peptidase. See, for example, International Patent Application Publication No. WO 2010/077643 to Tegopharm Corp. Masking ligands may contain or be derived from the antigen to which the antibody is intended to bind, or may be formed independently. Such masking ligands are useful in the treatment of cancers in which protease levels are significantly increased in the tumor microenvironment compared to non-tumor tissues. Selective cleavage of the cleavable linker in the tumor microenvironment allows the two binding domains to dissociate from each other, reducing the avidity of the antigen-binding surfaces of the antibody. The resulting dissociation of the masking ligand from the antibody allows selective binding of the antigen in the tumor rather than in peripheral tissues where antigen binding can cause undesirable side effects.

Fc and modified Fc.

In addition to the activity of the therapeutic antibody resulting from the binding of the antigen-binding domain to the antigen (eg, blocking the cognate ligand or receptor protein in the case of antagonistic antibodies or induced signaling in the case of agonistic antibodies), the Fc portion of the antibody interacts with the immune system, usually in complex ways. for any number of biological effects. Effector functions, such as the immunoglobulin Fc region, are responsible for many important antibody functions, such as antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP), leading to the killing of target cells, albeit through different mechanisms. There are five major classes or isotypes of the heavy chain constant region (IgA, IgG, IgD, IgE, IgM), each with characteristic effector functions. These isotypes can be further divided into subclasses, for example, IgG is divided into four subclasses known as IgG1, IgG2, IgG3 and IgG4. IgG molecules interact with three classes of Fcy receptors (FcyR) specific for the IgG class of antibodies, namely FcyRI, FcyRII and FcyRIII. Important sequences for IgG binding to FcyR receptors have been reported to be in the CH2 and CH3 domains. The serum half-life of an antibody depends on the ability of the antibody to bind to the neonatal Fc receptor (FcRn).

Anti-OX40 antibodies described herein may contain the variable domains of the present invention in combination with constant domains containing various Fc regions selected based on the biological activity (if any) of the antibody for the intended use. Salfeld (2007) Nat. Biotechnol. 25:1369 For example, human IgG can be divided into four subclasses: IgG1, IgG2, IgG3 and IgG4, and each of them contains an Fc region having a unique profile for binding to one or more Fcy receptors (activating receptors FcyRI (CD64), FcyRIIA, FcyRIIC (CD32), FcyRIIIA and FcyRIIIB (CD16) and inhibitory receptor FcyRIIB) and for the first complement component (C1q). Human IgG1 and IgG3 bind to all Fcy receptors; IgG2 binds FcyRIIA _H131 and, with lower affinity, FcyRIIA _R131 and FcyRIIIA _V158 ; IgG4 binds to FcyRI, FcyRIIA, FcyRIIB, FcyRIIC and FcyRIIIA _V158 ; and the inhibitory receptor FcyRIIB has lower affinity for IgG1, IgG2 and IgG3 than all other Fcy receptors. Bruhns et al. (2009) Blood 113:3716. Studies have shown that FcyRI does not bind to IgG2, and FcyRIIIB does not bind to IgG2 or IgG4. In general, with respect to ADCC activity, human IgG1 = IgG3 >> IgG4 = IgG2. As a consequence, for example, an IgG1 constant domain rather than IgG2 or IgG4 may be selected for use in a drug where ADCC is desired; IgG3 can be selected to activate FcyRIIIA-expressing NK cells, monocytes, or macrophages; and IgG4 may be selected if the antibody is to be used to desensitize allergic patients. IgG4 can also be selected if it is desired that the antibody lack all effector function.

Accordingly, the anti-OX40 variable regions described herein may be linked (e.g., covalently linked or fused) to an Fc, e.g., IgG1, IgG2, IgG3, or IgG4 Fc, which may be of any allotype or isoalotype, e.g., for IgG1: Glm , Glm1(a), Glm2(x), Glm3(f),

- 42 040561

Glm17(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1),

G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v). See, for example, Jefferis et al. (2009) mAbs 1:1). The choice of allotype can be influenced by potential immunogenicity problems, for example, to minimize the formation of antibodies to drugs.

In certain embodiments, the anti-OX40 variable regions described herein are associated with an Fc that binds to one or more activating Fc receptors (FcyI/CD64, FcyIIa/CD32, or FcyIIIa/CD16) and thereby stimulates ADCC and can cause T depletion. -cells. In specific embodiments, the anti-OX40 variable regions described herein are linked to an Fc that causes depletion. In other embodiments, the anti-OX40 variable regions described herein are linked to a human IgG1 or IgG3 Fc, ie. antibodies are characterized by the isotype IgG1 or IgG3. In other embodiments, the anti-OX40 antibodies are debilitating antibodies. For example, they can deplete T _reg cells that are in the tumor microenvironment (and thereby enhance antitumor activity), but not significantly deplete Tf cells that are in the tumor microenvironment and mediate the antitumor effect, and/or not significantly deplete T _reg cells and T _eff that are outside the tumor, for example, on the periphery. In other embodiments, anti-OX40 antibodies are characterized by an isotype (either naturally occurring or not occurring (e.g., including mutation(s)) that promotes depletion or elimination of T _reg cells at the tumor site and concomitant activation of T _eff cells.

In other embodiments, anti-OX40 antibodies create an increased ratio of Tef to T _reg at the tumor site, indicative of strong antitumor activity and preferably without significant depletion of T _reg and T _eff · cells that are outside the tumor, eg, in the periphery.

In some embodiments, anti-OX40 antibodies block the immunosuppressive activity of T _reg . In other embodiments, the anti-OX40 antibodies comprise an Fc receptor with reduced or eliminated binding to FcRs, such as reduced binding to activating FcRs. In some embodiments, the anti-OX40 antibodies comprise an Fc that binds to or improves binding to FcRIIb, which can provide enhanced agonism. See, for example, International Application Publication WO 2012/087928; Li & Ravetch (2011) Science 333:1030; Wilson et al. (2011) Cancer Cell 19:101; White et al. (2011) J. Immunol. 187:1754.

The anti-OX40 variable regions described herein can be associated with a non-naturally occurring Fc region, e.g., an effector-deficient or substantially effector-deficient Fc (e.g., human IgG2 or IgG4) or, alternatively, an Fc with improved binding to one or more activating Fc receptors (FcyI, FcyIIa or Fcyllla), such as enhancing T _reg depletion in the tumor environment.

The variable regions described herein can be linked to an Fc containing one or more modifications, typically to change one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, an antibody described herein may be chemically modified (eg, one or more chemical moieties may be attached to the antibody), or it may be modified to alter its glycosylation in order to alter one or more of the functional properties of the antibody. Each of these embodiments is described in more detail below. The numbering of residues in the Fc region refers to the EU index in Kabat. Sequence variants disclosed herein are provided by reference to the residue number followed by the amino acid that is substituted for the natural amino acid, optionally preceding the natural residue at that position. If multiple amino acids can be present at a given position, for example if the sequences differ between naturally occurring isotypes or if multiple mutations can be substituted at a position, they are separated by a slash (eg X/Y/Z).

For example, modifications can be made in the Fc region to produce an Fc variant with (a) increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complementary-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q and/or (d) increased or decreased affinity for the Fc receptor relative to the parent Fc. Such Fc region variants will typically contain at least one amino acid modification in the Fc region. It is believed that a combination of amino acid modifications is particularly desirable. For example, a variant Fc region may include two, three, four, five, and so on. substitutions, for example, at certain positions in the Fc region identified herein. Exemplary Fc sequence variants are disclosed herein and are also presented in US Pat. Nos. 5,624,821; 6277375; 6737056; 6194551; 7317091; 8101720; PCT Patent Publications WO 00/42072; WO 01/58957; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; W005/070963; WO 05/040217, WO 05/092925 and WO 06/020114.

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Decreased effector function.

ADCC activity can be reduced by changing the Fc region. In some embodiments, sites that affect binding to Fc receptors can be removed, preferably sites other than recycling receptor binding sites. In other embodiments, the Fc region may be modified to remove the ADCC site. ADCC sites are known in the art; see, for example, Sarmay et al. (1992) Molec. Immunol. 29 (5): 633-9 for ADCC sites in IgG1. In one embodiment, the G236R and L328R human IgG1 variant effectively abolishes FcyR binding. Horton et al. (2011) J. Immunol. 186:4223 and Chu et al. (2008) Mol. Immunol. 45:3926. In other embodiments, the Fc having reduced FcyR binding consisted of the amino acid substitutions L234A, L235E and Gross et al. (2001) Immunity 15:289.

CDC activity can also be reduced by modifying the Fc region. Mutations at positions D270, K322, P329 and P331 of IgG1, in particular alanine mutations D270A, K322A, P329A and P331A, significantly reduce the ability of the corresponding antibody to bind C1q and activate complement. Idusogie et al. (2000) J. Immunol. 164:4178; WO 99/51642. Modification of position 331 in IgG1 (eg P331S) has been shown to decrease complement fixation. Tao et al. (1993J J. Exp. Med. 178:661 and Canfield & Morrison (1991) J. Exp. Med. 173:1483. In another example, one or more amino acid residues at amino acid positions 231 to 239 are altered in such a way as to reduce the ability antibodies to fix complement WO 94/29351.

In some embodiments, the Fc with restored complementary fixation contains the amino acid substitutions A330S and P331S. Gross et al. (2001) Immunity 15:289.

For applications in which the effector function should be avoided altogether, for example where antigen binding alone is sufficient to produce the desired therapeutic effect and the effector function only leads to (or increases the risk of) unwanted side effects, IgG4 antibodies or antibodies or fragments can be used. devoid of the Fc region or a substantial portion thereof, or the Fc may be mutated to completely eliminate glycosylation (eg, N297A). Alternatively, a hybrid construct of human IgG2 (CH1 domain and hinge region) and human IgG4 ( _CH2 and CH3 domains) has been created that lacks effector function, lacks the ability to bind to FcyR (e.g., IgG2), and is unable to activate complement (e.g., IgG4). Rother et al. (2007) Nat. Biotechnol. 25:1256. See also Mueller et al. (1997) Mol. Immunol. 34:441; Labrijn et al. (2008) Curr. Op. Immunol. 20:479 (discussion of Fc modifications to reduce effector function in general).

In other embodiments, the Fc region is altered by replacing at least one amino acid residue with another amino acid residue to reduce the overall effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 may be substituted with a different amino acid residue such that the antibody has reduced affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. . The effector ligand for which the affinity is changed can be, for example, the Fc receptor (residues 234, 235, 236, 237, 297) or the d component of complement (residues 297, 318, 320, 322). US patents No. 5624821 and 5648260, both in the name of Winter et al.

One early patent application proposed modifications in the IgG Fc region to reduce binding to FcyRI to reduce ADCC (234A; 235E; 236A; G237A) or to block binding to complement component C1q to eliminate CDC (E318A or V/K320A and K322A/Q ). WO 88/007089. See also Duncan & Winter (1988) Nature 332:563; Chappel et al. (1991) Proc. Nat'l Acad. sci. (USA) 88:9036 and Sondermann et al. (2000) Nature 406:267 (discussion of the effect of these mutations on FcyRIII binding).

Fc modifications that reduce effector function also include substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, such as 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R. The Fc variant may contain 236R/328R. Other modifications to reduce FcyR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P and 234V. These and other modifications are discussed in Strohl (2009) Current Opinion in Biotechnology 20:685-691. Effector functions (both ADCC and complement activation) can be reduced while maintaining neonatal FcR binding (maintaining half-life) by mutating IgG residues at one or more positions 233-236 and 327-333, such as E233P, L234V, L235A, optional G236A, A327G, A330S and P331S in IgG1; E233P, F234V, L235A, optionally G236A in IgG4; and A330S and P331S in IgG2. See Armor et al. (1999) EUR. J. Immunol. 29:2613; publication of international application WO 99/58572. Other mutations that reduce effector function include L234A and L235A in IgG1 (Alegre et al. (1994) Transplantation 57:1537); V234A and G237A in IgG2 (Cole et al. (1997) J. Immunol. 159:3613, see also US Pat. No. 5,834,597); and S228P and L235E for IgG4 (Reddy et al. (2000) J. Immunol. 164:1925). Another combination of mutations to reduce effector function in human IgG1 includes L234F, L235E, and

P331S. Oganesyan et al. (2008) Acta Crystallogr. D Biol. Crystallogr. 64:700. See in general Labrijn et gal.

(2008) Curr. Op. Immunol. 20:479. Additional mutations found to reduce effector function in the context of the Fc (IgG1) fusion protein (abatacept) are C226S, C229S and P238S (EC residue numbering). Davis et al. (2007) J. Immunol. 34:2204.

Other Fc variants with reduced ADCC and/or CDC are described in Glaesner et al. (2010) Diabetes Metab. Res. Rev. 26:287 (F234A and L235A to reduce ADCC and ADCP in IgG4); Hutchins et al. (1995) Proc. Nat'l Acad. sci. (USA) 92:11980 (F234A, G237A and E318A in IgG4); An et al. (2009) MAbs 1:572 and US Patent Application Publication 2007/0148167 (H268Q, V309L, A330S and P331S in IgG2); McEarchern et al. (2007) Blood 109:1185 (C226S, C229S, E233P, L234V, L235A in IgG1); Vafa et al. (2014) Methods 65:114 (V234V, G237A, P238S, H268A, V309L, A330S, P331S in IgG2).

In some embodiments, an Fc is selected that has essentially no effector function, i.e. it is characterized by reduced FcyR binding and reduced complement fixation. An exemplary Fc, for example, an IgG1 Fc that lacks effector function, contains the following five mutations: L234A, L235E, G237A, A330S, and P331S. Gross et al. (2001) Immunity 15:289. Exemplary heavy chains containing these mutations are listed in the sequence listing as described in Table 1. 23 (for example, SEQ ID NO: 11). These five substitutions can be combined with N297A to eliminate glycosylation.

Strengthening the effector function.

Alternatively, ADCC activity can be increased by modifying the Fc region. With respect to ADCC activity, IgG1 = IgG3 >> IgG4 = human IgG2, therefore the constant domain of IgG1 rather than IgG2 or IgG4 may be selected for use in a drug where ADCC is desired. Alternatively, the Fc region can be modified to increase antibody-dependent cellular cytotoxicity (ADCC) and/or to increase affinity for the Fcy receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245,

247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,

286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325,

326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416,

419, 430, 433, 434, 435, 436, 437, 438 or 439. See International Patent Publication WO 2012/142515; see also WO 00/42072. Exemplary replacements include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Illustrative variants include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F/324T. For example, human IgG1 Fcs containing the G236A variant, which can optionally be combined with I332E, have been shown to increase the FcyIIA/FcyIIB binding affinity ratio by approximately 15-fold. Richards et al. (2008) Mol. cancer therapy. 7:2517; Moore et al. (2010) mAbs 2:181. Other modifications to improve FcyR and complement interactions include, without limitation, substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I, and 396L. These and other modifications are discussed in Strohl (2009) Current Opinion in Biotechnology 20:685-691. Specifically, both ADCC and CDC can be enhanced by changes in the E333 position in IgG1, eg E333A. Shields et al. (2001) J. Biol. Chem. 276:6591. The use of P247I and A339D/Q mutations to increase effector function in IgG1 is described in WO 2006/020114, and D280H, K290S±S298D/V is disclosed in WO 2004/044455. Variants K326A/W and E333A/S have been shown to increase effector function in human IgG1 and E333S in IgG2. Idusogie et al. (2001) J. Immunol. 166:2571.

In particular, human IgG1 binding sites for FcyR1, FcyRII, FcyRIII and FcRn have been described and variants with improved binding have been described. Shields et al. (2001) J. Biol. Chem. 276:6591-6604. Specific mutations at positions 256, 290, 298, 333, 334 and 339 have been shown to improve binding to FcyRIII, including the combination mutants T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A (with improved binding FcyRIIIa and ADCC activity). Other IgG1 variants with highly enhanced FcyRIIIa binding have been identified, including those with S239D/I332E and S239D/I332E/A330L mutations, which showed the greatest increase in FcyRIIIa affinity, reduced FcyRIIb binding, and strong cytotoxic activity in cynomolgus monkeys. Lazar et al. (2006) Proc. Nat'l Acad Sci. (USA) 103:4005; Awan et al. (2010) Blood 115:1204; Desjarlais & Lazar (2011) Exp. Cell Res. 317:1278. The introduction of triple mutations in antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2/neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) resulted in significantly increased ADCC activity in vitro, and the S239D/I332E variant showed an increased ability to deplete B-cells in monkeys. Lazar et al. (2006) Proc. Nat'l Acad Sci. (USA) 103:4005. In addition, IgG1 mutants containing L235V, F243L, R292P, Y300L, V305I, and P396L mutations have been identified that exhibit increased binding to FcyRIIIa and concomitantly increased ADCC activity in transgenic mice expressing human FcyRIIIa in B-cell and malignant tumor models. mammary gland - 45 040561 zy. Stavenhagen et al. (2007) Cancer Res. 67:8882; US patent No. 8652466; Nordström et al. (2011) Breast

Cancer Res. 13:R123.

Different isotypes of IgG also show different CDC activity (IgG3>IgG1>>IgG2®IgG4). Dangl et al. (1988) EMBO J. 7:1989. For applications in which an improved CDC is desired, mutations can also be introduced that increase binding to C1q. The ability to capture complement (CDC) can be enhanced by mutations in K326 and/or E333 in IgG2, such as K326W (which reduces ADCC activity) and E333S, to increase binding to C1q, the first component of the complement cascade. Idusogie et al. (2001) J. Immunol. 166:2571. The introduction of S267E/H268F/S324T (alone or in any combination) into human IgG1 enhances C1q binding. Moore et al. (2010) mAbs 2:181. The Fc region of the IgG1/IgG3 113F hybrid isotype in Natsume et al. (2008) Cancer Res. 68:3863 (see FIG. 1) also gives enhanced CDC. See also Michaelsen et al. (2009) Scand. J. Immunol. 70:553 and Redpath et al. (1998) Immunology 93:595.

Additional mutations that can increase or decrease effector function are described in Dall'Acqua et al. (2006) J. Immunol. 177:1129. See also Carter (2006) Nat. Rev. Immunol. 6:343; Presta (2008) Curr. Op. Immunol. 20:460.

Fc variants that increase affinity for the inhibitory FcyRIIb receptor can also be used, for example, to enhance apoptosis-inducing or adjuvant activity. Li & Ravetch (2011) Science 333:1030; Li & Ravetch (2012) Proc. Nat'l Acad. Sci (USA) 109:10966; U.S. Patent Application Publication 2014/0010812. Such variants may provide an antibody with immunomodulatory activity associated with FcyRIIb ⁺ cells, including, for example, B cells and monocytes. In one embodiment, the Fc variants confer selectively enhanced affinity for FcyRIIb for one or more activating receptors. Modifications to alter binding to FcyRIIb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, according to index EU. Exemplary FcyRIIb affinity enhancing substitutions include, without limitation, 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 268DE, 268DE, 2 , 327D, 327E, 328F, 328W, 328Y and 332E. Exemplary replacements include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing FcyRIIb binding include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E and 267E/328F. In particular, variants S267E, G236D, S239D, L328F and I332E, including the double variant S267E + L328F of human IgG1, are of particular importance for specific enhancement of affinity for the inhibitory FcyRIIb receptor. Chu et al. (2008) Mol. Immunol. 45:3926; US Patent Application Publication 2006/024298; publication of international application WO 2012/087928. Increased specificity for FcyRIIb (as opposed to FcyRIIaR ¹ ) can be obtained by adding a P238D substitution. Mimoto et al. (2013) Protein. Eng. Des. & Selection 26:589; publication of international application WO 2012/115241.

In some embodiments, an antibody is modified to increase its biological half-life. Various approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region for FcRn. In one embodiment, the antibody is altered in the CH1 or CL region to contain a recycling receptor-binding epitope taken from two loops of the CH2 domain of the IgG Fc region, as described in US Pat. Other exemplary Fc variants that increase FcRn binding and/or improve pharmacokinetic properties include substitutions at positions 259, 308, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y and 434M. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry, 2001, 276(9): 6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y/254T/256E, 433K/434F/436H (Dall Acqua et al. Journal of Immunology, 2002, 169: 5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281:23514-23524). See US Pat. No. 8,367,805.

Modification of certain conserved residues in IgG Fc (I253/H310/Q311/H433/N434), such as the N434A variant (Yeung et al., 2009) J. Immunol., 182:7663), has been proposed as a way to increase FcRn affinity, thereby increasing the half-life of the antibody in the circulation. W098/023289. A combination Fc variant containing M428L and N434S has been shown to increase FcRn binding and increase serum half-life up to five-fold. Zalevsky et al. (2010) Nat. Biotechnol. 28:157. The combined Fc variant containing modifications T307A, E380A and N434A also prolongs the half-life of IgG1 antibodies. Petkova et al. (2006) Int. Immunol. 18:1759. In addition, combination Fc variants containing M252Y/M428L, M428L/N434H, M428L/N434F, M428L/N434Y, M428L/N434A, M428L/N434M and M428L/N434S variants have also been shown to prolong half-life. WO 2009/086320.

In addition, the combined Fc variant containing M252Y, S254T, and T256E increased the half-life by almost 4-fold. Dall'Acqua et al. (2006) J. Biol. Chem. 281:23514. A related IgG1 modification providing increased FcRn affinity but reduced pH dependence (M252Y/S254T/T256E/H433K/N434F) was used to generate an IgG1 construct (MST-HN Abdeg) for use as a competitor to prevent other antibodies from binding to FcRn , resulting in increased clearance of that other antibody, either endogenous IgG (eg in an autoimmune setting) or another exogenous (therapeutic) monoclonal antibody. Vaccaro et al. (2005) Nat. Biotechnol. 23:1283; WO 2006/130834.

Other modifications to increase FcRn binding are described in Yeung et al. (2010) J. Immunol. 182:7663-7671; 6277375; 6821505; publications of international applications WO 97/34631; WO2002/060919.

In some embodiments, hybrid IgG isotypes can be used to increase FcRn binding and potentially increase half-life. For example, an IgG1/IgG3 fusion variant can be constructed by replacing IgG1 positions in the CH2 and/or CH3 region with amino acids from IgG3 at positions where the two isotypes differ. Thus, an IgG fusion antibody can be constructed that contains one or more substitutions, for example, 274Q, 276K, 30oF, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R, and 436F. In other embodiments described herein, an IgG1/IgG2 fusion variant can be constructed by replacing IgG2 positions in the CH2 and/or CH3 region with amino acids from IgG1 at positions where the two isotypes differ. Thus, an IgG antibody fusion variant can be constructed that contains one or more substitutions, such as one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (refers to the glycine insertion at position 236) and 327A. See US Pat. No. 8,629,113. A fusion of IgG1/IgG2/IgG4 sequences has been created that is expected to increase serum half-life and improve expression. U.S. Patent No. 7,867,491 (serial number 18 therein).

The serum half-life of the antibodies of the present invention can also be increased by pegylation. The antibody can be pegylated, for example, to increase the biological (eg, serum) half-life of the antibody. To PEGylate an antibody, the antibody or antibody fragment is typically reacted with a polyethylene glycol (PEG) reagent, such as a reactive ester or aldehyde derivative of PEG, under conditions where one or more PEG moieties are attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy or aryloxy polyethylene glycol or polyethylene glycol maleimide. In some embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylated proteins are known in the art and can be applied to the antibodies described herein. See, for example, EP 0154316 to Nishimura et al. and European patent EP 0401384 to Ishikawa et al.

Alternatively, under some circumstances it may be desirable to decrease the half-life of an antibody rather than increase it. Modifications such as I253A (Hornick et al. (2000) J. Nucl. Med. 41:355) and H435A/ RI253A or H310A (Kim et al. (2000) Eur. J. Immunol. 29:2819) in Fc in human IgG1 can reduce FcRn binding, thus reducing the half-life (increasing clearance) for use in situations where rapid clearance is preferred, such as medical imaging. See also Kenanova et al. (2005) Cancer Res. 65:622. Other methods for increasing clearance include formatting the antigen-binding domains of the present invention as antibody fragments that do not bind to FcRn, such as Fab fragments. Such a modification can reduce the circulating half-life of an antibody from a couple of weeks to several hours. Selective PEGylation of antibody fragments can then be used to improve (increase) the half-life of antibody fragments, if desired. Chapman et al. (1999) Nat. Biotechnol. 17:780. Antibody fragments can also be fused to human serum albumin, for example in a fusion protein construct, to increase half-life. Yeh et al. (1992) Proc. Nat'l Acad. sci. 89:1904. Alternatively, the bispecific antibody can be constructed with a first antigen-binding domain of the present invention and a second antigen-binding domain that binds to human serum albumin (HSA). See International Patent Application Publication WO 2009/127691 and the patent references cited therein. Alternatively, specialized polypeptide sequences can be added to antibody fragments to increase half-life, such as XTEN polypeptide sequences. Schellenberger et al. (2009) Nat. Biotechnol. 27:1186; publication of international patent application WO 2010/091122.

Additional options Fc.

When using an IgG4 constant domain, it is preferable to include an S228P substitution that mimics the hinge sequence in IgG1 and thereby stabilizes IgG4 molecules, e.g.

- 47 040561 reducing the Fab-arm exchange between the therapeutic antibody and endogenous IgG4 in the patient being treated. Labrijn et al. (2009) Nat. Biotechnol. 27:767; Reddy et al. (2000) J. Immunol. 164:1925.

A potential protease cleavage site at the hinge of IgG1 constructs can be removed with modifications to D221G and K222S, increasing antibody stability. Publication of international application WO 2014/043344.

The affinities and binding properties of an Fc variant for its ligands (Fc receptors) can be determined using various in vitro assay methods (biochemical or immunological assays) known in the art, including, but not limited to, equilibrium methods (e.g., enzyme immunosorbent assay). assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g. SPR BIACORE® assay) and other methods such as indirect binding assays, competitive inhibitory assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g. gel filtration). These and other methods may use a label on one or more components of interest and/or use a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinity and kinetics can be found in Paul W.E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on the antibody-immunogen interaction.

In other embodiments, the glycosylation of the antibody is modified to increase or decrease effector function. For example, an aglycosylated antibody that does not have full effector function can be generated by mutating a conserved asparagine residue at position 297 (eg, N297A), thereby abolishing complement binding and FcyRI. Bolt et al. (1993) Euro. J. Immunol. 23:403. See also Tao & Morrison (1989) J. Immunol. 143:2595 (using N297Q in IgG1 to eliminate glycosylation at position 297).

Although aglycosylated antibodies generally lack effector function, mutations can be introduced to restore this function. Aglycosylated antibodies, such as those resulting from N297A/C/D/ or H mutations or produced in systems (eg E. coli) that are not glycosylated proteins, can be further mutated to restore FcyR binding, such as S298G and/or T299A/G/or H (International Application Publication WO 2009/079242) or E382V and M428I (Jung et al. (2010) Proc. Nat'l Acad. Sci (USA) 107:604).

In addition, an ADCC-enhanced antibody can be obtained by altering glycosylation. For example, removal of fucose from Asn297-linked heavy chain oligosaccharides has been shown to enhance ADCC based on improved binding to FcyRIIIa. Shields et al. (2002) JBC 277:26733; Niwa et al. (2005) J. Immunol. Methods 306: 151; Cardarelli et al. (2009) Clin. Cancer Res.15:3376 (MDX1401); Cardarelli et al. (2010) Cancer Immunol. Immunotherap. 59:257 (MDX-1342) Such low fucose antibodies can be generated, for example, in knockout Chinese Hamster Ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng. 87 :614), or in other cells that produce acidolized antibodies. See, for example, Zhang et al. (2011) mAbs 3:289 and Li et al. (2006) Nat. Biotechnol. 24:210 (both describe antibody production in glycoengineered Pichia pastoris); Mossner et al. (2010) Blood 115:4393; Shields et al. (2002) J. Biol. Chem. 277:26733; Shinkawa et al. (2003) J. Biol. Chem. 278:3466; European patent EP 1176195 B1. ADCC can also be enhanced, as described in PCT publication WO 03/035835, which discloses the use of a variant of the CHO cell line, Lec13, with reduced ability to attach fucose to Asn(297)-bound carbohydrates, which also leads to hypofucosylation of antibodies expressed in this host cell (see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). Alternatively, fucose analogs can be added to the culture medium during antibody production to inhibit fucose incorporation into carbohydrate on the antibody (see, for example, WO 2009/135181).

An increase in GlcNac skew structures in antibody-attached oligosaccharides also enhances ADCC. PCT Publication WO 99/54342 from Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., e(1,4)-Nacetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased GlcNac skew structures resulting in increased ADCC activity of the antibody (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

Additional glycosylation variants have been developed that lack galactose, sialic acid, fucose, and xylose residues (so-called GNGN glycoforms), which exhibit enhanced ADCC and ADCP but reduced CDC, and others that lack sialic acid, fucose, and xylose (so-called G1/G2 glycoforms) that show enhanced ADCC, ADCP and CDC. Publication of US Patent Application No. 2013/0149300. Antibodies characterized by these glycosylation patterns are optionally produced in genetically modified N. benthamiana plants in which endogenous xylosyl and fucosyl transferase genes have been knocked out.

Glycoengineering can also be used to modify anti-inflammatory

- 48 040561 properties of the IgG construct by changing the content of sialyl α2,6 in the carbohydrate chains attached to Asn297 Fc regions, with an increased proportion of α2,6-sialylated forms leading to increased anti-inflammatory effects. See Nimmerjahn et al. (2008) Ann. Rev. Immunol. 26:513. Conversely, reducing the proportion of antibodies containing α6,6-sialylated carbohydrates may be beneficial in cases where anti-inflammatory properties are not needed. Methods for modifying the content of α2,6 silylation of antibodies, for example, by selective purification of α2,6-sialylated forms or by enzymatic modification, are described in US Patent Application Publication No. 2008/0206246. In other embodiments, the amino acid sequence of the Fc region can be modified to mimic the effects of a2,6-sialylation, for example by including the F241A modification (see, for example, WO 2013/095966).

VIII. Physical properties of antibodies.

The antibodies described herein may contain one or more glycosylation sites in the light or heavy chain variable region. Such glycosylation sites can lead to increased antibody immunogenicity or a change in antibody pK due to altered antigen binding (Marshall et al. (1972) Annu. Rev. Biochem. 41:673-702; Gala and Morrison (2004) J. Immunol. 172:5489 -94 Wallick et al (1988) J Exp Med 168:1099-109 Spiro (2002) Glycobiology 12:43R56R Parekh et al (1985) Nature 316:452-7 Mimura et al ( 2000) Mol Immunol 37:697-706). Glycosylation is known to occur in motifs containing the N-X-S/T sequence. In some embodiments, the anti-OX40 antibody does not contain variable region glycosylation. This can be achieved either by selecting antibodies that do not contain a glycosylation motif in the variable region, or by mutating residues in the glycosylation region.

In some embodiments, the antibodies described herein do not contain asparagine isomerization sites. Deamidation of asparagine can occur at the N-G or D-G sequences and lead to the formation of an isoaspartic acid residue, which introduces a kink into the polypeptide chain and reduces its stability (the effect of isoaspartic acid). For example, if the Asp-Gly amino acid sequence is present in the heavy and/or light chain CDR sequences of an antibody, the sequence is replaced with an amino acid sequence that does not undergo isomerization. In one embodiment, the antibody comprises a heavy chain variable region CDR2 sequence as set forth in SEQ ID NO: 76, but wherein Asp or Gly in the Asp-Gly sequence (LISYDGSRKHYADSVKG; SEQ III NO: 76) is replaced with an amino acid sequence that does not undergo isomerization, e.g. the Asp-Ser or Ser-Gly sequence. In another embodiment, the antibody comprises the heavy chain variable region CDR2 sequence shown in SEQ ID NO: 88, but wherein the Asp or Gly in the Asp-Gly sequence (AIDTDGGTFYADSVRG; SEQ ID NO: 88) is replaced with an amino acid sequence that is not isomerized, e.g. , Ser-Gly, Asp-Ala or Ser-Thr sequence.

Each antibody will have a unique isoelectric point (pI), which is typically in the pH range of 6 to 9.5. The pI for an IgG1 antibody is typically in the pH range of 7-9.5, and the pI for an IgG4 antibody is typically in the pH range of 6-8. It has been suggested that antibodies with pIs outside the normal range may exhibit some unfolding and instability in vivo. Thus, it is preferable to have an anti-OX40 antibody that has a pI value that falls within the normal range. This can be achieved either by selecting antibodies with a pI in the normal range or by mutating charged surface residues.

Each antibody will have a characteristic melting point with a higher melting point indicating greater overall in vivo stability (Krishnamurthy R. and Manning MC (2002) Curr. Pharm. Biotechnol. 3:361-71). Generally, it is preferred that T _M1 (initial unfolding temperature) is greater than 60°C, preferably greater than 65°C, even more preferably greater than 70°C. The melting point of an antibody can be measured using differential scanning calorimetry (Chen et al. (2003) Pharm. Res. 20:1952-60; Ghirlando et al. (1999) Immunol. Lett. 68:47-52) or circular dichroism (Murray et al (2002) J Chromatogr Sci 40:343-9).

In a preferred embodiment, antibodies are selected that do not rapidly degrade. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander A.J. and Hughes D.E. (1995) Anal. Chem. 67:3626-32). In some embodiments, anti-OX40 antibodies (eg, OX40.21 antibody) described herein are characterized by increased stability.

Accordingly, according to other embodiments, antibodies are chosen that have minimal aggregation effects, which may lead to the triggering of an undesirable immune response and/or altered or adverse pharmacokinetic properties. Generally, antibodies are acceptable with an aggregation of 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less, and even more preferably 5% or less. Aggregation can be measured in several ways, including gel permeation chromatography (SEC), high performance liquid chromatography (HPLC), and light scattering.

IX. Methods for constructing antibodies.

As discussed herein, anti-OX40 antibodies comprising the VH and V _L sequences disclosed herein can be used to generate novel anti-OX40 antibodies by modifying the VH and/or V _L sequences or antibody constant region(s). Thus, in another embodiment, the structural features of the anti-OX40 antibodies described herein, e.g., 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 2 and 20C1 are used to generate structurally related anti-OX40 antibodies that retain at least one functional property of the antibodies described herein, such as binding to human OX40 and cynomolgus monkey OX40. For example, one or more CDRs in 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1, or mutations thereof, may be recombinantly combined with known framework regions and/or other CDRs to generate additional, recombinantly engineered anti-OX40 antibodies as discussed above. Other types of modifications include those described in the previous section. The starting material for the design method is one or more V _H and/or V _L sequences provided herein, or one or more CDR regions. In order to create an engineered antibody, it is not necessary to actually produce (ie, express as a protein) an antibody having one or more of the VH and/or VL sequences provided herein or one or more of its CDR regions. Rather, the information contained in the sequence(s) is used as starting material to create a second generation sequence(s) derived from the original sequence(s), and then the second generation sequence(s) are obtained and expressed as a protein.

Accordingly, provided herein are methods for producing anti-OX40 antibodies comprising:

(a) providing: (i) an antibody heavy chain variable region sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 11, 19, 31, 39, 51, 59, 67, 75 and 87, a CDR2 sequence, selected from the group consisting of SEQ ID NOs: 12, 20, 32, 40, 52, 60, 68, 76, 88 and 317, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 13, 21, 33, 41, 53, 61, 69, 77 and 89; and (ii) an antibody light chain variable region sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 14, 22, 25, 34, 42, 45, 54, 62, 70, 78, 81, and 90, the sequence A CDR2 selected from the group consisting of SEQ ID NOs: 15, 23, 26, 35, 43, 46, 55, 63, 71, 79, 82 and 91 and/or a CDR3 sequence selected from the group consisting of SEQ ID NO: 16, 24, 27, 36, 44, 47, 56, 64, 72, 80, 83 and 92;

(b) modifying at least one amino acid residue in the antibody heavy chain variable region sequence and/or the antibody light chain variable region sequence to create at least one altered antibody sequence; and (c) expressing the altered antibody sequence as a protein. Standard molecular biology techniques can be used to generate and express altered antibody sequences.

Preferably, the antibody encoded by the altered antibody sequence(s) is an antibody that retains one, some, or all of the functional properties of the anti-OX40 antibodies described herein, which include:

(1) binding to soluble human OX40, eg, a KD of 10 nM or less (eg, 0.01 to 10 nM), eg, as measured by Biacore;

(2) binding to human membrane-bound OX40, eg EC ₅₀ 1 nM or less (eg 0.01 to 1 nM), eg as measured by FACS;

(3) binding to cynomolgus monkey OX40, eg, binding to membrane-bound cynomolgus monkey OX40, eg, with an EC ₅₀ of 10 nM or less (eg, 0.01 to 10 nM), eg, as measured by FACS;

(4) inducing or enhancing T cell activation as evidenced by (i) increased production of IL-2 and/or IFN-γ in OX40 expressing T cells and/or (ii) increased T cell proliferation;

(5) inhibition of OX40 ligand binding to OX40, eg with an EC ₅₀ of 1 nM or less, as measured by FACS, eg in an assay with hOX40-293 cells;

(6) binding to an epitope on the extracellular portion of mature human OX40 (SEQ ID NO: 2), such as an epitope in the DVVSSKPCKPCTWCNLR region (SEQ ID NO: 178), or

DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179);

(7) competition for human OX40 binding to 3F4, 14B6-1, 14B6-2, 23H3, 18E9, 8B11, 20B3 and 20C1;

- 50 040561 (8) competition for binding to human OX40 with 6E1-1, 6E1-2, 14A2-1 and 14A2-2. The modified antibody may exhibit one or more, two or more, three or more, four or more, five or more, six or all of the functional properties listed under (1) to (7) above. The functional properties of the modified antibodies can be assessed using standard assays available in the art and/or described herein, such as those exemplified (eg, ELISA, FACS).

In some embodiments of the methods for constructing antibodies described herein, mutations can be introduced randomly or selectively along all or part of the coding sequence of an anti-OX40 antibody, and the resulting modified anti-OX40 antibodies can be screened for binding activity and/or other functional properties, as described in this document. Mutation methods have been described in the present technical field. For example, PCT publication WO 02/092780 from Short describes methods for generating and screening for antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT publication WO 03/074679 by Lazar et al. describes how to use computational screening methods to optimize the physicochemical properties of antibodies.

X. Nucleic acid molecules.

Also provided herein are nucleic acid molecules that encode the antibodies described herein. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is isolated or reduced to a substantially pure form when purified from other cellular components or other contaminants, such as other cellular nucleic acids (for example, other chromosomal DNA, such as chromosomal DNA that is associated with isolated DNA in nature) or proteins, by standard methods, including alkali/SDS treatment, CsCl rinse, column chromatography, restriction enzymes, agarose gel electrophoresis, and others well known in the art. See F. Ausubel et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid may be, for example, DNA or RNA, and may or may not contain intron sequences. In some embodiments, the nucleic acid is a cDNA molecule.

The nucleic acids provided herein can be prepared using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas derived from transgenic mice carrying human immunoglobulin genes as described below), cDNAs encoding the hybridoma-derived antibody light and heavy chains can be obtained using standard PCR cloning or cDNA cloning procedures. For antibodies derived from an immunoglobulin gene library (eg, using phage display techniques), the nucleic acid encoding the antibody can be retrieved from the library.

Preferred nucleic acid molecules are those encoding the VH and VL sequences at 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and 20C1. Exemplary DNA sequences encoding the VH sequences at 3F4, 14B6, 23H3, 6E1, 18E9, 8B11, 20B3, 14A2, and 20C1 are shown in SEQ ID NOs: 126, 128, 131, 133, 136, 138, 140, 142, and 145 respectively. Exemplary DNA sequences encoding the VL sequences at 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1 are shown in SEQ ID NO: 127, 129, 130, 132, 134, 135, 137, 139, 141, 143, 144 and 146 respectively. Exemplary DNA sequences encoding heavy chain sequences are shown in SEQ ID NOs: 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169171, 173, 175, 176 and 177. Exemplary DNA sequences , coding sequences for the light chain are shown in SEQ ID NOS: 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172 and 174.

Exemplary nucleotide sequences encoding the mature VH and VL domains of an anti-OX40 antibody are shown in SEQ ID NOs: 145 and 146, respectively. An exemplary nucleotide sequence encoding an anti-OX40 antibody heavy chain is shown in SEQ ID NO: 177. An exemplary nucleotide sequence encoding an OX40 antibody light chain is shown in SEQ ID NO: 168.

Methods for producing anti-OX40 antibodies provided herein may include expression of the heavy chain and light chains in a cell line containing nucleotide sequences encoding heavy and light chains with a signal peptide. Host cells containing these nucleotide sequences are also provided herein.

Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further processed by standard recombinant DNA techniques, for example, to convert variable region genes to full length antibody chain genes, Fab fragment genes, or scFv gene. In these manipulations, a DNA fragment encoding a VL or VH is operably linked to another DNA fragment encoding a different protein, such as an antibody constant region.

- 51 040561 or flexible linker. As used in this context, the term operably linked refers to the joining of two DNA fragments such that the amino acid sequences they encode remain in frame.

An isolated VH coding region can be converted into a full length heavy chain gene by operably linking the VH coding DNA to another DNA molecule encoding the heavy chain constant regions (hinge, CH1, CH2 and/or CH3). Human heavy chain constant region gene sequences are known in the art (see, e.g., Kabat E.A. el al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242), and DNA fragments spanning these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, such as an IgG1 region. For a Fab fragment heavy chain gene, the VH-encoding DNA may be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

An isolated VL coding region can be converted into a full length light chain gene (as well as a Fab light chain gene) by operably linking the VL coding DNA to another DNA molecule encoding the light chain constant region, CL. Human light chain constant region gene sequences are known in the art (see, for example, Kabat E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242), and DNA fragments spanning these regions can be obtained by standard NDP amplification. The light chain constant region may be the k or λ constant region.

To create the scFv gene, the VH and VL encoding DNA fragments are operably linked to another flexible linker encoding fragment, for example encoding the amino acid sequence (Gly4-Ser) ₃ , so that the VH and VL sequences can be expressed as a continuous single strand protein with VL and VL regions. VH connected by a flexible linker (see, for example, Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al. ., (1990) Nature 348:552-554).

Also provided herein are nucleic acid molecules encoding VH and VL sequences that are homologous to the monoclonal antibody sequences 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1 , 14A2-2 and 20C1. Exemplary nucleic acid molecules encode VH and VL sequences that are at least 70% identical, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to nucleic acid molecules encoding the VH and VL sequences of monoclonal antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and 20C1. Also provided herein are nucleic acid molecules with conservative substitutions (ie, substitutions that do not change the resulting amino acid sequence when the nucleic acid molecule is translated), for example, for codon optimization.

XI. Obtaining antibodies.

Anti-OX40 antibodies described herein can be generated using a variety of known methods, such as the standard somatic cell hybridization method described by Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle other methods of obtaining monoclonal antibodies can be used, for example, viral or oncogenic transformation of B-lymphocytes, phage display technique using human antibody gene libraries.

The preferred animal system for producing hybridomas is the murine system. The production of hybridoma in mice is a very well established procedure. Immunization protocols and methods for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg, murine myeloma cells) and fusion methods are also known.

The chimeric or humanized antibodies described herein can be derived from the sequence of a mouse monoclonal antibody prepared as described above. DNA encoding immunoglobulin heavy and light chains can be obtained from a mouse hybridoma of interest and engineered to contain non-mouse (eg, human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, mouse variable regions can be linked to human constant regions using methods known in the art (see, for example, US Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody, mouse CDR regions can be inserted into a human scaffold using methods known in the art (see, for example, US Pat. No. 5,225,539 to Winter and US Pat. al.).

In one embodiment, the antibodies described herein are human monoclonal antibodies. Such human monoclonal antibodies directed against OX40 can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as human Ig mice.

HuMAb mouse® (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged sequences for the heavy (μ and γ) and light k chains of human immunoglobulin together with targeted mutations that inactivate the endogenous μ and k chain loci (see, e.g., Lonberg et al (1994) Nature 368(6474): 856-859). Accordingly, mice show reduced expression of mouse IgM or k, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce a high-affinity human IgGK monoclonal antibody (Lonberg N. et al. (1994), supra; in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101 Lonberg N. and Huszar D. (1995) Intern Rev. Immunol 13:65-93 and Harding F. and Lonberg N. (1995) Ann NY Acad Sci 764:536-546). The production and use of HuMab mice and the genomic modifications tolerated by such mice are further described in Taylor L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor L. et al. (1994) International Immunology 6: 579-591 and Fishwild D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of which are incorporated herein by reference in their entirety. See also US Pat. Nos. 5,545,806; 5569825; 5625126; 5633425; 5789650; 5877397; 5661016; 5814318; 5874299 and 5770429, all addressed to Lonberg and Kay; US Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all addressed to Lonberg and Kay; and PCT Publication No. WO 01/14424 to Korman et al.

In some embodiments, the antibodies described herein are grown using a mouse that carries human immunoglobulin sequences on transgenes and transhomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as KM mice, are described in detail in PCT publication WO 02/43478 to Ishida et al.

In addition, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to grow the anti-OX40 antibodies described herein. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used; such mice are described, for example, in US patent No. 5939598; 6075181; 6114598; 6150584 and 6169663, to Kucherlapati et al.

Moreover, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to build up the anti-OX40 antibodies described herein. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as TC mice, can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. sci. USA 97:722-727. In addition, cows carrying human heavy and light chain transchromosomes are described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to build up the anti-OX40 antibodies described herein.

Additional murine systems for raising human antibodies, e.g., anti-human OX40 antibodies, described in the art include (i) the VelocImmune® mouse (Regeneron Pharmaceuticals, Inc.) in which endogenous mouse heavy and light chain variable regions are replaced by a homologous recombination with human heavy and light chain variable regions operably linked to endogenous mouse constant regions such that chimeric antibodies (human V/mouse C) are grown in mice and then converted to fully human antibodies using standard recombinant DNA techniques; and (ii) a MeMo® mouse (Merus Biopharmaceuticals, Inc.), wherein the mouse contains unrearranged human heavy chain variable regions but one rearranged common human light chain variable region. Such mice and their use for raising antibodies are described, for example, in WO 2009/15777, US 2010/0069614, WO 2011/072204, WO 2011/097603, WO 2011/163311, WO 2011/163314, WO 2012/148873, US 2012 /0070861 and US 2012/0073004.

The human monoclonal antibodies described herein can also be generated using phage display methods to screen human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are well established in the art. See, for example: US Pat. Nos. 5,223,409; 5403484; and 5571698 to Ladner et al; US Pat. Nos. 5,427,908 and 5,580,717 to Dower et al; US Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al. and US Pat. Nos. 5,885,793; 6521404; 6544731; 6555313; 6582915 and 6593081 to Griffiths et al.

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The human monoclonal antibodies described herein can also be generated using SCID mice in which human immune cells have been reconstituted such that a human antibody response can be obtained upon immunization. Such mice are described, for example, in US patent No. 5476996 and 5698767 in the name of Wilson et al.

Immunization.

To generate fully human anti-OX40 antibodies, transgenic or transchromosomal mice containing human immunoglobulin genes (e.g., HCo12, HCo7, or KM mice) can be immunized with a purified or enriched preparation of the OX40 antigen and/or cells expressing OX40 or a fragment thereof, as described. for other antigens, for example, Lonberg et al. (1994) Nature 368(6474): 856-859; Fishwild et al (1996) Nature Biotechnology 14: 845-851 and international publication WO 98/24884. Alternatively, mice can be immunized with DNA encoding human OX40 or a fragment thereof. Preferably, the mice will be 6-16 weeks old after the first infusion. For example, a purified or enriched preparation (5-50 μg) of recombinant OX40 antigen can be used to immunize HuMAb mice intraperitoneally. In the event that immunization with a purified or enriched OX40 antigen preparation does not result in antibodies, mice can also be immunized with OX40 expressing cells, such as a cell line, to stimulate immune responses. Exemplary cell lines include the OX40 overexpressing stable CHO and Raji cell lines.

Accumulated experience with various antigens has shown that HuMAb transgenic mice respond best when initially immunized intraperitoneally (IP) or subcutaneously (SC) with antigen in Ribi adjuvant, followed by IP/SC immunization every week (up to 10) with antigen in Ribi adjuvant. The immune response can be monitored during the course of the immunization protocol, with plasma samples obtained via retroorbital bleeding. Plasma can be screened by ELISA and FACS (as described below), and mice with sufficient anti-human OX40 immunoglobulin titers can be used for fusions. Mice can be immunized intravenously with antigen 3 days before sacrifice and removal of the spleen and lymph nodes. It is expected that 2-3 fusions may need to be performed for each immunization. 6 to 24 mice are typically immunized for each antigen. As a rule, strains HCo7, HCo12 and KM are used. In addition, both the HCo7 and HCo12 transgene can be co-produced in the same mouse having two different human heavy chain transgenes (HCo7/HCo12).

Generation of hybridomas producing anti-OX40 monoclonal antibodies To obtain hybridomas producing the antibodies described herein, splenocytes and/or lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for antigen-specific antibodies. For example, single chain suspensions of splenic lymphocytes from immunized mice can be fused to non-secreting Sp2/0 mouse myeloma cells (ATCC, CRL 1581) with 50% PEG. Cells are plated at approximately 2x105 in flat-bottomed plates, followed by a 2-week incubation in selective media containing 10% fetal clone serum, 18% 653 conditioned medium, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamicin and 1x HAT (Sigma). After approximately two weeks, the cells can be cultured in medium in which HAT is replaced with HT. Individual wells can then be screened by ELISA for human IgM and IgG monoclonal antibodies. After extensive growth of the hybridoma, the environment can be observed, as a rule, after 10-14 days. Antibody-secreting hybridomas can be replicated, screened again and, if still positive for human IgG, monoclonal antibodies can be subcloned at least twice by limited dilution. The stable subclones can then be cultured in vitro to produce small amounts of antibody in tissue culture medium for characterization.

For antibody purification, selected hybridomas can be grown in 2 liter flasks with agitation to purify monoclonal antibodies. Supernatants can be filtered and concentrated prior to protein A-sepharose affinity chromatography (Pharmacia, Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged for PBS and the concentration can be determined by OD280 using an extinction coefficient of 1.43. Then the antibodies can be divided into aliquots and stored at -80°C.

Production of transfectomes producing anti-OX40 monoclonal antibodies Antibodies can be generated in the transfectome of a host cell using, for example, a combination of recombinant DNA techniques and gene transfection techniques, as is well known in the art (Morrison, S. (1985) Science 229: 1202 ).

For example, for the expression of antibodies or antibody fragments, DNA encoding incomplete or full-length light and heavy chains can be prepared by standard molecular biology techniques (eg, NDP amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and The DNA can be inserted into expression vectors so that the genes are operably linked to transcription and translation control sequences. In this context, the term operably linked means that the antibody gene is ligated into the vector such that transcription and translation control sequences within the vector serve their intended function of regulating transcription and translation of the antibody gene. Expression vectors and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into a separate vector, or both genes are inserted into the same expression vector. The antibody genes are introduced into the expression(s) vector(s) by standard methods (eg, ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt end ligation if there are no restriction sites). The light and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding the heavy chain constant regions and light chain constant regions of the desired isotype, such that the VH segment is operably linked to the CH segment(s) within the vector, and the V _L segment is operably linked to the C _L segment within the vector.

Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein). Exemplary signal sequences for use in antibody heavy and light chains include signal sequences originally found in the anti-OX40 antibodies described herein.

In addition to the antibody chain genes, recombinant expression vectors can carry regulatory sequences that control the expression of the antibody chain genes in the host cell. The term regulatory sequence is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Those skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of the desired protein, and so on. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), simian 40 virus (SV40), adenovirus (e.g. , adenovirus major late promoter (AdMLP) and polyoma.Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the β-globin promoter can be used.In addition, regulatory elements are composed of sequences from different sources, such as the SRa promoter system, which contains the sequences from the SV40 early promoter and the human T cell leukemia virus type 1 long terminal repeat (Takebe Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to antibody chain genes and regulatory sequences, recombinant expression vectors may carry additional sequences, such as sequences that regulate the vector's replication in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, US Pat. For example, typically, a selectable marker gene provides resistance to drugs such as G418, hygromycin, or methotrexate in the host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into the host cell by standard methods. The various forms of the term transfection are intended to encompass a wide range of methods typically used to introduce exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the antibodies described herein in prokaryotic or eukaryotic host cells, the expression of antibodies in eukaryotic cells and most preferably mammalian host cells is most preferred because such eukaryotic cells, and in particular mammalian cells, are more similar than prokaryotic cells. cells

- 55 040561 for the assembly and secretion of properly packaged and immunologically active antibodies. Prokaryotic expression of antibody genes has been reported to be inefficient in obtaining high yields of active antibodies (Boss M.A. and Wood C.R. (1985) Immunology Today 6:12-13). The antibodies described herein can also be produced in glycoengineered strains of the yeast Pichia pastoris.

Li et al. (2006) Nat. Biotechnol. 24:210.

Preferred mammalian host cells for expressing the recombinant antibodies described herein include Chinese hamster ovary (CHO cells) (including the dhfr-CHO cells described in Urlaub and Chasm, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 used with a DHFR selectable marker, for example as described in R. J. Kaufman and P. A. Sharp (1982) Mol Biol 759:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338841. When recombinant expression vectors encoding antibody genes are administered into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be isolated from the culture medium using standard protein purification methods.

The N- and C-terminus of the polypeptide chains of the antibodies of the present invention may differ from the expected sequence due to commonly observed post-translational modifications. For example, C-terminal lysine residues are often absent from antibody heavy chains. Dick et al. (2008) Biotechnol. Bioeng. 100:1132. N-terminal glutamine residues and, to a lesser extent, glutamate residues are often converted to pyroglutamate residues in both the light and heavy chains of therapeutic antibodies. Dick et al. (2007) Biotechnol. Bioeng. 97:544; Liu et al. (2011) JBC 28611211; Liu et al. (2011) J. Biol. Chem. 286:11211.

XII. Analyzes.

Anti-OX40 antibodies described herein can be assayed for binding to OX40, for example, by a standard ELISA assay. Briefly, microtiter plates are coated with purified OX40 at 1-2 μg/ml in PBS and then blocked with 5% bovine serum albumin in PBS. Antibody dilutions (eg, plasma dilutions from OX40 immunized mice) are added to each well and incubated for 1-2 hours at 37°C. The plates are washed with PBS/Tween and then incubated with a secondary reagent (eg, for human antibodies, goat polyclonal reagent specific for human Fc IgG) conjugated with horseradish peroxidase (HRP) for 1 hour at 37°C. After washing, the plates are developed with ABTS support (Moss Inc, product: ABTS-1000) and analyzed with a spectrophotometer at OD 415-495. Sera from immunized mice are then further screened by flow cytometry for binding to a cell line expressing human OX40, but not to a control cell line that does not express OX40. Briefly, anti-OX40 antibody binding is assessed by incubating OX40-expressing CHO cells with anti-OX40 antibody at a 1:20 dilution. Cells are washed and binding is detected with PE-labeled anti-human IgG. Flow cytometric analyzes were performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Preferably, mice that produce the highest titers will be used for fusions.

The ELISA assay described above can be used to screen for antibodies and thus hybridomas that produce antibodies that show positive reactivity with the OX40 immunogen. Hybridomas that produce antibodies that bind, preferably with high affinity, to OX40 can then be subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the original cells (by ELISA) can then be selected for cell bank generation and antibody purification.

For OX40 antibody purification, selected hybridomas can be grown in 2 liter monoclonal antibody purification bottles. Supernatants can be filtered and concentrated prior to protein A-sepharose affinity chromatography (Pharmacia, Piscataway, NJ). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged for PBS and the concentration can be determined by OD ₂₈₀ using an extinction coefficient of 1.43. Monoclonal antibodies can be divided into aliquots and stored at -80°C.

To determine if selected OX40 antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Binding of biotinylated monoclonal antibodies can be detected using a streptavidin-labeled probe. Competitive studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using OX40 coated ELISA plates as described above.

To determine the isotype of purified antibodies, isotype ELISA assays can be performed with

- 56 040561 using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of microtiter plates can be coated with 1 μg/ml of anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, the plates are reacted with 1 µg/ml or less of the tested monoclonal antibodies or purified isotype controls at ambient temperature for 1-2 hours. The wells can then be reacted with either IgG1-specific or human IgM-specific alkaline phosphatases. probes. The plates are developed and analyzed as described above.

To investigate the binding of antibodies to live cells expressing OX40, you can use flow cytometry, as described in the examples. Briefly, cell lines expressing membrane-bound OX40 (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4°C for 1 hour. After washing, the cells are reacted with fluorescein labeled antibody. to IgG under the same conditions as the staining of the primary antibody. Samples can be analyzed with the FACScan instrument using light and side scatter properties to generate gates on individual cells and determine binding of labeled antibodies. An alternative analysis using fluorescence microscopy can be used in addition to or instead of the flow cytometry method. Cells can be stained exactly as described above and examined using fluorescence microscopy. This method allows visualization of individual cells, but may be characterized by reduced sensitivity depending on the density of the antigen.

Anti-OX40 antibodies can be further tested for reactivity with the OX40 antigen by Western blotting. Briefly, cell extracts from cells expressing OX40 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes blocked with 20% mouse serum and examined with the monoclonal antibodies to be tested. IgG binding can be detected using anti-IgG alkaline phosphatase and developed using BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

Methods for assaying binding affinity, cross-reactivity, and binding kinetics include standard assays known in the art, for example, the SPR BIACORE® assay using the SPR BIACORE® 2000 instrument (Biacore AB, Uppsala, Sweden).

In some embodiments, the anti-OX40 antibody specifically binds to the extracellular region of human OX40. For example, an antibody can specifically bind to a specific domain (eg, a functional domain) within the extracellular domain of OX40. In a specific embodiment, the antibody specifically binds to a site on OX40 to which OX40-L binds. In other embodiments, the antibody specifically binds to the extracellular region of human OX40 and the extracellular region of cynomolgus monkey OX40.

XIII. Immunoconjugates, derivatives and diagnostics of antibodies.

The anti-OX40 antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging, and for this purpose, the antibody (or binding fragment thereof) can be conjugated with a suitable detection agent to form an immunoconjugate. For diagnostic purposes, detectable labels are suitable, which include radioisotopes for whole body imaging, and radioisotopes, enzymes, fluorescent labels, and other suitable antibody tags for examining samples.

The detectable labels can be any of the various types currently used in the field of in vitro diagnostics, including particulate labels, including metal sols such as colloidal gold, isotopes such as I ¹²⁵ or Tc ⁹⁹ , for example, a peptide chelating agent such as N ₂ S ₂ , N3S or N ₄ , chromophores including fluorescent markers, biotin, luminescent markers, phosphorescent markers, and the like, as well as label enzymes that convert a given substrate into a detectable marker, and polynucleotide labels that are detected after amplification, such as polymerase chain reaction. The biotinylated antibody can then be detected by binding avidin or streptavidin. Suitable label enzymes include horseradish peroxidase, alkaline phosphatase, and the like. For example, the label may be an alkaline phosphatase enzyme detected by measuring the presence or formation of chemiluminescence after conversion of 1,2-dioxetane substrates such as adamantylmethoxyphosphoryloxyphenyldioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decan}-4-yl)phenyl phosphate (CSPD) as well as CDP and CDPstar® or other luminescent substrates well known to those skilled in the art, such as chelates suitable lanthanides such as terbium(III) and europium(III) The means of detection is determined by the chosen label The manifestation of the label or its reaction products can be achieved with the naked eye, in the case where the label is a particle and accumulates at appropriate levels, or using instruments such as spectrophotometer, luminometer, fluorimeter, etc., all in accordance with standard practice.

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Preferably, conjugation methods result in bonds that are essentially (or nearly) non-immunogenic, eg peptide (ie amide), sulfide, (hindered), disulfide, hydrazone and ether bonds. These bonds are almost non-immunogenic and exhibit reasonable stability in serum (see, for example, Senter P.D., Curr. Opin. Chem. Biol. 13 (2009) 235-244, International Application Publications WO 2009/059278, WO 95/17886).

Depending on the biochemical nature of the fragment and the antibody, different conjugation strategies can be used. In case the fragment is a natural or recombinant polypeptide containing from 50 to 500 amino acids, the manuals describe the chemicals for the synthesis of protein conjugates, which can be easily performed by a person skilled in the art (see, for example, Hackenberger, C.P.R., and Schwarzer D., Angew Chem Int Ed Engl 47 (2008) 10030-10074). In one embodiment, the reaction of a maleimido group with a cysteine residue within an antibody or fragment is used. This is a particularly suitable coupling chemistry in the case of, for example, a Fab or Fab' fragment of an antibody. Alternatively, according to one embodiment, coupling is performed to the C-terminal end of the antibody or fragment. C-terminal modification of a protein, eg a Fab fragment, can, for example, be performed as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

In general, site-specific reaction and covalent coupling are based on the transformation of a natural amino acid into an amino acid with a reactivity that is orthogonal to the reactivity of other functional groups present. For example, a particular cysteine in the context of a rare sequence can be enzymatically converted to an aldehyde (see Frese M.A., and Dierks T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain the desired amino acid modification using the specific enzymatic reactivity of certain naturally occurring amino acid enzymes in a specific sequence context (see, for example, Taki M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier A. et al Chem Biol 15 (2008) 128-136 and protease-catalyzed C--N bond formation is described in Bordusa F., Highlights in Bioorganic Chemistry (2004) 389-403).

Site-specific reaction and covalent coupling can also be obtained by selective reaction of terminal amino acids with appropriate modifying reagents.

The reactivity of N-terminal cysteine with benzonitriles (see Ren et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent coupling.

Primary chemical ligation can also rely on C-terminal cysteine residues (Taylor E. Vogel; Imperiali B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96). EP 1074563 describes a conjugation method based on a faster reaction of cysteine within the negatively charged amino acid region than cysteine located at the positively charged amino acid region.

This fragment may also be a synthetic peptide or a peptide mimic. In the case of chemical synthesis of a polypeptide, amino acids with orthogonal chemical reactivity can be included during this synthesis (see, for example, de Graaf et al., Bioconjug. Chem. 20 (2009) 1281-1295). Because a wide variety of orthogonal functionalities are at stake and can be incorporated into a synthetic peptide, conjugation of such a peptide to a linker is standard chemistry.

To obtain a monolabeled polypeptide, the 1:1 stoichiometry conjugate can be separated by chromatography from other conjugation by-products. This procedure can be facilitated by using a dye-labeled member of the binding pair and a charged linker. Using this type of labeled and highly negatively charged member of the binding pair, monoconjugated polypeptides are easily separated from unlabeled polypeptides and polypeptides that carry more than one linker because the difference in charge and molecular weight can be used for separation. A fluorescent dye can be used to purify the complex from unbound components, such as a labeled monovalent binder.

In one embodiment, the fragment attached to the anti-OX40 antibody is selected from the group consisting of a binding fragment, a labeled fragment, and a biologically active fragment.

Anti-OX40 antibodies described herein can also be conjugated to a therapeutic agent to form an immunoconjugate such as an antibody drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binding agents, DNA intercalators, DNA crosslinking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors. , antibiotics and antimimetic agents. In ADC, the antibody and therapeutic agent are preferably conjugated via a cleavable linker such as a peptidyl, disulfide or hydrazone linker. More preferably, the linker is a peptidyl

- 58 040561 linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 180), Ala-Asn-Val,

Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu. ADC can be obtained as described in US patent No. 7087600; 6989452 and 7129261; PCT publications WO 02/096910; WO 07/038658; WO

07/051081; WO 07/059404; WO 08/083312 and WO 08/103693; US Patent Publications 20060024317;

20060004081 and 20060247295; the disclosure of which is incorporated herein by reference.

More specifically, in an ADC, an antibody is conjugated to a drug, wherein the antibody functions as a targeting agent to target the ADC to a target cell expressing the antigen, such as a cancer cell. Preferably, the antigen is a tumor-associated antigen, ie one that is uniquely expressed or overexpressed by the cancer cell. The drug is then released either within the target cell or in its vicinity to act as a therapeutic agent. For a review of the mechanism of action and use of ADCs in cancer therapy, see Schrama et al., Nature Rev. drug disc. 2006, 5, 147.

For the treatment of cancer, the drug is preferably a cytotoxic drug that causes death of the targeted cancer cell. Cytotoxic drugs that can be used in ADC include the following types of compounds and their analogs and derivatives:

(a) enediines such as calicheamicin (see, for example, Lee et al., J. Am. Chem. Soc. 1987, 109, 3464 and 3466) and uncialamycin (see, for example, Davies et al., WO 2007 /038868 A2 (2007) and Chowdari et al., US 8709431 B2 (2012));

(b) tubulisins (see, for example, Domling et al., US 7778814 B2 (2010); Cheng et al., US 8394922 B2 (2013) and Cong et al., US No. 14/177376, filed Feb. 11, 2014 .));

(c) CC-1065 and duocarmycin (see, for example, Boger, US 6545853θ B1 (2003); Sufi et al., US 8461117 B2 (2013) and Zhang et al., US 2012/0301490 A1 (2012));

(d) epothilones (see, for example, Vite et al., uS 2007/0275904 A1 (2007) and US RE42930E(2011));

(e) auristatins (see, for example, Senter et al., US 684869 B2 (2005) and Doronina et al., US 7498298 B2 (2009));

(f) Pyrrolobezodiazepine (PBD) dimers (see, for example, Howard et al., US 2013/0059800 A1 (2013), US 2013/0028919 A1 (2013) and WO 2013/041606 A1 (2013)); and also (g) maytansinoids such as DM1 and DM4 (see, for example, Chari et al., US 5202020 (1993) and Amphlett et al., US 7374762 B2 (2008)).

In the ADC, the linker covalently binds the antibody and the drug. Typically, there is one drug molecule attached to each linker, but the linker can be branched, allowing multiple drug molecules to be attached to increase the drug payload delivered to the ADC. In addition, each antibody may contain more than one attached linker. The number of drug molecules carried on the ADC is referred to as the drug to antibody ratio (DAR). For example, if each antibody heavy chain is attached to it by one linker, which is in turn attached to one drug molecule, the DAR is 2. Preferably, the DAR is 1 to 5, more preferably 2 to 4. Those skilled in the art it will also be appreciated that although the antibody is conjugated to an integer number of drug molecules in each individual ADC, in general, ADC production can be analyzed for a non-integer DAR reflecting a statistical average. Thus, the ADC architecture can be represented by the following formula:

[Antibody]-[Linker-(Drug)^ where m is typically 1, 2, 3, 4, 5, or 6 (preferably 2, 3, or 4) and n is 1, 2, or 3.

In some embodiments, the linker contains a leaving group that is cleaved within or near the target cell to release the drug. In other embodiments, the linker does not contain a leaving group, but rather the ADC relies on antibody catabolism to release the drug.

One type of cleavable group is a pH sensitive group. The pH value in blood plasma is slightly above neutral, while the pH value inside the lysosome, where most ADCs end up after internalization inside the target cell, is acidic, approximately 5. Thus, a cleavable group whose cleavage is catalyzed by acid will be cleaved at a rate several orders of magnitude higher inside the lysosome than in blood plasma. Examples of acid sensitive groups include cis-aconithylamides and hydrazones as described in Shen et al., US 4,631,190 (1986); Shen et al. US 5144011 (1992); Shen et al., Biochem. Biophys. Res. commun. 1981, 102, 1048 and Yang et al., Proc. Nat'l Acad. Sci (USA), 1988, 85, 1189; the disclosure of which is incorporated herein by reference.

In another embodiment, the cleavable group is a disulfide. Disulfides can be cleaved by a thiol-disulfide exchange mechanism at a rate dependent on the concentration of the surrounding thiol. Since the intracellular concentration of glutathione and other thiols is higher than their serum concentration, the rate of disulfide degradation will be higher intracellularly, ie after ADC internalization. In addition, the rate of thiol-disulfide exchange can be modulated by adjusting the steric and electronic characteristics of the disulfide (e.g., alkylarylaryl disulfide versus alkylalkyl disulfide, substitution on the aryl ring, etc.), allowing the design of disulfide bonds that enhance serum stability or specific splitting rate. For more information regarding disulfide cleavable groups in conjugates, see, for example, Thorpe et al., Cancer Res. 1988, 48, 6396; Santi et al. US 7541530 B2 (2009); Ng et al., US 6989452 B2 (2006); Ng et al., WO 2002/096910 A1 (2002); Boyd et al., US 7691962 B2 (2010) and Sufi et al., US 2010/0145036 A1 (2010); the disclosure of which is incorporated herein by reference.

A preferred cleavable group is a peptide that is selectively cleaved by a protease within the target cell, as opposed to a serum protease. Typically, the cleavable peptide group contains 1 to 20 amino acids, preferably 1 to 6 amino acids, more preferably 1 to 3 amino acids. The amino acid(s) may be a natural and/or non-natural amino acid. Natural amino acids are those encoded by the genetic code, as well as amino acids derived from them, such as hydroxyproline, γ-carboxyglutamate, citrulline and O-phosphoserine. In this context, the term amino acid also includes amino acid analogs and mimetics. Analogues are compounds having the same general structure H ₂ N(R)CHCO ₂ H of a naturally occurring amino acid, except that the R group does not occur among naturally occurring amino acids. Examples of analogs include homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. An amino acid mimetic is a compound that has a structure that is different from the general chemical structure of an α-amino acid, but functions similarly to that. An amino acid can be characterized by the stereochemistry L of the genetically encoded amino acids as well as the enantiomeric stereochemistry D.

Preferably, the cleavable peptide group contains an amino acid sequence that is a cleavage recognition sequence for a protease. Many split recognition sequences are known in the art. See, for example, Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244:175 (1994); Thornberry, Meth. Enzymol. 244:615 (1994); Weber et al. Meth. Enzymol. 244:595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994) and Bouvier et al. Meth. Enzymol. 248:614 (1995); the disclosure of which is incorporated herein by reference.

More preferably, the cleavable peptide group contains an amino acid sequence selected for cleavage by an endosomal or lysosomal protease, especially the latter. Examples of such proteases include cathepsins B, C, D, H, L, and S, especially cathepsin B. Cathepsin B preferentially cleaves peptides in the sequence -AA2-AA1-, where AA1 is a basic or highly hydrogen-binding amino acid (such as lysine , arginine, or citrulline) and AA2 is a hydrophobic amino acid (such as phenylalanine, valine, alanine, leucine, or isoleucine), such as Val-Cit (where Cit is citrulline) or Val-Lys, written N to C. For more information on groups cleaved by cathepsin, see Dubowchik et al., Biorg. Med. Chem. Lett. 1998, 8, 3341; Dubowchik et al., Bioorg. Med. Chem. Lett, 1998, 8, 3347 and Dubowchik et al., Bioconjugate Chem. 2002, 13, 855; the disclosure of which is incorporated by reference. Another enzyme that can be used to cleave peptidyl linkers is legumen, a lysosomal cysteine protease that preferentially cleaves Ala-Ala-Asn.

In a preferred embodiment, the linker in the ADC contains a di- or tripeptide that is preferably cleaved by a protease located within the target cell. Preferably the di- or tripeptide is cleaved with cathepsin B, more preferably the Val-Cit or ValLys dipeptide.

Single amino acid cleavable peptide groups can also be used as described in Chen et al., US 2010/0113476 A1 (2010), the disclosure of which is incorporated herein by reference.

For conjugates that are not intended to be internalized by the cell, the cleavable group may be chosen to be cleavable by a protease present in the extracellular matrix near the target cell, such as a protease released by neighboring dying cells or a tumor-associated protease. Illustrative extracellular tumor-associated proteases are matrix metalloproteases (MMPs), plasmin, thymetic oligopeptidase (TOP), and CD10. See, for example, Trouet et al., US 5961216 (1999) and US 7402556 B2 (2008); Dubois et al. US 7425541 B2 (2008) and Bebbington et al. US 6897034 B2 (2005); the disclosure of which is incorporated herein by reference.

The linker may serve other functions in addition to covalently linking the antibody and drug. For example, the linker may contain poly(ethylene glycol) (PEG) groups that increase solubility either during conjugation chemistry or in the final ADC product.

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The linker may further include a self-destructive fragment adjacent to the peptide group to be cleaved off. The self-destructive group serves as a spacer that prevents the antibody and/or drug component from sterically interfering with protease cleavage of the peptide group, but then spontaneously releases (ie, self-destructs) so as not to interfere with the action of the drug. See Carl et al., J. Med. Chem. 1981, 24(3), 479; Carl et al., WO 81/01145 (1981); Dubowchik et al., Pharmacology & Therapeutics 1999, 83, 67; Firestone et al., US 6214345 B1 (2001); Toki et al., J. Org. Chem. 2002.67, 1866; Doronina et al., Nature Biotechnology 2003, 21 (7), 778 (erratum, p. 941); de Groot et al.,. Org. Chem. 2001, 66, 8815; Boyd et al., US 7691962 B2 (2010); Boyd et al., US 2008/0279868 A1 (2008); Sufi et al., WO 2008/083312 A2 (2008); Feng, US 7375078 B2 (2008); Jeffrey, US 8039273 B2 (2011) and Senter et al., US 2003/0096743 A1 (2003); the disclosure of which is incorporated by reference.

A preferred self-destructive group is a p-aminobenzyloxycarbonyl (PABC) group, the structure and mechanism of action of which is shown below: protease cleavage ₀

[medicine]

PABC

PABC

HN

_n ^[drug]

-CO ₂

Thus, according to a preferred embodiment, the ADC comprises a linker containing a di- or tripeptide that is preferentially cleaved by a protease located within the target cell and a self-destructive group adjacent to the di- or tripeptide. Preferably, the di- or tripeptide is cleaved with cathepsin B. Preferably, the self-destructive group is a PABC group.

Numerous methods can be used to conjugate an antibody and a drug. Preferably, the s-amino group in the side chain of a lysine residue in the antibody is reacted with 2-iminothiolane to introduce a free thiol group (-SH). The thiol group can react with a maleimide or other nucleophilic acceptor group to effect conjugation as shown below:

Thiolane antibody

[| m-[Linker]-[Drug]

ABOUT

NH _p

N—[Linker]—[Drug]

[Linker]-[Medication]

ABOUT

ADC

Typically, a thiolation level of two to three thiols per antibody is achieved. For a typical procedure, see Chowdari et al., US 8709431 B2 (2014), the disclosure of which is incorporated herein by reference. Thus, in one embodiment, an antibody of the present invention contains one or more lysine residues (preferably two or three) modified by reaction with iminothiolane.

An alternative conjugation technique uses copper-free click chemistry in which an azide group is added through a deformed cyclooctyne alkyne bond to form a 1,2,3triazole ring. See, for example, Agard et al., J. amer. Chem. soc. 2004.126, 15046; Best, Biochemistry 2009, 48, 6571, the disclosure of which is incorporated herein by reference. The azide may be located on the antibody and the cyclooctyne on the drug moiety, or vice versa. A preferred cyclooctyne group is dibenzocyclooctyne (DIBO). Various reagents containing a DIBO group are available from Invitrogen/Molecular Probes, Eugene, Oregon. The reaction below illustrates click chemistry conjugation for a case in which a DIBO group is attached to an antibody.

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In an ADC made using this technique, the linker contains a 1,2,3-triazole ring.

Yet another conjugation method involves introducing a non-natural amino acid into an antibody, wherein the non-natural amino acid provides the functionality for conjugation to a reactive functional group on the drug moiety. For example, a non-natural amino acid p-acetylphenylalanine can be incorporated into an antibody or other polypeptide as described in Tian et al., WO 2008/030612 A2 (2008). The ketone group in p-acetylphenyalanine may be a conjugation site by forming an oxime with a hydroxylamino group on the linker drug moiety. Alternatively, a non-natural amino acid p-azidophenylalanine can be incorporated into an antibody to provide an azide functional group for conjugation via clickchemistry, as discussed above. Non-natural amino acids can also be incorporated into an antibody or other polypeptide using cell-free methods as described in Goerke et al., US 2010/0093024 A1 (2010) and Goerke et al., Biotechnol. Bioeng. 2009, 102(2), 400-416. The above disclosures are incorporated herein by reference. Thus, in one embodiment, the antibody comprises one or more amino acids substituted with a non-natural amino acid, which is preferably p-acetylphenylalanine or p-azidophenylalanine, more preferably p-acetylphenylalanine.

Yet another conjugation method uses the enzyme transglutaminase (preferably bacterial transglutaminase or BTG) as described in Jeger et al., Angew. Chem. Int. Ed. 2010, 49, 9995. BTG forms an amide bond between the glutamine side chain carboxamide and an alkyleneamino group, which may be, for example, a lysine s-amino group or a 5-amino-n-pentyl group. In a typical conjugation reaction, the glutamine residue is located on the antibody, while the alkyleneamino group is located on the drug linker moiety, as shown below:

Gin—(CH ₂ ) ₂ -C-NH ₂

Antibody

BTG

N “[Linker] - [Drug - remedy]

Gin-I

ABOUT

II (C H ₂ ) ₂ -C - N- [Linker] - [Drug] H

ADC

The positioning of the glutamine residue on the polypeptide chain has a major impact on its susceptibility to BTG-mediated transamidation. None of the glutamine residues on an antibody is typically a BTG substrate. However, if the antibody is deglycosylated, then the glycosylation site, which is asparagine 297 (N297) - the nearest glutamine 295 (Q295), is successfully exposed to BTG. Alternatively, the antibody can be synthesized without the glycoside by introducing an N297A mutation in the constant region to eliminate the N297 glycosylation site. In addition, it has been shown that the N297Q substitution in the antibody not only abolishes glycosylation, but also introduces a second glutamine residue (at position 297), which is also a receptive BTG-mediated transamidation. Thus, in one embodiment, the anti-OX40 antibody is deglycosylated. In another embodiment, the anti-OX40 antibody contains the N297Q substitution. Those skilled in the art will appreciate that deglycosylation by post-synthetic modification or by introducing the N297A mutation results in two BTG-reactive glutamine residues per antibody (one per heavy chain at position 295), while an antibody with an N297Q substitution will contain four BTG-reactive glutamine residues (two per heavy chain, at positions 295 and 297).

In addition, another conjugation technique uses the sortase A enzyme as described in Levary et al., PLoS One 2011, 6(4), e18342; Proft, Biotechnol. Lett. 2010, 32, 1-10; Ploegh et al., WO 2010/087994 A2 (2010) and Mao et al., WO 2005/051976 A2 (2005), the disclosure of which is incorporated herein by reference. A sortase A recognition motif (typically LPXTG (SEQ ID NO: 181) where X is any naturally occurring amino acid) can be attached to the antibody and a nucleophilic acceptor motif (typically GGG) can be located on the drug moiety, or vice versa .

Anti-OX40 antibodies described herein can also be used to detect

- 62 040561 OX40, such as human OX40, eg human OX40, in tissues or tissue samples. The antibodies can be used, for example, in an ELISA assay or in flow cytometry. In some embodiments, the anti-OX40 antibody is contacted with cells, such as cells in a tissue, for a time suitable for specific binding, and then a reagent is added, such as an antibody that detects the anti-OX40 antibody. Illustrative assays are provided in the examples. An anti-OX40 antibody may be a fully human antibody, or may be a chimeric antibody, such as an antibody containing human variable regions and mouse constant regions, or a portion thereof. Exemplary methods for detecting OX40, e.g., human OX40, in a sample (cell or tissue sample) involve (i) contacting the sample with an anti-OX40 antibody for a time sufficient to allow the anti-OX40 antibody to specifically bind to OX40 in the sample, and (2) contacting the sample with a detection reagent, for example an antibody that specifically binds to an anti-OX40 antibody, such as the Fc region of an anti-OX40 antibody, to thereby detect OX40 bound by the anti-OX40 antibody. Washing steps may be included after incubation with the antibody and/or detection reagent. Anti-OX40 antibodies for use in these methods need not be associated with a label or detection means, as a separate detection tool may be used.

XIV. bispecific molecules.

Anti-OX40 antibodies described herein can be used to form bispecific molecules. For example, an antibody or antigen-binding portions thereof may be derivatized or linked to another functional molecule, such as a different peptide or protein (e.g., a different antibody or ligand for a receptor) to form a bispecific molecule that binds to at least two different binding sites, or target molecules. In one embodiment, an anti-OX40 antibody can be linked to an antibody or scFv that specifically binds to any protein that can be used as potential targets for combination treatments, such as the proteins described herein (e.g., anti-PD-1, PD-L1 or LAG-3). Alternatively, an antibody can be derivatized or linked to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and/or target molecules. Such multispecific molecules should also be covered by the term bispecific molecule as used herein. To create the bispecific molecule described herein, an anti-OX40 antibody may be operably linked (e.g., by chemical bonding, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, so that a bispecific molecule is obtained.

Accordingly, provided herein are bispecific molecules comprising at least one first binding specificity for OX40 and a second binding specificity for a second target epitope. In one embodiment, the bispecific molecule is multispecific, eg, the molecule further includes a third binding specificity.

In some embodiments, the bispecific molecules comprise at least one antibody or antibody fragment as binding specificity, including, for example, Fab, Fab', F(ab') ₂ , Fv, or single chain Fv (scFv). The antibody can also be a light chain or heavy chain dimer or any minimal fragment thereof such as Fv or a single chain construct as described in Ladner et al., US Pat. No. 4,946,778, the contents of which are expressly incorporated by reference.

While human monoclonal antibodies are preferred, other antibodies may be used in the bispecific molecules described herein, including, for example, murine, chimeric, and humanized antibodies.

Provided herein bespecifically molecules can be obtained by conjugation of specific binding components using methods known in the present field of technology. For example, each binding specificity of a bispecific molecule can be obtained separately and then conjugated to each other. When the specific binding components are proteins or peptides, various binding or cross-linking agents can be used for covalent conjugation. Examples of crosslinkers include protein A, carbodiimide, N-succinimidyl-S-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3 -(2-pyridyldithio)propionate (SPDP) and sulfosuccinimidyl-4-(N-maleimidomethyl)cycloxane-1-carboxylate (sulfo-SMCC) (see e.g. Karpovsky et al. (1984) J. Exp. Med. 160 :1686; Liu M. A. et al (1985) Proc Natl Acad Sci USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. no. 78, 118-132; Brennan et al. (1985) Science 229:81-83) and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Preferred conjugants are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

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When the specific binding components are antibodies, they can be conjugated by a sulfhydryl bond between the C-terminal hinge regions of the two heavy chains. According to a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both specific binding components can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, mAb x (scFv)2, Fab x F(ab')2 fusion protein, or a Fab x ligand. The bispecific antibody may comprise an antibody containing an scFv at the C-terminus of each heavy chain. The bispecific molecule described herein can be a single chain molecule containing one single chain antibody and a binding determinant, or a single chain bispecific molecule containing two binding determinants. Bispecific molecules may contain at least two single chain molecules. Methods for obtaining bispecific molecules are described, for example, in US patent No. 5260203; US patent No. 5455030; US Pat. No. 4,881,175; US patent No. 5132405; US Pat. No. 5,091,513; US patent No. 5476786; US patent No. 5013653; U.S. Patent No. 5,258,498 and U.S. Patent No. 5,482,858.

Binding of bispecific molecules to their specific targets can be confirmed using methods recognized in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS assay, bioassay (eg, growth inhibition), or Western blot assay. Each of these assays typically detects the presence of protein-antibody complexes of particular interest using a labeled reagent (eg, antibody) specific for the complex of interest.

XV. Compositions.

Further contemplated are compositions, eg pharmaceutical compositions, comprising one or more anti-OX40 antibodies, alone or in combination with antibodies to other targets, formulated with a pharmaceutically acceptable carrier. Such compositions may include one or a combination (eg, two or more different) antibodies or immunoconjugates, or bispecific molecules described herein. For example, a composition may contain a combination of antibodies (or immunoconjugates or bispecific antibodies) described herein that bind to different epitopes on OX40 or that have complementary activities.

In some embodiments, the composition comprises an anti-OX40 antibody at a concentration of at least 1, 5, 10, 50, 100, 150, 200, 1-300, or 100-300 mg/mL.

The pharmaceutical compositions described herein may also be administered in combination therapy, ie. in combination with other means. For example, combination therapy may include administering an anti-OX40 antibody described herein in combination with at least one other antitumor and/or T-cell stimulating (ie, activating) agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the use of antibodies described herein.

In some embodiments, the therapeutic compositions disclosed herein include other compounds, drugs, and/or agents used to treat cancer. Such compounds, drugs and/or agents may include, for example, chemotherapeutic drugs, small molecule drugs, or antibodies that stimulate an immune response to a given cancer. In some cases, therapeutic compositions may include, for example, one or more of the following: an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PDL-1 antibody, an anti-GITR antibody, an anti-CD137 antibody, or an anti-LAG-3 antibody.

As used herein, the term pharmaceutically acceptable carriers includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active substance, i.e. the antibody, immunoconjugate, or bispecific molecule may be coated with a material to protect the compound from acids and other environmental conditions that may inactivate the compound.

The pharmaceutical compositions described herein may include one or more pharmaceutically acceptable salts. A pharmaceutically acceptable salt refers to a salt that retains the desired biological activity of the parent compound and does not produce any undesirable toxicological effects (see, for example, Berge S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphoric, and the like, as well as from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids. acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as from non-toxic organic amines such as N,N'-dibenzylethylenediamine, Nmethylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

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

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

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of the presence of microorganisms can be ensured both by the sterilization procedures above and by the inclusion of various antibacterial and antifungal agents, eg paraben, chlorobutanol, phenolic sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of an injectable pharmaceutical form may be caused by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for preparing sterile immediate injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except that any conventional medium or agent is incompatible with the active compound, their use in the pharmaceutical compositions described herein is contemplated. The pharmaceutical composition may or may not contain a preservative. Additional active compounds may be included in the compositions.

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

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or more of the ingredients listed above, followed by sterilizing microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains the basic dispersion medium and the required other ingredients listed in the present invention. In the case of sterile powders for the preparation of sterile injectable solutions, vacuum drying and freeze-drying (lyophilization) are the preferred methods of preparation, which yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution.

The amount of active ingredient which may be combined with the carrier material to form a unit dosage form will vary depending on the subject to be treated and the particular route of administration. The amount of active ingredient that can be

- 65 040561 combined with the carrier material to obtain a unit dosage form, as a rule, will be such an amount of the composition that gives a therapeutic effect. Typically, out of one hundred percent, this amount will be from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient in combination with pharmaceutically acceptable carrier.

Dosing regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally reduced or increased as required by the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and dosage uniformity. As used herein, the dosage form refers to physically discrete units suitable as unit doses for the patients to be treated; each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect, in combination with the required pharmaceutical carrier. The specification of the unit dosage forms described herein is dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the scope of mixing such an active compound for the treatment of sensitivity in individuals.

For administration of an anti-OX40 antibody, the dosage is about 0.0001 to 100 mg/kg, about 0.01 to 5 mg/kg, about 0.01 to 10 mg/kg, about 0.1 to about 1 mg/kg. kg, from about 0.1 to about 0.5 mg/kg, or from about 0.5 to 0.8 mg/kg of the body weight of the host. For example, dosages may be 0.2 mg/kg body weight, 0.3 mg/kg body weight, 0.5 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within 1-10 mg/kg. In some embodiments, the dosage is 0.2 mg/kg. In some embodiments, the dosage is 0.25 mg/kg. In other embodiments, the dosage is 0.5 mg/kg. An exemplary treatment regimen is administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months. Exemplary dosing regimens for the antibodies described herein include 1 mg/kg body weight or 3 mg/kg body weight intravenously, with the antibody administered using one of the following dosing schedules: (i) every four weeks until six doses, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight followed by 1 mg/kg body weight every three weeks.

In some embodiments, for combination treatment with an anti-OX40 antibody and an anti-PD-1 or anti-CTLA-4 antibody, the antibodies are administered at a fixed dose. Accordingly, in some embodiments, the anti-OX40 antibody is administered at a fixed dose of about 25 to about 320 mg, such as about 25 to about 160 mg, about 25 to about 80 mg, about 25 to about 40 mg, about 40 to about 320 mg, about 40 to about 160 mg, about 40 to about 80 mg, about 80 to about 320 mg, about 30 to about 160 mg, or about 160 to about 320 mg. In one embodiment, the anti-OX40 antibody is administered at a dose of 20 mg, or about 20 mg. In another embodiment, the anti-OX40 antibody is administered at a dose of 40 mg, or about 40 mg. In another embodiment, the anti-OX40 antibody is administered at a dose of 80 mg, or about 80 mg. In another embodiment, the anti-OX40 antibody is administered at a dose of 160 mg, or about 160 mg. In another embodiment, the anti-OX40 antibody is administered at a dose of 320 mg, or about 320 mg.

In some embodiments, the anti-PD-1 antibody is administered at a fixed dose of about 100 to 300 mg. For example, the dosage of the immuno-oncological agent may be 240 mg or about 240 mg, 360 mg or about 360 mg or 480 mg or about 480 mg. In some embodiments, the dose of anti-PD1 antibody is in the range of about 0.0001 to 100 mg/kg, and more typically 0.01 to 5 mg/kg of host body weight. For example, dosages may be 0.3 mg/kg body weight, or about 0.3 mg/kg body weight, 1 mg/kg body weight, or about 1 mg/kg body weight, 3 mg/kg body weight, or about 3 mg/kg. kg body weight, 5 mg/kg body weight or about 5 mg/kg body weight or 10 mg/kg body weight or about 10 mg/kg body weight or in the range of 1-10 mg/kg. In some embodiments, the dosage of the anti-PD-1 antibody is 240 mg, or about 240 mg, administered once every 2 weeks (Q2W). This dose may be adjusted proportionately (at 120 mg per week) for a longer or shorter period, such as 360 mg given once every 3 weeks (Q3W) or 480 mg given once every 4 weeks (Q4W).

In some embodiments, the anti-CTLA-4 antibody is administered at a dose of about 0.1 mg/kg to about 10 mg/kg. For example, dosages may be 1 mg/kg or about 1 or 3 mg/kg or about 3 mg/kg of host body weight.

Exemplary dosage regimens for combination treatment with anti-OX40 and anti-PD-1 or anti-CTLA-4 antibody are provided below in the Use and Methods section.

In some embodiments, the anti-OX40 antibody is administered to the patient over an infusion time of about 15 minutes to about 60 minutes, such as about 30 minutes.

In some embodiments, an anti-PD-1 antibody (e.g., nivolumab) is administered to a patient with an infusion duration of about 15 minutes to about 60 minutes, such as about 30 minutes, at a dose of 3 mg/kg (0.1 mg/kg/min ). In some embodiments, the anti-PD-1 antibody is administered to the patient with an infusion duration of about 45 to 75 minutes, such as about 60 minutes, when administered at a dose of 10 mg/kg.

In some embodiments, an anti-CTLA-4 antibody (eg, ipilimumab) is administered to a patient over an infusion time of about 15 to 120 minutes, eg, about 30 minutes, at a dose of 3 mg/kg. In some embodiments, the anti-CTLA-4 antibody is administered to the patient with an infusion duration of 15 to 120 minutes, such as 90 minutes, when administered at a dose of 10 mg/kg.

In some embodiments, for same-day administration, the anti-OX40 antibody is administered before the anti-PD-1 or anti-CTLA-4 antibody. In some embodiments, for same-day administration, the anti-OX40 antibody is administered after the anti-PD-1 or anti-CTLA-4 antibody. In some embodiments, when administered on the same day, the anti-OX40 antibody is administered simultaneously with the anti-PD1 or anti-CTLA-4 antibody.

In some embodiments, for same-day administration, the anti-OX40 antibody is administered approximately 15 to 45 minutes (eg, approximately 30 minutes) prior to the anti-PD-1 or anti-CTLA-4 antibody. In some embodiments, for same-day administration, the anti-OX40 antibody is administered approximately 15-45 minutes (eg, approximately 30 minutes) after the anti-PD-1 or anti-CTLA-4 antibody.

Alternatively, the anti-OX40 antibodies provided herein can be administered at a constant dose (fixed dose regimen).

In some cases, two or more monoclonal antibodies with different binding specificities are administered simultaneously so that the dosage of each antibody administered is within the above ranges. In addition, antibodies are typically administered multiple times. Dosing intervals may be, for example, one week, one month, three months or one year. The intervals may also be irregular, as indicated by measuring the level of antibodies to the target antigen in the patient. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 µg/ml, and in some methods about 25-300 µg/ml.

Anti-OX40 antibodies described herein can be administered with another antibody in a different antibody dosing regimen. For example, an anti-OX40 antibody can be administered with an anti-PD-1 antibody such as nivolumab (OPDIVO) every two weeks as an intravenous infusion for 60 minutes until disease progression or unacceptable toxicity. Alternatively, an anti-OX40 antibody can be administered with pembrolizumab (KEYTRUDA) every 3 weeks as an intravenous infusion over 30 minutes until disease progression or unacceptable toxicity.

Antibodies can be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency will vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively short intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage is sometimes required at relatively short intervals until the progression of the disease is reduced or stopped, and preferably until the patient shows a partial or complete improvement in the symptoms of the disease. After that, the patient can enter a prophylactic treatment regimen.

Actual dosage levels of active ingredients in the pharmaceutical compositions described herein may vary so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and route of administration, without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the potency of the particular compositions described herein or their ester, salt, or amide, route of administration, time of administration, rate of release of the particular compound employed, duration of treatment, other drugs, compounds, and /or materials used in combination with the specific compositions used, age, sex, weight, condition, general health and previous medical history of the patient to be treated, and similar factors well known in medicine.

Therapeutically effective doses of the antibodies described herein preferably result in a reduction in the severity of the symptoms of the disease, an increase in the frequency and duration of periods without symptoms of the disease, or the prevention of deterioration or disability due to the disease. In the context of cancer, a therapeutically effective dose preferably results in increased survival and/or prevention of further worsening of the physical symptoms associated with the cancer. Symptoms of cancer are well known in the art and include, for example, unusual features of the mole, change in the appearance of the mole, including asymmetry, border, color and/or diameter, area of newly pigmented skin, abnormal mole, darkened area under the nails, lumps in the chest, nipple changes, breast cysts, chest pain, death, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, worsening shortness of breath, coughing up blood, blood in the urine, blood in the stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, stomach fullness, bloating, abdominal fluid, vaginal bleeding, constipation, abdominal distention, colonic perforations, acute peritonitis (infection, fever, pain), pain, severe sweating, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, colon metastases, metastases in the lungs, bladder metastases, liver metastases, bone metastases, kidney metastases and pancreas metastases, difficulty in swallowing, and the like.

A therapeutically effective dose may prevent or delay the onset of cancer, which may be required, for example, when early or early signs of the disease are present. Laboratory tests used in the diagnosis of cancer include chemical analysis (including OX40 measurement), hematology, serology, and radiology. Accordingly, any clinical or biochemical assay that controls any of the foregoing can be used to determine whether a particular exposure is a therapeutically effective dose for treating cancer. Those skilled in the art can determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the selected formulation or route of administration.

The antibodies and compositions described herein may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and/or route of administration will vary depending on the desired results. Preferred routes of administration of the antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral route of administration, for example, by injection or infusion. As used herein, the phrase parenteral administration means routes of administration other than enteral and topical administration, typically by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intraarticular injection and infusion.

Alternatively, the antibody can be administered via a non-parenteral route, such as a topical, epidermal, or mucosal route, such as intranasally, orally, vaginally, rectally, sublingually, or topically.

Active compounds can be formulated with carriers that will protect the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such compositions are patented or known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Antibody compositions can be administered using medical devices known in the art. For example, in one embodiment, the composition is administered using a needleless hypodermic injector such as the devices disclosed in US Pat. Nos. 5,399,163; 5383851; 5312335; 5064413; 4941880; 4,790,824 or 4,596,556. Examples of well-known implants and modules for use in antibody administration include: US Patent No. 4,487,603, which discloses an implantable microinfusion pump for drug delivery at a controlled rate; US patent No. 4486194, which discloses a therapeutic device for the introduction of drugs through the skin; US Pat. No. 4,447,233, which discloses a drug infusion pump for drug delivery at a high infusion rate; US patent

- 68 040561 No. 4447224, which discloses an implantable infusion system with a flow regulator for continuous drug delivery; US Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US Pat. No. 4,475,196 which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.

In some embodiments, anti-OX40 antibodies are formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that antibodies cross the BBB (if desired, eg in brain cancer), they can be formulated, eg, in liposomes. For methods of making liposomes, see, for example, US Pat. Nos. 4,522,811; 5,374,548 and 5,399,331. Liposomes may contain one or more moieties that are selectively transferred to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, V.V. Ranade (1989) J. Clin. Pharmacol. 29:685). Illustrative target fragments include folate or biotin (see, for example, US patent No. 5416016 in the name of Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346:123; I.J. killion; I.J. Fidler (1994; Immunomethods 4:273.

XVI. Application and methods.

The anti-OX40 antibodies and compositions described herein have numerous in vitro and in vivo uses, including, for example, enhancing the immune response by activating OX40 signaling or detecting OX40. In a preferred embodiment, the antibodies are human antibodies. For example, anti-OX40 antibodies described herein can be contacted with cells in culture, in vitro or ex vivo, or administered to humans, for example, in vivo, to enhance immunity in various diseases. Accordingly, provided herein are methods for modifying an immune response in a subject, comprising administering to the subject an antibody described herein, or an antigen-binding portion thereof, such that the subject's immune response is modified. Preferably, the response is enhanced, stimulated or regulated.

Preferred subjects include human patients in whom an enhanced immune response would be desirable. The methods are particularly suitable for treating human patients with a disorder that can be treated by enhancing an immune response (eg, T-cell mediated immune response, eg, antigen-specific T-cell response). In a specific embodiment, the methods are particularly suitable for treating cancer in vivo. To achieve antigen-specific immunity enhancement, the anti-OX40 antibodies described herein may be co-administered with the antigen of interest, or the antigen may already be present in the subject to be treated (eg, tumor-bearing or virus-bearing subject). When antibodies to OX40 are administered together with another agent, they can be administered separately or simultaneously.

Also covered are methods for detecting the presence of human OX40 antigen in a sample, or measuring the amount of human OX40 antigen, comprising contacting the sample and a control sample with the anti-OX40 antibodies (or antigen-binding portions thereof) described herein under conditions that allow the formation of a complex between the antibody and human OX40. Complex formation is then detected, with the difference between complex formation with the sample compared to the control sample indicating the presence of human OX40 antigen in the sample. Anti-OX40 antibodies described herein can also be used to purify human OX40 by immunoaffinity purification.

Given the ability of the anti-OX40 antibodies described herein to stimulate or co-stimulate T cell responses, such as antigen-specific T cell responses, also provided herein are in vitro and in vivo methods of using antibodies to stimulate, enhance, or up-regulate antigen-specific T-cell responses. cells, such as antitumor T-cell responses. In some embodiments, CD3 stimulation is also included (eg, by co-incubation with a CD3 membrane-expressing cell), and stimulation can be provided at the same time, before or after stimulation with anti-OX40 antibody. In one embodiment, the method comprises contacting T cells with an anti-OX40 antibody as described herein, and optionally with an anti-CD3 antibody, such that an antigen-specific T cell response is stimulated. Any suitable indicator of antigen specific T cell response can be used to measure antigen specific T cell response. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of an antibody and/or an increase in cytokine production in the presence of an antibody. In a preferred embodiment, the production of interleukin-2 and/or interferon-y by the antigen-specific T cell is stimulated.

T cells that can be boosted or co-stimulated by anti-OX40 antibodies include

CD4+ T cells and CD8+ T cells. T cells may be T _eff cells, e.g. CD4+ cells

T _eff , CD8+ T _eff cells, T-helper (Th) cells and T-cytotoxic (T _c ) cells.

Also provided are methods of stimulating an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering to the subject a therapeutically effective amount of an anti-OX40 antibody described herein such that an immune response (e.g., an antigen-specific T cell response) is stimulated in the subject. In a preferred embodiment, the subject is a subject with a tumor and is stimulated to have an immune response against the tumor. The tumor may be a solid tumor or a liquid tumor, such as a hematological malignancy. In some embodiments, the tumor is an immunogenic tumor. In some embodiments, the tumor is non-immunogenic. In some embodiments, the tumor is positive for PD-L1. In some embodiments, the tumor is PD-L1 negative. The subject may also be a carrier of the virus and an immune response against the virus is stimulated.

Further provided are methods for inhibiting tumor cell growth in a subject, comprising administering to the subject a therapeutically effective amount of an anti-OX40 antibody described herein such that tumor growth in the subject is inhibited. Also provided are methods of treating a viral infection in a subject, comprising administering an anti-OX40 antibody described herein to the subject such that the viral infection is treated in the subject.

Also covered herein are methods for depleting Treg cells from the tumor microenvironment of a subject with a tumor, e.g., cancer, comprising administering to the subject a therapeutically effective amount of an anti-OX40 antibody described herein that contains an Fc that stimulates _Treg depletion in the tumor microenvironment. An Fc may be, for example, an Fc with effector function or enhanced effector function, such as binding or having improved binding to one or more activating Fc receptors. In a preferred embodiment, T _reg depletion occurs without significant depletion or inhibition of T _eff in the tumor microenvironment and without significant depletion or inhibition of T _eff cells and T _reg cells outside the tumor microenvironment, eg, in the periphery. In some embodiments, the subject has a higher OX40 content on Treg cells than on Teff cells, for example, in a tumor microenvironment.

In some embodiments, the subject is treated with an anti-OX40 antibody containing an Fc that enhances agonism, eg, binds or improves binding to an inhibitory FcRIIb. Anti-OX40 antibodies can deplete Treg in tumors and/or Treg in tumor infiltrating lymphocytes (TILs).

In some embodiments, the anti-OX40 antibody is provided to the subject as an adjunctive therapy. Treatment of cancer subjects with an anti-OX40 antibody may result in long-term survival, eg, a long-term sustained response relative to current standard of care; long-term survival of at least 3 months, 6 months, 9 months, 1, 2, 3, 4, 5, 10 years or more, or disease-free survival of at least 3, 6, 9 months, 1, 2, 3, 4, 5, or 10 or more years. In some embodiments, treating a subject with cancer with an anti-OX40 antibody prevents cancer recurrence or delays cancer recurrence, e.g., by 3, 6, 9 months, 1, 2, 3, 4, 5, or 10 or more years. Anti-OX40 antibody treatment can be used as a first, second or third line treatment.

In preferred embodiments, the anti-OX40 antibody is not significantly toxic. For example, the antibody is not significantly toxic to a human organ, such as one or more of: liver, kidney, brain, lung, and heart, as determined, for example, in clinical trials. In some embodiments, the antibody has no significant effect on an unwanted immune response, such as autoimmunity or inflammation.

In some embodiments, treatment of a subject with an anti-OX40 antibody does not overstimulate the immune system to the point that the subject's immune system then attacks the subject itself (eg, an autoimmune response) or does not result in, for example, anaphylaxis. Thus, the antibodies preferably do not cause anaphylaxis.

In some embodiments, treatment of a subject with an anti-OX40 antibody does not cause significant inflammatory responses, such as immune-mediated pneumonitis, immune-mediated colitis, immune-mediated hepatitis, immune-mediated nephritis or renal dysfunction, immune-mediated hypophysitis, immune-mediated hypothyroidism and hyperthyroidism, or others. immune-mediated adverse reactions.

In some embodiments, an anti-OX40 antibody provides synergistic antitumor effects when combined with another cancer treatment, such as a compound that stimulates the immune system (e.g., an immuno-oncological agent), such as a compound described herein, or a compound modulating the target described herein.

These and other methods described herein are discussed in more detail below.

Malignant tumor.

Activation of OX40 by anti-OX40 antibodies can enhance the immune response to malignant cells in a patient. Accordingly, provided herein are methods of treating a subject with a cancer comprising administering to the subject anti-OX40 antibodies described herein such that the subject is treated, such as such that the growth of cancerous tumors is inhibited or reduced and/or that the tumors regress, and/or that long-term survival is achieved. Anti-OX40 antibody can be used alone to inhibit the growth of malignant tumors. Alternatively, an anti-OX40 antibody may be used in combination with another agent, such as another immunogenic agent, standard cancer treatment, or another antibody, as described below.

Accordingly, provided herein are methods of treating cancer, for example, by inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of the anti-OX40 antibodies described herein. The antibody may be a human antibody. Additionally or alternatively, the antibody may be a chimeric or humanized antibody.

Also contemplated herein are combination therapies comprising administering an anti-OX40 antibody and an anti-PD-1 antibody or an anti-CTLA-4 antibody to treat subjects with tumors (eg, advanced solid tumors).

In some embodiments, provided herein are methods for treating cancer, wherein an anti-OX40 antibody and an anti-PD-1 antibody or an anti-CTLA-4 antibody are administered to a patient with a tumor (e.g., advanced solid tumor) according to a specific clinical dosing regimen. In some embodiments, the anti-OX40 antibody is OX40.21. In some embodiments, the anti-PD-1 antibody is BMS-936558 (nivolumab). In some embodiments, the anti-CTLA-4 antibody is ipilimumab (Yervoy®). In some embodiments, dosing regimens are adjusted to provide the optimal desired response (eg, effective response).

Used herein, additional or combined administration (co-administration) provides for the simultaneous administration of compounds in the same or different dosage form, or separate administration of the compounds (eg, sequential administration). Thus, an anti-OX40 antibody and an anti-PD-1 antibody or an anti-CTLA-4 antibody can be administered simultaneously in the same formulation. Alternatively, the anti-OX40 antibody and the anti-PD-1 antibody or anti-CTLA-4 antibody can be formulated for separate administration and administered simultaneously or sequentially (eg, one antibody is administered within about 30 minutes prior to the administration of the second antibody).

For example, an anti-PDI antibody or an anti-CTLA-4 antibody can be administered first and then (eg, immediately after) an anti-OX40 antibody, or vice versa. In some embodiments, an anti-PD-1 antibody or an anti-CTLA-4 antibody is administered prior to administration of the anti-OX40 antibody. In another embodiment, the anti-PD-1 antibody or anti-CTLA-4 antibody is administered after administration of the anti-OX40 antibody. In another embodiment, the anti-OX40 antibody and the anti-PD-1 antibody or anti-CTLA-4 antibody are administered simultaneously. Such simultaneous or sequential administration preferably results in both antibodies being simultaneously present in treated patients.

Cancers whose growth can be inhibited by anti-OX40 antibodies or combination therapy of an anti-OX40 antibody and an anti-PD-1 antibody or an anti-CTLA-4 antibody include those that typically respond to immunotherapy and those that typically do not respond to immunotherapy. The malignant tumors can be solid tumor malignancies or blood malignancies (fluid tumors). Non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, kidney cancer (e.g., clear cell carcinoma), malignant ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (eg, renal cell carcinoma (RCC)), prostate cancer (hormone-resistant prostate cancer), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast jelly cancer 71 040561 malignant tumor of the head and neck (or carcinoma), malignant tumor of the stomach, embryonic cell tumor, childhood sarcoma, sinus natural killer, melanoma (for example, metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma) , bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, cancer endocrine system, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra cancer, penis cancer, pediatric solid tumors, ureter cancer, kidney carcinoma renal pelvis, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, malignant brain tumor, brain glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid malignant tumor, squamous cell malignant tumor, T-cell lymphoma, environmentally induced malignancies, including asbestos-induced, virus-associated malignancies or malignant tumors of viral origin (for example, human papillomavirus (HPV) or related tumors)) and hematological malignancies derived from two major blood cell lines, i.e. myeloid cell line (which produces granulocytes, erythrocytes, platelets, macrophages and mast cells) or lymphoid cell line (which produces B, T, NK and plasma cells) such as all types of leukemias, lymphomas and myelomas, eg acute, chronic , lymphocytic and/or myelogenous leukemia diseases such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), undifferentiated AML (M0), myeloid leukemia (M1), myeloid leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megacariblastic leukemia (M7), isolated granulocytic sarcoma and chloroma; lymphomas such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematologic malignancy, eg B-cell lymphomas, T-cell lymphomas, lymphoplasmic lymphoma, monocytoid B-cell lymphoma, mucosal associated lymphoma (MALT) , anaplastic (eg, Ki 1+) large cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, progenitor lymphoblastic lymphoma T-cell, T-lymphoblastic lymphoma and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, histiocytic true lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, B -cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of the lymphoid line, acute lymphoblastic l leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, B-cell progenitor lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycoses fungoides or Cesari's syndrome), and lymphoplasmic lymphoma (LPL) with Waldenström's macroglobulinemia; myeloma such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called silent myeloma), single plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of the myeloid line, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nervous system, including astrocytoma, schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoskarma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminomia, follicular thyroid cancer, and teratocarcinoma, hematopoietic tumors of the lymphoid lineage, e.g., T-cell and B-cell tumors, including, but not limited to, T- cells such as T-prolymphocytic leukemia (T-PLL), including small cell type and medullary cell type; large granular lymphocyte (LGL) leukemia, preferably of the T cell type; a/d T-NHL lymphoma of the liver and spleen; peripheral/postthymic T-cell lymphoma (pleomorphic and immunoblastic subtype); angiocentric (nasal) T-cell lymphoma; head or neck cancer, renal cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, as well as any combination of these types of malignant tumors. The methods described herein may also be applicable to the treatment of metastatic cancers, unresectable and/or refractory cancers (e.g., cancers resistant to previous immunotherapy, e.g., blocking antibody CTLA-4 or PD-1), and recurrent cancers. tumors.

In some embodiments, the patient who is being treated with the antibody

- 72 040561 to OX40 or a combination of an antibody to OX40 and an antibody to PD-1 or to CTLA-4, contains a widespread solid tumor. For example, in one embodiment, the patient to be treated is characterized by the presence of cervical cancer. According to another embodiment, the patient to be treated is characterized by the presence of a malignant tumor of the colon and rectum (CRC). In another embodiment, the patient to be treated is characterized by the presence of a bladder cancer (eg, inoperable local advanced or metastatic bladder cancer). In another embodiment, the patient to be treated is characterized by the presence of an ovarian cancer (eg, unresectable local advanced or metastatic ovarian cancer).

In one embodiment, the patient being treated with an anti-OX40 antibody or a combination of an anti-OX40 antibody and an anti-PD-1 or anti-CTLA-4 antibody is characterized by the presence of non-small cell lung cancer (NSCLC). In another embodiment, the patient to be treated is characterized by the presence of a head and neck squamous cell carcinoma (SCCHN). In another embodiment, the patient to be treated is characterized by the presence of non-Hodgkin's B-cell lymphoma (B-NHL). In another embodiment, the patient to be treated is characterized by the presence of myeloma. In another embodiment, the patient is characterized by the presence of melanoma. In another embodiment, the patient to be treated is characterized by the presence of diffuse large B-cell lymphoma (DLBCL).

In some embodiments, an anti-OX40 antibody is administered to patients with a cancer that has shown an inadequate response to a prior course of treatment, such as prior treatment with an immuno-oncology drug, or to patients with a cancer that is refractory or resistant, or intrinsically refractory or resistant ( for example, resistant to an antagonist of the PD-1 pathway), or wherein a resistant or refractory state is acquired. For example, subjects who do not or do not adequately respond to a first treatment or who see disease progression after treatment, e.g., anti-PD-1 treatment, may be treated with anti-OX40 antibody alone or in combination with another treatment (e.g., , a method of treatment against PD-1).

In some embodiments, an anti-OX40 antibody is administered to patients who have not previously received (ie, not been treated with) an immuno-oncological agent, such as a PD-1 pathway antagonist.

In some embodiments, the anti-OX40 antibody may be administered with a standard of care (eg, surgery, radiation, and chemotherapy). In other embodiments, the anti-OX40 antibody may be administered as maintenance therapy, for example, for therapy that is intended to prevent the onset or recurrence of tumors.

In some embodiments, the anti-OX40 antibody may be administered with other treatment, such as radiation, surgery, or chemotherapy. For example, adjuvant anti-OX40 antibody therapy may be administered when there is a risk that micrometastases may be present and/or to reduce the risk of recurrence.

In some embodiments, the anti-OX40 antibody may be administered as monotherapy or as the sole immunostimulatory therapy. In other embodiments, an anti-OX40 antibody can also be combined with an immunogenic agent such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immunostimulatory cytokines (He et al. (2004) J Immunol 173:4919-28). Non-limiting examples of tumor vaccines that can be used include melanoma antigen peptides such as gp100 peptides, MAGE, Trp-2, MART1 and/or tyrosinase antigens, or tumor cells transfected to express the cytokine GM-CSF (discussed further below). ).

In humans, some tumors have been shown to be immunogenic, such as melanomas. By lowering the T cell activation threshold through OX40 activation, tumor responses in the host can be activated, allowing treatment of non-immunogenic tumors or those that have limited immunogenicity.

In some embodiments, an anti-OX40 antibody may be used in conjunction with a vaccination protocol. Many experimental tumor vaccination strategies have been developed (see Rosenberg S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis C., 2000, ASCO Educational Book Spring: 300-302; Khayat D. 2000 , ASCO Educational Book Spring: 414-428, Foon K. 2000, ASCO Educational Book Spring: 730-738, see also Restifo N. and Sznol M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one such strategy and these strategies, the vaccine is prepared using autologous or allogeneic tumor cells. These cell vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. It has been shown that GM-73 040561

CSF is a potent antigen presentation activator for tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90: 3539-43).

The study of gene expression and large-scale gene expression profiles in various tumors has led to the identification of so-called tumor-specific antigens (Rosenberg S.A. (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and in the cell from which the tumor originated, for example, gp100 melanocyte antigens, MAGE and Trp-2 antigens. More importantly, many of these antigens can be shown to be targets of tumor-specific T cells found in the host. OX40 activation can be used in combination with a set of recombinant proteins and/or peptides expressed in a tumor to generate an immune response to these proteins. These proteins are usually perceived by the immune system as self antigens and are therefore tolerant of them. The tumor antigen may include the protein telomerase, which is essential for the synthesis of chromosome telomeres and which is expressed in more than 85% of human malignant tumors and only in a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013).

The tumor antigen may also be neoantigens expressed in malignant cells due to somatic mutations that alter the protein sequence or create fusion proteins between two unrelated sequences (i.e. bcr-abl on the Philadelphia chromosome) or an idiotype from B-cell tumors.

Other tumor vaccines may include proteins from viruses involved in human cancers such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's sarcoma virus (KHSV). Another form of tumor specific antigen that can be used in conjunction with OX40 activation is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins from tumor cells and these HSPs are very efficient in delivering to antigen presenting cells to elicit tumor immunity (Suot & Srivastava (1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117 -120).

Dendritic cells (DC) are strong antigen-presenting cells that can be used for primary antigen-specific responses. DCs can be prepared ex vivo and loaded with various protein and peptide antigens as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be genetically transduced to express these tumor antigens. DCs have also been fused directly to tumor cells for immunization purposes (Kugler et al. (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunization can be effectively combined with OX40 activation to activate more potent antitumor responses.

Anti-OX40 antibodies described herein can also be combined with chemotherapy regimens. In these cases, it may be possible to reduce the dose of the chemotherapeutic agent (Mokyr et al. (1998) Cancer Research 58: 5301-5304). For example, an anti-OX40 antibody can be used in combination with decarbazine to treat melanoma. In another example, an anti-OX40 antibody can be used in combination with interleukin-2 (IL-2) to treat melanoma. The scientific rationale for the combined use of antibodies to OX40 and chemotherapy is that cell death, which is a consequence of the cytotoxic effect of most chemotherapeutic compounds, should lead to an increase in the content of tumor antigen in the antigen-presenting pathway. Other combination therapies that may result in synergy with anti-OX40 antibodies through cell death are radiation, surgery, and endocrine shutdown. Each of these protocols creates a source of tumor antigen in the host. Angiogenesis inhibitors can also be used in combination with an anti-OX40 antibody. Inhibition of angiogenesis results in the death of tumor cells, which can supply tumor antigen into the antigen-presenting pathways of the host.

Anti-OX40 antibodies described herein can also be used in combination with bispecific antibodies that target Fca or Fcy receptor-expressing effector cells to tumor cells (see, for example, US Pat. Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, Fc receptor/antitumor antigen bispecific antibodies (eg, Her-2/neu) have been used to target macrophages to tumor sites. This targeting can more effectively activate tumor-specific responses. The T cell arm of these responses will be complemented by OX40 activation. Alternatively, the antigen can be delivered directly to the DC using bispecific antibodies that bind to the tumor antigen and a dendritic cell-specific cell surface marker.

Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins that are expressed by tumors and that are immunosuppressive. Among them, TGF-β (Kehrl et al. (1986) J. Exp.

- 74 040561

Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200) and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies to each of these entities can be used in combination with antibodies to OX40 to counteract the effects of immunosuppressants and to support host tumor immune responses.

Other antibodies that activate the host's immune response may be used in combination with the anti-OX40 antibodies described herein. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies are capable of effectively displacing T-cell helper activity (Ridge et al. (1998) Nature 393: 474-478) and can be used in combination with anti-OX40 antibodies. Activation of antibodies to T-cell co-stimulatory molecules such as CTLA-4 (e.g. US Pat. No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997)) and ICOS (Hutloff et al. (1999) Nature 397: 262-266) may also provide increased levels of T cell activation. PD1 or PD-L1 inhibitors may also be used in combination with anti-OX40 antibodies.

Bone marrow transplantation is currently used to treat various tumors of hematopoietic origin. While graft-versus-host disease is a consequence of this treatment, there may be therapeutic benefit from graft-versus-tumor disease. Anti-OX40 antibodies can be used to improve the efficiency of donor-grafted tumor-specific T cells.

There are also several experimental treatment protocols that include ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells to recipients to stimulate antigen-specific T cells against the tumor (Greenberg & Riddell (1999) Science 285: 546-51). These methods can also be used to activate T cell responses to infectious pathogens such as CMV. Ex vivo activation in the presence of anti-OX40 antibodies can increase the frequency and activity of adoptively transferred T cells.

Infectious diseases.

Also provided herein are methods for treating patients exposed to certain toxins or pathogens. Accordingly, provided herein are methods for treating an infectious disease in a subject comprising administering to the subject anti-OX40 antibodies described herein such that the subject is treated for the infectious disease. In some embodiments, the anti-OX40 antibody is a chimeric or humanized antibody.

Similar to use in tumors as discussed above, OX40 antibodies can be used alone or as an adjuvant in combination with vaccines to stimulate an immune response to pathogens, toxins, and self antigens. Examples of pathogens for which this therapeutic approach may be particularly applicable are pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are less effective. These include, but are not limited to, HIV, hepatitis (A, B and C), influenza, herpes, jardia, malaria, leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. Anti-OX40 antibodies may be useful against established infections by agents such as HIV, which are altered antigens during infections. These new epitopes are recognized as foreign at the time of administration of the anti-OX40 antibody, which elicits a strong T cell response.

Some examples of pathogenic viruses that cause infections amenable to treatment by the methods described herein include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV , Epstein-Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus , papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arbovirus encephalitis virus.

Some examples of pathogenic bacteria that cause infections amenable to treatment by the methods described herein include Chlamydia, Rickettsial bacteria, Mycobacteria, Staphylococci, Streptococci, Pneumococci, Meningococci and Gonococci, Klebsiella, Proteus, Seracia, Pseudomonas, Legionella, Diphtheria, Salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis and Lyme disease bacteria.

Some examples of pathogenic fungi that cause infections treatable by the methods described herein include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites that cause infections amenable to treatment by the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii,

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Nippostrongylus brasiliensis

In all of the above methods, anti-OX40 antibodies can be combined with other forms of immunotherapy, such as cytokine treatment (e.g., interferons, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy, which provides improved presentation of tumor antigens (see below). eg Holliger (1993) Proc Natl Acad Sci USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

autoimmune reactions.

Antibodies to OX40 can provoke and enhance autoimmune responses. Indeed, the induction of antitumor responses using tumor cells and peptide vaccines shows that many antitumor responses involve autoimmune reactivities (van Elsas et al. (2001) J. Exp. Med. 194:481-489; Overwijk, et al. ( 1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987; Hurwitz, (2000) supra; Rosenberg & White (1996) J. Immunother Emphasis Tumor Immunol 19 (1): 81-4). Thus, anti-OX40 antibodies can be used in combination with various self proteins to develop vaccination protocols to effectively elicit immune responses against these self proteins to treat diseases. For example, Alzheimer's disease involves the non-physiological accumulation of the Ae peptide in amyloid deposits in the brain; amyloid antibody responses are able to clear these amyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

Other self proteins can also be used as targets, such as IgE for allergy and asthma, and TNFc for rheumatoid arthritis. Finally, antibody responses to various hormones can be elicited by the use of antibodies to OX40. Neutralizing antibody responses to reproductive hormones can be used for contraception. Neutralizing antibody responses to hormones and other soluble factors that are required for the growth of specific tumors can also be considered as possible targets for vaccination.

Similar methods described above for the use of antibodies to OX40 can be used to induce therapeutic autoimmune responses to treat patients with non-physiological accumulation of other self antigens, such as amyloid deposits, including Ae in Alzheimer's disease, cytokines such as TNFa, and IgE.

Vaccines.

Anti-OX40 antibodies described herein can be used to stimulate antigen-specific immune responses by co-administering the antibody with an antigen of interest (eg, a vaccine). Accordingly, provided herein are methods for enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) an antigen; and (ii) antibodies to OX40 so that the subject's immune response to the antigen is enhanced. The antibody may be an anti-human OX40 antibody (such as any anti-human OX40 antibody described herein). In other embodiments, the antibody may be a chimeric or humanized antibody. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include those discussed in the sections above, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from the viruses, bacteria, or other pathogens described above.

In some embodiments, a peptide or fusion protein containing an epitope to which an anti-OX40 antibody binds is used as a vaccine instead of or in addition to an anti-OX40 antibody.

Suitable routes of administration of the compositions of the antibodies described herein (eg, human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates), in vivo and in vitro, are well known in the art and can be selected by those skilled in the art. For example, antibody compositions can be administered by injection (eg, intravenous or subcutaneous). Suitable doses of the molecules used will depend on the age and weight of the subject, on the concentration and/or composition of the antibody composition.

As described above, the anti-OX40 antibodies described herein may be co-administered with one or more other therapeutic agents, for example, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent. The antibody may be associated with the agent (as an immunocomplex) or may be administered separately from the agent. In the latter case (separate administration), the antibody may be administered before, after, or simultaneously with the agent, or may be administered in conjunction with other known treatments, such as anticancer therapy, such as radiation. Such therapeutic agents include, inter alia, antitumor agents such as doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and hydroxyurea cyclophosphamide, which are themselves effective only at levels that are toxic or subtoxic to the patient. Cisplatin is administered intravenously at a dose of 100 mg/ml once every four weeks, and adriamycin is administered intravenously at a dose of 60-75 mg/ml once every 21 days.

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Co-administration of the anti-OX40 antibodies described herein, or antigen-binding fragments thereof, with chemotherapeutic agents provides two antitumor agents that act through different mechanisms that confer a cytotoxic effect on human tumor cells. Such co-administration can solve problems associated with the development of drug resistance or changes in the antigenicity of tumor cells that render them inactive with the antibody.

Also provided herein are kits containing compositions of anti-OX40 antibodies described herein (eg, human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use. The kit may further comprise at least one additional reagent or one or more additional human antibodies described herein (eg, a human antibody having additional activity that binds to an epitope in OX40 other than the first human antibody). Kits typically include a label indicating the intended use of the contents of the kit. The term label means any recording or recorded material supplied on or with a kit, or otherwise attached to a kit.

treatment protocols.

Suitable protocols for treating a solid tumor (e.g., an advanced solid tumor) in a human patient include, for example, administering to the patient an effective amount of an anti-OX40 antibody comprising the heavy chain variable region CDR1, CDR2, and CDR3 domains with the sequence shown in SEQ ID NO : 318, and the CDR1, CDR2 and CDR3 domains of the light chain variable region with the sequence shown in SEQ ID NO: 94, and the method involves at least one cycle of administration, and the cycle is a period of two weeks (Q2W), and for each from at least one cycle, at least one dose of anti-OX40 antibody is administered at a dose of 1 mg/kg of body weight; fixed dose of 20, 40, 80, 160 or 320 mg; a dose of approximately 1 mg/kg body weight or a fixed dose of approximately 20, 40, 80, 160 or 320 mg.

Another suitable protocol for treating a solid tumor in a human patient includes, for example, administering to the patient an effective amount of each of:

(a) an anti-OX40 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 318 and the light chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 94 ; and (b) an anti-PD-1 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 301 and the light chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 301. NO: 302, wherein the method comprises at least one administration cycle, where the cycle is a period of two weeks, wherein for each of the at least one cycle, at least one dose of anti-OX40 antibody is administered at a dose of 1 mg/kg of body weight; fixed dose of 20, 40, 80, 160 or 320 mg; a dose of about 1 mg/kg body weight or a fixed dose of about 20, 40, 80, 160 or 320 mg, and at least one dose of anti-PD-1 antibody is administered at a constant dose of 240 mg or a constant dose of about 240 mg. In some embodiments, the anti-PD-1 antibody is administered once every three weeks (q3w) at a fixed dose of 360 mg or once every four weeks (q4w) at a dose of 480 mg.

Another suitable protocol for treating a solid tumor in a human patient includes, for example, administering to the patient an effective amount of each of:

(a) an anti-OX40 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 318 and the light chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 94 ; and (b) an anti-CTLA-4 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 309 and the light chain variable region CDR1, CDR2 and CDR3 domains of the sequence shown in SEQ ID NO: 310, wherein the method comprises at least one cycle of administration, wherein the cycle is a period of three weeks (q3w), wherein for each of the at least one cycle, at least one dose of anti-OX40 antibody is administered at a dose of 1 mg/kg body weight; fixed dose of 20, 40, 80, 160 or 320 mg; dose of approximately 1 mg/kg body weight or a fixed dose of approximately 20, 40, 80, 160, or 320 mg, and at least one dose of anti-CTLA-4 antibody administered at a constant dose of 1 mg/kg body weight or a constant dose of approximately 1 mg /kg of body weight. In one embodiment, the anti-OX40 antibody is co-administered with the anti-CTLA-4 antibody for at least one cycle, followed by monotherapy with the anti-OX40 antibody for at least one cycle. In some embodiments, the anti-OX40 antibody is co-administered with ipilimumab for the initial four cycles, followed by anti-OX40 antibody monotherapy for subsequent cycles.

In some embodiments, the anti-OX40 antibody and anti-PD-1 antibody are administered at the following doses:

(a) 1 mg/kg anti-OX40 antibody and 240 mg, 360 or 480 mg anti-PD-1 antibody;

(b) 20 mg anti-OX40 antibody and 240 mg, 360 or 480 mg anti-PD-1 antibody;

- 77 040561 (c) 40 mg anti-OX40 antibody and 240 mg, 360 or 480 mg anti-PD-1 antibody;

(d) 80 mg anti-OX40 antibody and 240 mg, 360 or 480 mg anti-PD-1 antibody;

(e) 160 mg of anti-OX40 antibody and 240 mg, 360 or 480 mg of anti-PD-1 antibody; or (f) 320 mg of antibody to

OX40 and 240 mg, 360 or 480 mg anti-PD-1 antibody. In some embodiments, the anti-OX40 antibody and anti-CTLA-4 antibody are administered at the following doses:

(a) 1 mg/kg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(b) 20 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(c) 40 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(d) 80 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(e) 160 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody; or (f) 320 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody.

In one embodiment, the dose of anti-OX40 antibody and/or anti-PD-1 antibody or anti-CTLA-4 antibody is calculated on a body weight basis, eg, mg/kg body weight. In another embodiment, the dose of the anti-OX40 and/or anti-PD-1 or anti-CTLA-4 antibody is a constant fixed dose. In another embodiment, the dose of the anti-OX40 and/or PD-1 or anti-CTLA-4 antibody varies over time. For example, an anti-OX40 and/or anti-PD-1 or anti-CTLA-4 antibody may be initially administered at a high dose and may be tapered over time. In another embodiment, the anti-OX40 and/or anti-PD-1 or anti-CTLA-4 antibody is initially administered at a low dose and increased over time.

In another embodiment, the amount of anti-OX40 and/or anti-PD-1 or anti-CTLA-4 antibody administered is constant for each dose. In another embodiment, the amount of antibody administered varies with each dose. For example, the maintenance (or follow-up) dose of the antibody may be greater than or the same as the initial administration dose. In another embodiment, the maintenance dose of the antibody may be lower than or the same as the loading dose.

In some embodiments, the anti-OX40 and/or anti-PD-1 or anti-CTLA-4 antibody is formulated for intravenous administration. In some embodiments, an anti-OX40 antibody or an anti-OX40 antibody and an anti-PD-1 antibody or an anti-CTLA-4 antibody are administered on the first day of each cycle.

In some embodiments, the anti-OX40 and/or anti-PD-1 or anti-CTLA-4 antibody is administered weekly, biweekly, every three weeks, or every four weeks or until clinical benefit is observed or until complete response, evidenced by progressive disease or unmanaged toxicity.

In one embodiment, the administration cycle is two weeks, which can be repeated as needed. In another embodiment, the cycle is three weeks. In some embodiments, the treatment consists of eight cycles. In other embodiments, the treatment consists of 12 cycles.

In one embodiment, one dose of each anti-OX40 antibody and anti-PD-1 antibody is administered over a two week cycle. In another embodiment, one dose of each anti-PD-1 antibody and anti-OX40 antibody is administered over a three week cycle. In another embodiment, one dose of each anti-PD-1 antibody and anti-OX40 antibody is administered over a four week cycle.

In one embodiment, one dose of each anti-OX40 antibody and anti-CTLA4 antibody is administered over a three week cycle. In some embodiments, one dose of each anti-OX40 antibody and anti-CTLA-4 antibody is administered over a three week cycle for the first four cycles, followed by anti-OX40 antibody monotherapy for the fifth through eighth cycles.

In another embodiment, an anti-OX40 antibody and an anti-PD-1 or anti-CTLA-4 antibody are administered as first line treatment (eg, initial or first treatment). In another embodiment, an anti-OX40 antibody and an anti-PD-1 or anti-CTLA-4 antibody are administered as a second line treatment (eg, after initial or first treatment, including after relapse and/or first treatment failure).

According to another aspect, the present invention relates to any of the above embodiments wherein an anti-PD-1 antibody is substituted or combined with an anti-PD-L1 or anti-PD-L2 antibody.

In some embodiments, the human patient is characterized by having a cancer selected from the group consisting of cervical cancer, bladder cancer, colon cancer, and ovarian cancer.

In some embodiments, the anti-OX40 antibody comprises a heavy chain variable region CDR1 comprising the sequence set forth in SEQ ID NO: 87, a heavy chain variable region CDR2 comprising the sequence set forth in SEQ ID NO: 317, a heavy chain variable region CDR3 comprising the sequence presented in SEQ ID

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NO: 89, a light chain variable region CDR1 containing the sequence shown in SEQ ID NO: 90, a light chain variable region CDR2 containing the sequence shown in SEQ ID NO: 91, and a light chain variable region CDR3 containing the sequence shown in SEQ ID NO: 92. In some embodiments, the anti-OX40 antibody comprises heavy and light chain variable regions comprising the sequences shown in SEQ ID NO: 318 and 94, respectively. In some embodiments, the anti-OX40 antibody comprises heavy and light chain sequences comprising the sequences shown in SEQ ID NOs: 124 and 116, respectively.

In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region CDR1, CDR2, and CDR3 containing the sequences set forth in SEQ ID NO: 303305, respectively, and a light chain variable region CDR1, CDR2, and CDR3 containing the sequences set forth in SEQ ID NO: 306-308 respectively. In some embodiments, the anti-PD-1 antibody comprises the heavy and light chain variable region sequences set forth in SEQ ID NOs: 301 and 302, respectively. In some embodiments, the anti-PD-1 antibody comprises the heavy and light chain sequences shown in SEQ ID NOs: 299 and 300, respectively.

In some embodiments, the anti-CTLA-4 antibody comprises a heavy chain variable region CDR1, CDR2, and CDR3 containing the sequences set forth in SEQ ID NOs: 311-313, respectively, and a light chain variable region CDR1, CDR2, and CDR3 containing the sequences set forth in SEQ ID NO: 311-313, respectively. in SEQ ID NO: 314-316, respectively. In some embodiments, the anti-CTLA-4 antibody comprises the heavy and light chain variable region sequences set forth in SEQ ID NOs: 309 and 310, respectively.

Results.

With regard to targeted lesions, responses to treatments may include

Complete response (CR) (RECIST VI.1) Disappearance of all target lesions. Any pathological lymph nodes (both target and non-target) should be characterized by a reduction in the short axis to < 10 mm. Partial Response (PR) (RECIST VI.1) At least 30% reduction in the sum of target lesion diameters, taking into account the original sums of diameters. Disease Progression (PD) (RECIST VI.1) An increase of at least 20% in the sum of target lesion diameters, taking into account the minimum sum at the time of examination (this includes the original sum if it is the smallest during research). In addition to a relative increase of 20% of the amount must also be an absolute increase of at least 5 mm. (Note: the appearance of one or more new damage also counts as progression). Stable disease (SD) (RECIST VI.1) Not reducing enough to qualify for a PR, or

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enough magnification to qualify for PD by taking as a reference the smallest sums of diameters during the study. Immune System Related Complete Response (irCR) (irRECIST) Disappearance of all target lesions. Any pathological lymph nodes (both target and non-target) should be characterized by a reduction in the short axis to < 10 mm. Immune System Related Partial Response (irPR) (irRECIST) At least a 30% reduction in the sum of target lesion diameters and all newly measured lesions (i.e., percentage change in tumor mass), taking into account the original total diameters. Note: The appearance of new measurable lesions counts towards the total sum of tumor masses, but does not automatically qualify as disease progression until the sum of diameters increases by >20% above the lower threshold. Immune System Related Disease Progression (irPD) (irRECIST) At least 20% reduction in tumor mass (i.e. the sum of target lesion diameters and all newly measured lesions), taking into account the smallest sum at the time of the study (this includes the original sum if it is the smallest in the study) . In addition to the relative increase of 20% of the sum, there must also be an absolute increase of at least 5 mm. Tumor scores using immune criteria for disease progression includes the contribution of newly measured lesions. Each percentage change in tumor mass in the assessment takes into account the size and growth kinetics of both old and new lesions as they appear. Immune system-associated stable disease (rSO) (irRECIST) Not decreasing enough to qualify for irPR or increasing enough to qualify qualify for irPD by taking as a reference the smallest sums of diameters during the study.

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With regard to off-target lesions, responses to treatments may include

Complete response (CR) (RECIST V1.1) Disappearance of all non-target lesions. All lymph nodes should be non-pathological in size (short axis <10 mm). non-CR/He-PD Persistence of one or more non-target (RECIST V1.1) defeats. Disease Progression (PD) Unambiguous Progression (RECIST V1.1) existing non-target lesions. The appearance of one or more new lesions is also considered a progression. associated with the immune system Disappearance of all non-target lesions. complete response (irCR) All lymph nodes should be (irRECIST) non-pathological in size (short axis <10 mm). Related to the immune system Increasing the number or size disease progression (irPD) non-target injury(s) does not represent (irRECIST) is disease progression if/until tumor mass increases by 20% (ie, the sum of the diameters at the lower threshold of the target lesions and any new measurable lesions increase by the required amount). Off-target lesions are not considered in the definition of Stable Disease and Partial Response.

Patients treated according to the methods described herein preferably experience improvement in at least one sign of cancer. In one embodiment, improvement is measured by a reduction in the number and/or size of measurable tumor lesions. In another embodiment, the lesions can be measured on chest radiographs or on CT or MRI films. In another embodiment, cytology or histology can be used to evaluate response to therapy.

In one embodiment, the treated patient exhibits a complete response (CR), a partial response (PR), stable disease (SD), an immune system-associated complete response (irCR), an immune system-associated partial response (irPR), or an immune system-associated response. stable disease (irSD). In another embodiment, the treated patient experiences tumor shrinkage and/or a decrease in growth rate, i. suppression of tumor growth. In another embodiment, unwanted cell proliferation is reduced or inhibited. According to another embodiment, one or more of the following may occur: the number of malignant cells may decrease; tumor size may decrease; infiltration of malignant cells into peripheral organs may be inhibited, retarded, slowed down, or stopped; tumor metastases may be slowed down or inhibited; tumor growth may be inhibited; tumor recurrence can be prevented or delayed; one or more cancer-related symptoms may be alleviated to some extent.

In other embodiments, administering effective amounts of an anti-OX40 antibody and an anti-PD-1 or anti-CTLA-4 antibody according to any of the methods provided herein produces at least one therapeutic effect selected from the group consisting of reduction in tumor size, reduction number of metastatic lesions occurring over time, complete remission, partial remission, or stable disease. In other embodiments, the treatments result in a comparable rate of clinical efficacy (CBR = CR + PR + SD >>> 6 months) better than that achieved by anti-OX40 or anti-PD-1 antibody or anti-CTLA-4 antibody alone. In other embodiments, the improvement in clinical efficacy rate is approximately 20%, 30%, 40%, 50%, 60%, 70%, 80% or more compared to the antibody alone.

- 81 040561 to OX40 or to PD-1 or an antibody to CTLA-4.

Combined therapy.

In addition to the combination therapy described above, the anti-OX40 antibodies described herein may be used in the combination therapy described below.

Combination therapies include those in which an anti-OX40 antibody or a combination of an anti-OX40 antibody and an anti-PD-1 antibody or an anti-CTLA-4 antibody is co-administered with one or more additional agents, such as small molecule drugs, antibodies, or antigen-binding portions thereof, and which are effective in stimulating immune responses, thereby further enhancing, stimulating or upregulating immune responses in a subject. For example, as shown in the examples, administration of an anti-OX40 antibody and an anti-PD-1 antibody antagonist to mice can result in a synergistic effect in inhibiting tumor growth.

An anti-OX40 antibody can be combined with (i) an agonist of a stimulatory (eg, costimulatory) molecule (eg, receptor or ligand) and/or (ii) an antagonist of an inhibitory signal or molecule (eg, receptor or ligand) on immune cells such as T- cells, both of which lead to enhanced immune responses such as antigen-specific T-cell responses. In some aspects, the immuno-oncological agent is (i) an agonist of a stimulatory (including a co-stimulatory) molecule (e.g., a receptor or ligand) or (ii) an antagonist of an inhibitory (including a co-inhibitory) molecule (e.g., a receptor or ligand) on cells involved in innate immunity, such as NK cells, and wherein the immuno-oncological agent enhances innate immunity. Such immuno-oncological agents are often referred to as immune checkpoint regulators, such as an immune checkpoint inhibitor or an immune checkpoint stimulant.

In some embodiments, an anti-OX40 antibody is administered by an agent that targets a stimulatory or inhibitory molecule that is a member of the immunoglobulin superfamily (IgSF). For example, an anti-OX40 antibody can be administered to a subject with an agent that targets a member of the IgSF family to increase the immune response. In other embodiments, an anti-OX40 antibody can be administered with an agent that targets (or specifically binds to) a member of the B7 family of membrane-bound ligands, which includes B7-1, B7-2, B7-H1 (PD-L1), B7- DC (PD-L2), B7H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA) and B7-H6 or a co-stimulatory or co-inhibitory receptor that specifically binds to a member of the B7 family.

An anti-OX40 antibody can also be administered by an agent that targets a member of the TNF and TNFR family of molecules (ligands or receptors), such as CD40 and CD40L, GITR, GITR-L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 TRAIL/Apo2-L TRAILR1/DR4 TRAILR2/DR5 TRAILR3 TRAILR4 OPG RANK RANKL TWEAKR/Fn14 TWEAK BAFFR EDAR XEDAR TACI APRIL BCMA ΙΐβΚ LIGHT DcR3 HVEM , VEGI/TL1A, TRAMP/DR3, EDA1, EDA2, TNFR1, lymphotoxin α/TNFe, TNFR2, TNFa, LTeR, lymphotoxin α 1β2, FAS, FASL, RELT, DR6, TROY and NGFR (see e.g. Tansey (2009 ) Drug Discovery Today 00:1).

T cell responses can be stimulated by a combination of anti-OX40 antibodies and one or more of the following:

(1) an antagonist (inhibitor or blocking agent) of a protein that inhibits T cell activation (eg, immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, and LAG-3, as described above , and any of the following proteins: TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1 and TIM-4; and/or (2) a protein agonist that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, GITR, GITR-L , CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents that modulate one of the above proteins and can be combined with an anti-OX40 antibody for cancer treatment include: Yervoy™ (ipilimumab) or tremelimumab (anti-CTLA-4), galiximab (anti-B7.1), BMS- 936558 (to PD-1), MK-3475 (to PD-1), AMP224 (to B7DC), BMS-936559 (to B7-H1), MPDL3280A (to B7H1), MEDI-570 (to ICOS), AMG557 ( to B7H2), MGA271 (to B7H3), IMP321 (to LAG-3), BMS-663513 (to CD137), PF-05082566 (to CD137), CDX-1127 (to CD27), atacicept (to TACI), CP- 870893 (anti-CD40), lucatumumab (anti-CD40), dacetuzumab (anti-CD40), muromonab-CD3 (anti-CD3), ipilimumab (anti-CTLA-4).

Anti-OX40 antibodies can also be given with pidilizumab (CT-011).

Other molecules that can be combined with an anti-OX40 antibody for the treatment of cancer include inhibitory receptor antagonists on NK cells or activating receptor agonists on NK cells. For example, an anti-OX40 antibody can be combined with KIR antagonists (eg, lirilumab).

T cell activation is also regulated by soluble cytokines, and OX40 antibodies can be administered to a subject, eg, cancer, with cytokine antagonists that inhibit T cell activation, or cytokine agonists that stimulate T cell activation.

In some embodiments, anti-OX40 antibodies may be used in combination with (i) antagonists (or inhibitors or blockers) of IgSF or B7 or TNF family proteins that inhibit T cell activation, or cytokine antagonists that inhibit T cell activation. (e.g., IL-6, IL-10, TGF-β, VEGF, immunosuppressive cytokines) and/or (ii) stimulatory receptor agonists of the IgSF family, B7 family, or TNF family, or cytokines that stimulate T cell activation to stimulate an immune response , for example, for the treatment of proliferative diseases such as cancer.

Other combination therapies include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1R antagonists such as CSF-1R antibody antagonists, including RG7155 (WO 11/70024, WO 11 /107553, WO 11/131407, WO 13/87699, WO 13/119716, WO 13/132044) or FPA-008 (WO 11/140249, WO 13169264, WO 14/036357).

Anti-OX40 antibodies can also be administered with agents that inhibit TGF-β signaling.

Additional agents that may be combined with the anti-OX40 antibodies described herein include agents that improve tumor antigen presentation, such as dendritic cell vaccines, GM-CSF secreting cell vaccines, CpG oligonucleotides, and imiquimod, or therapeutic agents that enhance immunogenicity. tumor cells (eg anthracyclines).

Other treatments that can be combined with anti-OX40 antibodies include treatments that deplete or block Treg cells, such as an agent that specifically binds to CD25.

Another treatment that can be combined with anti-OX40 antibodies is a treatment that inhibits a metabolic enzyme such as indolamindioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase.

Another class of agents that can be used with OX40 antibodies includes agents that inhibit adenosine formation or inhibit the adenosine A2A receptor.

Other therapies that can be combined with anti-OX40 antibodies to treat cancer include therapies that reverse/prevent T cell tolerance or depletion and therapies that trigger innate immune activation and/or inflammation at the tumor site. .

An anti-OX40 antibody may be combined with more than one immuno-oncological agent and may, for example, be combined with a combinatorial approach that targets multiple elements of the immune pathway, such as one or more of the following: a treatment that enhances tumor antigen presentation (eg, dendritic cell vaccine, GM-CSF secreting cell vaccines, CpG oligonucleotides, imiquimod); a method of treatment that inhibits immune downregulation, for example by inhibiting the CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or depleting or blocking Treg or other immunosuppressive cells; a method of treatment that stimulates positive immune regulation, for example with agonists that stimulate the CD-137 and/or GITR pathway and/or stimulate T cell effector function; a method of treatment that systemically increases the frequency of antitumor T cells; a method of treatment that depletes or inhibits Tregs, such as Tregs in a tumor, for example using a CD25 antagonist (eg, daclizumab) or by ex vivo depletion of anti-CD25 granules; a method of treatment that affects the function of suppressor myeloid cells in the tumor; a method of treatment that enhances the immunogenicity of tumor cells (eg, anthracyclines); adoptive transfer of T cells or NK cells, including genetically modified cells, such as cells modified with chimeric antigen receptors (CAR-T therapy); a method of treatment that inhibits a metabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase; a method of treatment that reverses/prevents T-cell tolerance or depletion; a method of treatment that induces innate immune activation and/or inflammation at a tumor site; administration of immunostimulatory cytokines or blocking of immunoreceptive cytokines.

Anti-OX40 antibodies can be used in conjunction with one or more agonistic agents that ligate positive costimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, antagonists, and one or more agents that systemically increase the frequency of antitumor T cells, agents that overcome various immune suppressive pathways in the tumor microenvironment (eg, block inhibitory receptor interactions (eg, PD-L1/PD-1 interactions), deplete or inhibit Treg (eg, using an anti-CD25 monoclonal antibody (eg, daclizumab), or ex vivo aHTu-CD25 granule depletion), inhibit metabolic enzymes such as IDO, or reverse/prevent T cell tolerance or depletion), and agents that induce innate immune activation and/or inflammation at tumor sites.

In some embodiments, an anti-OX40 antibody is administered to a subject along with a BRAF inhibitor if the subject is positive for the BRAF V600 mutation.

In some embodiments, the anti-OX40 antibody is administered together with another immunostimulatory antibody.

Provided herein are methods of stimulating an immune response in a subject, comprising administering to the subject an anti-OX40 antibody and one or more additional immunostimulatory antibodies, such as an anti-PD-1 antagonist, e.g. to CTLA-4, eg, an antagonist antibody and/or an antagonist to LAG3, eg, an antagonist antibody, such that an immune response is stimulated in the subject, eg, to inhibit tumor growth or to stimulate an antiviral response. In one embodiment, an anti-OX40 antibody and an anti-PD-1 antagonist antibody are administered to the subject. In one embodiment, an anti-OX40 antibody and an anti-PD-L1 antagonist antibody are administered to the subject. In one embodiment, an anti-OX40 antibody and an anti-CTLA-4 antagonist antibody are administered to the subject. In one embodiment, the anti-OX40 antibody is a human antibody. Alternatively, the OX40 antibody may be, for example, a chimeric or humanized antibody. In one embodiment, at least one additional immunostimulatory antibody (e.g., PD-1 antagonist antibody, PD-L1 antagonist antibody, CTLA-4 antagonist antibody, and/or LAG3 antagonist antibody) is a human antibody. Alternatively, the at least one additional immunostimulatory antibody may be, for example, a chimeric or humanized antibody (eg, derived from a mouse anti-PD-1, anti-PDL1, anti-CTLA-4, and/or anti-LAG3 antibody).

Provided herein are methods of treating a hyperproliferative disease (e.g., cancer) comprising administering to a subject an anti-OX40 antibody with an anti-PD-1 antagonist antibody, an anti-PD-L1 antagonist antibody, an anti-CTLA-4 antagonist antibody, or an anti-LAG3 antagonist antibody. In some embodiments, one or both antibodies are administered at a subtherapeutic dose. Also provided herein are methods for modifying an adverse event associated with treatment of a hyperproliferative disease with an immunostimulatory agent, comprising administering an anti-OX40 antibody and a subtherapeutic dose of an anti-PD-1, anti-PD-L1, anti-CTLA-4, or LAG3 antibody to a subject (e.g., human) . In some embodiments, the anti-OX40 antibody comprises the CDRs or variable regions 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2, and 20C1, or is another anti-OX40 agonist antibody described herein.

Suitable PD-1 antagonists for use in the methods described herein include, without limitation, ligands, antibodies (eg, monoclonal antibodies and bispecific antibodies), and multivalent agents. In one embodiment, the PD-1 antagonist is a fusion protein, such as an Fc fusion protein such as AMP-244. In one embodiment, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody.

An exemplary anti-PD-1 antibody is nivolumab (BMS-936558) or an antibody that contains the CDRs or variable regions of one of the antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4, and 4A11 described in International Application Publication WO 2006/121168. In some embodiments, the anti-PD1 antibody is MK-3475 (lambrolizumab) as described in International Application Publication WO 02012/145493; and AMP-514 as described in WO 2012/145493. Other known anti-PD-1 antibodies and other PD-1 inhibitors include those described in international application publications WO 2009/014708, WO 03/099196, WO 2009/114335, WO 2011/066389, WO 2011/161699, WO 2012/145493, U.S. Patent Nos. 7,635,757 and 8,217,149 and U.S. Patent Publication No. 2009/0317368. Any of the anti-PD-1 antibodies described in WO 023/173223 may also be used. An anti-PD-1 antibody that competes for binding and/or binds to the same epitope on PD-1 as one of these antibodies can also be used in combination treatments. Another approach to target the PD-1 receptor is a recombinant protein consisting of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called AMP-224. In some embodiments, the antibody is at least about 90% identical to the amino acid sequence of the variable region with the aforementioned antibodies.

In some embodiments, an anti-OX40 antibody is used in combination with nivolumab, which contains heavy and light chains containing the sequences shown in SEQ ID NOs: 299 and 300, respectively, or antigen-binding fragments and variants thereof. In some embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of nivolumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2 and CDR3 domains of a nivolumab VH containing the sequence shown in SEQ ID NO: 301 and the CDR1, CDR2 and CDR3 domains of a nivolumab VL containing the sequence shown in SEQ ID NO: : 302. In some embodiments, the antibody comprises CDR1, CDR2, and CDR3 domains containing the sequences shown in SEQ ID NO: 303-305, respectively, and CDR1, CDR2, and CDR3 domains containing the sequences shown in SEQ ID NO: 306- 308, respectively. In some embodiments, the antibody comprises VH and/or VL regions containing the amino acid sequences shown in SEQ ID NO: 301 and/or SEQ ID NO: 302, respectively. In some embodiments, the antibody variable region is at least about 90%, such as at least about 90%, 95%, or 99% identical to SEQ ID NO: 301 or SEQ ID NO: 302.

Exemplary anti-PD-L1 antibodies include BMS-936559 (designated 12A4 in WO 2007/005874 and US Pat. No. 7,943,743) or an antibody that contains CDRs or variable regions 3G10, 12A4, 10A5, 5F8, 10H10, 1B12 , 7H1, 11E6, 12B7, and 13G4, which are described in PCT Publication WO 07/005874 and US Pat. No. 7,943,743. as RG7446), MSB0010718C (International Application Publication WO 2013/79174) or rHigM12B7. Any of the anti-PD-L1 antibodies described in WO 02011/173223, WO 201/63638, WO 02012/145493, US Pat.

Exemplary anti-CTLA-4 antibodies include Yervoy™ (ipilimumab or antibody 10D1 described in PCT publication WO 01/14424), tremelimumab (formerly ticilimumab, CP-675,206), or an anti-CTLA-4 antibody described in any of the following publications: publications of international applications WO 98/42752; WO00/37504; US patent No. 6207156; Hurwitz et al. (1998) Proc. Natl. Acad. sci. USA 95(17):10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP675206) and Mokyr et al. (1998) Cancer Res. 58:5301-5304. Any of the anti-CTLA-4 antibodies described in WO 023/173223 may also be used.

Exemplary anti-LAG3 antibodies include those containing the CDRs or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2, or 17E5 as described in US Patent Publication No. US 02011/0150892, International Application Publications WO 10/19570 and WO 2014/008218 . In one embodiment, the anti-LAG-3 antibody is BMS-986016. Other anti-LAG-3 antibodies known in the art that may be used include IMP731 and IMP-321 as described in US 2011/007023, WO 08/132601 and WO 09/44273.

In some embodiments, an anti-OX40 antibody is used in combination with ipilimumab. In some embodiments, the antibody comprises the heavy and light chain CDRs or variable regions of ipilimumab.

Accordingly, in one embodiment, the antibody comprises the ipilimumab VH CDR1, CDR2, and CDR3 domains of SEQ ID NO: 309 and the ipilimumab VL CDR1, CDR2, and CDR3 domains of SEQ ID NO: 310. In some embodiments, embodiment, the antibody contains CDR1, CDR2 and CDR3 domains containing the sequences shown in SEQ ID NOs: 311-313, respectively, and CDR1, CDR2 and CDR3 domains containing the sequences shown in SEQ ID NOs: 314-316, respectively. In some embodiments, the antibody comprises VH and/or VL regions containing the amino acid sequences shown in SEQ ID NO: 309 and/or SEQ ID NO: 310, respectively. In some embodiments, the antibody variable region is at least about 90%, such as at least about 90%, 95%, or 99% identical to SEQ ID NO: 309 or SEQ ID NO: 310.

Administration of OX40 antibodies and antagonists, e.g. antagonist antibodies, to one or more target antigens such as LAG-3 and/or CTLA-4 and/or PD-1 and/or PD-L1 can enhance the immune response to malignant cells in a patient. Cancers whose growth can be inhibited using anti-OX40 antibodies include those that typically respond to immunotherapy and those that typically do not respond to immunotherapy. Typical examples of cancers for combination therapy treatment according to the present disclosure include those cancers listed herein.

In some embodiments, the combination of therapeutic antibodies discussed herein may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or simultaneously as separate compositions with each antibody in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic antibodies may be administered sequentially. In addition, if more than one dose of the combination therapy is administered sequentially, the order of the sequential administration may be reversed or kept in the same order at each administration time, and the sequential administrations may be combined with simultaneous administrations or any combination thereof. For example, the first input

- 85 040561 combination of anti-OX40 antibody and anti-PD1 antibody (and/or anti-CTLA-4 antibody and/or anti-PD-L1 antibody and/or anti-LAG-3 antibody) may be simultaneous, the second administration may be sequential with the first anti-PD1 antibody and a second anti-OX40 antibody, and the third administration may be sequential with the first anti-OX40 antibody and the second anti-PD1 antibody, and so on. Another exemplary dosing regimen is a first administration that is sequential with a first anti-OX40 antibody and a second anti-PD1 antibody (and/or an anti-CTLA-4 antibody and/or an anti-PD-L1 antibody and/or an anti-LAG-3 antibody), and then the introductions can be simultaneous.

In a specific embodiment, a subject with a disease that could benefit clinically from immune system stimulation, such as a cancer or infectious disease, is treated by administering an anti-OX40 antibody and an immuno-oncology agent to the subject. Examples of immuno-oncological agents include CD137 (4-1BB) agonists (eg, a CD137 agonist antibody such as urelumab or PF-05082566 (WO12/32433)); GITR agonists (eg, anti-GITR agonist antibody), CD40 agonists (eg, CD40 agonist antibody); CD40 antagonists (eg, a CD40 antagonist antibody such as lucatumumab (HCD122), dacetuzumab (SGN-40), Cp-870,893, or Chi Lob 7/4); CD27 agonists (eg CD27 agonist antibody such as varlilumab (CDX-1127)), MGA271 (up to B7H3) (WO 11/109400)); KIR antagonists (eg, lirilumab); IDO antagonists (e.g. INCB-024360 (WO 2006/122150, WO 07/75598, WO 08/36653, WO 08/36642), indoxymod, NLG-919 (WO 09/73620, WO 09/1156652, WO 11/56652 , WO12/142237) or F001287); Toll-like receptor agonists (eg, TLR2/4 agonists (eg, Bacillus Calmette-Guerin), TLR7 agonists (eg, hiltonol or imiquimod), TLR7/8 agonists (eg, resiquimod), or TLR9 agonists (eg, CpG7909)) and TGF-β inhibitors (eg GC1008, LY2157299, TEW7197 or IMC-TR1).

In one embodiment, the anti-OX40 antibody is administered prior to administration of the second agent, such as an immuno-oncological agent. In another embodiment, the anti-OX40 antibody is administered simultaneously with a second agent, such as an immuno-oncological agent. In yet another embodiment, the anti-OX40 antibody is administered after administration of the second agent. Administration of the two agents may begin, for example, 30, 60, 90, 120 minutes, 3, 6, 12, 24, 36, 48 hours, 3, 5, 7 days, or one or several weeks before, or the introduction of the second agent can begin, for example, after 30, after 60, after 90, after 120 minutes, after 3, after 6, after 12, after 24, after 36, after 48 hours, after 3 , 5, 7 days, or one or more weeks after the first remedy was administered.

In some embodiments, the anti-OX40 antibody and the second agent, such as an immuno-oncological agent, are administered to the patient simultaneously, such as simultaneously by infusion, such as over 30 or 60 minutes. The anti-OX40 antibody can be co-formulated with a second agent, such as an immuno-oncological agent.

Optionally, an anti-OX40 antibody as the sole immunotherapeutic agent, or a combination of an anti-OX40 antibody and one or more additional immunotherapeutic antibodies (e.g., blockade of CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3) can be further combined with an immunogenic agent such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune-stimulating cytokines (He et al. (2004 ) J Immunol 173:4919-28). Non-limiting examples of tumor vaccines that can be used include melanoma antigen peptides such as gp100 peptides, MAGE, Trp-2, MART1 and/or tyrosinase antigens, or tumor cells transfected to express the GMCSF cytokine (discussed further below). The combination of an anti-OX40 antibody and one or more additional antibodies (eg, blockades of CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3) can also be further combined with standard cancer treatments. For example, a combination of an anti-OX40 antibody and one or more additional antibodies (eg, blockade of CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3) can be effectively combined with chemotherapy regimens. In these cases, the dose of the other chemotherapeutic agent administered with the combination can be reduced (Mokyr et al. (1998) Cancer Research 58: 5301-5304). For example, such a combination may include an anti-OX40 antibody with an additional antibody (e.g., an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PD-L1 antibody and/or an anti-LAG-3 antibody), or without it, then in combination with decarbazine or interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale for combining an OX40 agonist antibody with a CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blocker with chemotherapy is that the cell death that results from the cytotoxic effects of most chemotherapeutic compounds , should lead to increased levels of tumor antigen in the antigen presentation pathway. Other combination therapies that may result in synergy with the combination of an anti-OX40 antibody with or without blockade of CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3, through cell death, include radiation, surgery, or endocrine shutdown. Each of these protocols creates a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with a combination of anti-OX40 antibodies and blockers

- 86 040561

CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3. Inhibition of angiogenesis results in tumor cell death, which may be the source of tumor antigen introduced into the antigen-presenting pathways of the host.

In some embodiments, an anti-OX40 antibody may be used as the sole immunotherapeutic agent, or a combination of an anti-OX40 antibody and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blocking antibodies may also be used in combination. with bispecific antibodies that target Fca or Fcy receptor-expressing effector cells to tumor cells (see, for example, US Pat. Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. The T cell response of these responses would be complemented by the use of a combination of an anti-OX40 antibody and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockers.

In another example, an anti-OX40 antibody can be used as the sole immunotherapeutic agent, or a combination of an anti-OX40 antibody and an additional immunostimulatory agent, e.g., an anti-CTLA-4 antibody and/or an anti-PD-1 antibody and/or an anti-PD-L1 antibody and/or a LAG-3 (eg, antibodies) can be used in combination with an antitumor antibody such as Rituxan® (rituximab), Herceptin® (trastuzumab), Bexxar® (tositumomab), Zevalin® (ibritumomab), Campath® (alemtuzumab), Lymphocide® (eprtuzumab), Avastin® (bevacizumab) and Tarceva® (erlotinib), etc. By way of example, and without wishing to be bound by theory, treatment with an antitumor antibody or an antitumor antibody conjugated to a toxin may result in the death of malignant cells (eg, tumor cells) that potentiate an immune response mediated by an immunostimulatory agent (eg, an OX40 agent, CTLA- 4, PD-1, PD-L1 or LAG-3, for example, an antibody). In an exemplary embodiment, treatment of a hyperproliferative disease (e.g., cancer) may include an antitumor agent (e.g., an antibody) in combination with an anti-OX40 antibody and optionally an additional immunostimulatory agent, e.g., an anti-CTLA-4 and/or anti-PD-1 agent. , and/or to PD-L1, and/or to LAG-3 (eg, by an antibody), simultaneously or sequentially, or any combination thereof, which can potentiate antitumor immune responses by the host.

Tumors evade host immune surveillance through a variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins that are expressed by tumors and that are immunosuppressive. These include, in particular, TGF-β (Kehrl et al. (1986) J. Exp. Med 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200) and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies to each of these entities can be further combined with an anti-OX40 antibody with or without an additional immunostimulatory agent, such as an agent against CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG- 3, such as an antibody, to counteract the effects of immunosuppressive agents and favor an antitumor immune response by the host.

Other agents (eg, antibodies) that can be used to activate the host's immune response may additionally be used in combination with an anti-OX40 antibody with or without an additional immunostimulatory agent, such as an anti-CTLA-4 and/or anti-PD-1 antibody. , and/or to PD-L1, and/or to LAG-3. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. An anti-CD40 antibody (Ridge et al., supra) can be used in combination with an anti-OX40 antibody and optionally an additional immunostimulatory agent, such as an anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 agent, and/ or to LAG-3, such as an antibody. Other activating antibodies to costimulatory T cell molecules Weinberg et al., supra, Melero et al., supra, Hutloff et al., supra, may also confer increased levels of T cell activation.

As discussed above, bone marrow transplantation is currently being used to treat various tumors of hematopoietic origin. Immunotherapy against OX40 alone or in combination with CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blockers can be used to increase the efficacy of tumor-specific T cells inoculated into a donor.

Several experimental treatment protocols involve ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells to anti-tumor antigen-specific T cell recipients (Greenberg & Riddell, supra). These methods can also be used to activate T cell responses to infectious pathogens such as CMV. Ex vivo activation in the presence of an anti-OX40 antibody with or without additional immunostimulatory therapy, such as anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies, would be expected , will increase the frequency and activity of adoptively transferred T cells.

Provided herein are methods for modifying an adverse event associated with the treatment of a hyperproliferative disease (e.g., cancer) with an immunostimulatory agent, comprising administering an anti-OX40 antibody with an anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 agent. , and/or to a LAG-3 (eg, antibody) subject. For example, those described in

- 87 040561 herein, the methods provide a method of reducing the incidence of immunostimulatory therapeutic antibody induced colitis or diarrhea by administering a non-absorbable steroid to a patient. As used herein, the term "non-absorbable steroid" is a glucocorticoid that exhibits extensive first pass metabolism such that after hepatic metabolism, the bioavailability of the steroid is low, i. less than about 20%. In one embodiment, the non-absorbable steroid is budesonide. Budesonide is a locally acting glucocorticosteroid that is extensively metabolized, primarily by the liver, following oral administration. ENTOCORT EC® (AstraZeneca) is a pH- and time-dependent oral formulation of budesonide designed to optimize drug delivery to the ileum and entire colon. ENTOCORT EC® is approved in the US for the treatment of mild to moderate Crohn's disease involving the ileum and/or ascending colon. The usual oral dose of ENTOCORT EC® for the treatment of Crohn's disease is 6 to 9 mg/day. ENTOCORT EC® is released in the intestine before absorption and is retained in the intestinal mucosa. After it passes through the tissue of the intestinal mucosa, ENTOCORT EC® is extensively metabolized by the cytochrome P450 system in the liver to metabolites with negligible glucocorticoid activity. Thus, bioavailability is low (about 10%). The low bioavailability of budesonide results in an improved therapeutic ratio compared to other glucocorticoids with less first pass metabolism. Budesonide results in fewer side effects, including a reduction in hypothalamic-pituitary suppression, than systemically acting corticosteroids. However, prolonged administration of ENTOCORT EC® may result in systemic glucocorticoid effects such as hypercorticism and adrenal suppression. See PDR ^58th ed. 2004; 608-610.

In other embodiments, an anti-OX40 antibody with a CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3 blocker (i.e. an anti-CTLA-4 and/or anti-PD-1 antibody, and/or to PD-L1 and/or to LAG-3) or without it in combination with a non-absorbable steroid can be further combined with salicylate. Salicylates include 5-ASA agents such as, for example, sulfasalazine (AZULFIDINE®, Pharmacia &UpJohn); olsalazine (DIPENTUM®, Pharmacia &UpJohn); balsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.); and mesalamine (ASACOL®, Procter & Gamble Pharmaceuticals, PENTASA®, Shire US, CANASA®, Axcan Scandipharm, Inc, ROWASA®, Solvay).

According to the methods described herein, salicylate administered in combination with an anti-OX40 antibody with or without anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies, and non-absorbable steroid may include any overlapping or sequential administration of salicylate and non-absorbable steroid in order to reduce the incidence of colitis caused by immunostimulatory antibodies. For example, methods for reducing the incidence of colitis caused by immunostimulatory antibodies described herein include administering salicylate and a nonabsorbable steroid simultaneously or sequentially (eg, salicylate is administered 6 hours after the nonabsorbable steroid) or any combination thereof. In addition, the salicylate and the non-absorbable steroid may be administered by the same route (eg, both are administered orally) or by different routes (eg, the salicylate is administered orally and the non-absorbable steroid is administered rectally), which may differ from the route(s) used for administration. antibodies to OX40; and antibodies to CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3.

The anti-OX40 antibodies and combination antibodies described herein may also be used in combination with other well known treatments that are selected for their particular condition being treated (eg, cancer). Combinations with OX40 antibodies can be used sequentially with known pharmaceutically acceptable agent(s).

For example, anti-OX40 antibodies and combination antibodies described herein can be used in combination (e.g., simultaneously or separately) with additional treatments such as radiation, chemotherapy (e.g., camptothecin (CPT-11), 5-fluorouracil (5FU) , cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-fu, or camptothecin + apo21/TRAIL (6X combo)), one or more proteasome inhibitors (eg, bortezomib or MG132 ), one or more Bcl-2 inhibitors (eg, BH3I-2' (bcl-xl inhibitor), indoleamine dioxygenase-1 inhibitor (eg, INCB24360, indoxymod, NLG-919 or F001287), AT-101 (R-(-) -gossypol derivative), ABT-263 (small molecule), GX-15-070 (obatoclax) or MCL-1 antagonists (myeloid leukemia cell differentiation protein 1)), iAP antagonists (apoptosis protein inhibitor) (e.g. smac7, smac4, low molecular weight smac-mimetic, synthetic smac peptides (see Fulda et al., Nat. Med. 2002;8:808-15), ISIS23722 (LY2181308) or AEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20-antibodies (eg, rituximab), angiogenesis inhibitors (eg, bevacizumab), anti-angiogenic agents , targeting VEGF and VEGFR (e.g. Avastin), synthetic triterpenoids (see Hyer et

- 88 040561 al., Cancer Research 2005;65:4799-808), c-FLIP (cellular FLICE inhibitory peptide) modulators (e.g. natural and synthetic PPARy ligands (peroxisome proliferator-activated receptor γ), 5809354 or 5569100), kinase inhibitors (e.g. sorafenib), trastuzumab, cetuximab, temsirolimus, mTOR inhibitors such as rapamycin and temsirolimus, bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, lenalildomide, GSK3P inhibitors, IAP inhibitors, and/or genotoxic drugs.

Anti-OX40 antibodies and combination antibodies described herein may additionally be used in combination with one or more antiproliferative cytotoxic agents. Classes of compounds that can be used as antiproliferative cytotoxic agents include, without limitation, the following.

Alkylating agents (including but not limited to nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas, and triazenes):

uracil mustard, chlormethine, cyclophosphamide (CYTOXAN™), phosphamide, melphalan, chlorambucil, piprobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentastatin, and gemcitabine.

Suitable antiproliferative agents for combination with OX40 antibodies are, but are not limited to, taxanes, paclitaxel (paclitaxel commercially available as TAXOL™), docetaxel, discodermolide (DDM), dictiostatin (DCT), pelaruside A, epothilones, epothilone A, epothilone B, epothilone C , epothilone D, epothilone E, epothilone F, furanoepothilone D, deoxyepothilone B1, [17]-dehydrodeoxyepothilone B, [18]dehydrodeoxyepothilones B, C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A, 9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone B10, discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (discodermolide), TZT -1027 (Soblidotin), ILX-651 (Tasidotin Hydrochloride), Chalichondrin B, Eribulin Mesylate (E7389), Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703, Maytansinoid Immunoconjugates (DM-1), MKC- 1, ABT-751, T1-38067, T-900607, SB-715992 (ispinezib), SB-743921, MK-0731, STA-5312, eleutherobin, 17-β-acetoxy-2-ethoxy-6-oxo-B -homo-estra-1 ,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-diethyl-(+)-discodermolides, and cryptothilon 1, in addition to other microtubule stabilizing agents known in the present technical field.

In cases where it is desirable to immobilize aberrantly proliferative cells in combination with or before anti-OX40 antibody treatment, hormones and steroids (including synthetic analogues) such as 17a-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostonolone propionate, testolactone, megestrol acetate, methylprednisolone, methylsterosterone, prednisolone, triamcinolone, chlortrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, ZOLADEX™ may also be administered to the patient. When using the methods or compositions described herein, other agents used to modulate tumor growth or metastasis in clinical settings, such as antimimetics, may also be administered as desired.

In some embodiments, the anti-OX40 antibody is administered in combination (simultaneously or separately) with nivolumab to treat a patient with cancer, eg, colon and rectal or bladder cancer.

In some embodiments, an anti-OX40 antibody is administered in combination (simultaneously or separately) with ipilimumab to treat a patient with cancer, eg, ovarian, bladder, or prostate cancer.

Those skilled in the art are aware of methods for safely and effectively administering chemotherapeutic agents. In addition, their administration is described in the standard literature. For example, the administration of many chemotherapeutic agents is described in the Physicians' Directory (PDR), eg, 1996 edition (Medical Economics Company, Montvale, NJ. 07645-1742, USA); the disclosure of which is incorporated herein by reference.

The chemotherapeutic agent(s) and/or radiation therapy may be administered according to therapeutic protocols well known in the art. Those skilled in the art will appreciate that the administration of the chemotherapeutic agent(s) and/or radiation therapy may vary depending on the disease being treated and the known effects of the chemotherapeutic agent(s) and/or radiation therapy on that disease. In addition, in accordance with the knowledge of the skilled physician, therapeutic protocols (eg, number of doses and time of administration) may vary depending on the observed effect of the administered therapeutic agents on the patient and taking into account the observed responses of the disease to the administered therapeutic agents.

The present disclosure is further illustrated by the following examples, which should not be construed as a further limitation. The contents of all figures and all references, Genbank sequences, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

XVII. Sets and unit dosage forms.

Also provided herein are kits that include a pharmaceutical composition comprising an anti-OX40 antibody (eg, OX40.21) and an anti-PD-1 (eg, nivolumab) or CTLA-4 (ipilimumab) antibody, and a pharmaceutically acceptable carrier, in a therapeutically effective amount adapted for use in the previous methods. Kits may optionally also include instructions, such as containing administration schedules, to allow a practitioner (eg, physician, nurse, or patient) to administer the composition contained therein to a patient with a malignant tumor (eg, a solid tumor). The kit may also include a syringe.

Optionally, the kits include multiple packs of single dose pharmaceutical compositions, each containing an effective amount of an anti-OX40 antibody or an anti-PD-1 or anti-CTLA-4 antibody for a single administration according to the methods described above. The tools or devices needed to administer the pharmaceutical composition(s) may also be included in kits. For example, the kit may contain one or more pre-filled syringes containing an amount of anti-OX40 antibody or anti-PD-1 or anti-CTLA-4 antibody.

In one embodiment, the present invention provides a kit for the treatment of a solid tumor in a human patient, the kit comprising a dose of an anti-OX40 antibody comprising the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of SEQ ID NO: 318 and the domains Light chain variable region CDR1, CDR2 and CDR3 with the sequence shown in SEQ ID NO: 94 and instructions for use in the methods described herein. In some embodiments, the kit further comprises (a) a dose of an anti-PD-1 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 301 and the light chain variable region CDR1, CDR2 and CDR3 domains with the sequence shown in SEQ ID NO: 302, or (b) a dose of an anti-CTLA-4 antibody containing the CDR1, CDR2 and CDR3 domains of the heavy chain variable region with the sequence shown in the sequences of SEQ ID NO: 309, and the CDR1, CDR2 domains and a light chain variable region CDR3 with the sequence shown in SEQ ID NO: 310.

Embodiments.

1. An isolated antibody or antigen-binding portion thereof that binds to human OX40 and has the following properties:

(a) binds to human membrane-bound OX40;

(b) binds to cynomolgus monkey OX40;

(c) binds to soluble human OX40;

(d) induces or enhances T cell activation;

(e) inhibits OX40 ligand binding to OX40;

(f) competes for binding to human OX40 with one or more of the antibodies 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3, 14A2-1, 14A2-2 and 20c1.

2. The antibody of embodiment 1, wherein the antibody does not bind to mouse and/or rat OX40.

3. An antibody or antigen-binding portion thereof according to embodiment 1 or 2, wherein the antibody stimulates an antitumor immune response.

4. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody stimulates an antigen-specific T cell response.

5. An antibody or antigen-binding portion thereof according to any of the previous embodiments, wherein the antibody increases the production of IL-2 and/or IFN-γ in OX40 expressing T cells.

6. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody increases T cell proliferation.

7. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody binds to Fc receptors.

8. An antibody or antigen-binding portion thereof according to embodiment 7, wherein the antibody binds to one or more activating FcyRs.

9. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody binds to soluble human OX40 with a K _D of about 1 nM or less, such as 0.5 nM or less or 0.1 nM or less, as measured by assay Biacore.

10. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody binds to membrane-bound human OX40 with an EC _{50 of} 50 nM or less, such as 10 nM or less or 1 nM or less, as measured by FACS.

11. An antibody or antigen-binding portion thereof according to any of the previous embodiments

- 90 040561, wherein the antibody binds to membrane-bound cynomolgus monkey OX40 with an EC ₅₀ of 50 nM or less, eg 10 nM or less or 1 nM or less as measured by FACS analysis.

12. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody induces or enhances T cell activation via multivalent crosslinking.

13. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody binds to the C1q component of the human complement.

14. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody induces NK cell-mediated lysis of activated CD4+ T cells.

15. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody promotes macrophage-mediated phagocytosis of OX40-expressing cells.

16. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody inhibits regulatory T cell-mediated suppression of CD4+ T cell proliferation.

17. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody binds to the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) of human OX40 (SEQ ID NO: 2).

18. An antibody or antigen-binding portion thereof according to any one of embodiments 1-16, wherein the antibody binds to the sequence DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179) of human OX40 (SEQ ID NO: 2).

19. An isolated monoclonal antibody, or antigen-binding portion thereof, that specifically binds to OX40 and contains three variable heavy chain CDRs and three variable light chain CDRs that are in variable heavy chain and variable light chain pairs selected from the group consisting of:

(a) SEQ ID NOs: 318 and 94;

(b) SEQ ID NOs: 17 and 18;

(c) SEQ ID NOs: 28 and 29;

(d) SEQ ID NOs: 28 and 30;

(e) SEQ ID NOs: 37 and 38;

(f) SEQ ID NOs: 48 and 49;

(g) SEQ ID NOs: 48 and 50;

(h) SEQ ID NOs: 57 and 58;

(i) SEQ ID NOs: 65 and 66;

(j) SEQ ID NOs: 73 and 74;

(k) SEQ ID NOs: 84 and 85;

(l) SEQ ID NOs: 84 and 86; and (m) SEQ ID NOs: 93 and 94.

20. An isolated monoclonal antibody or antigen-binding portion thereof that binds to OX40, containing:

(a) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 87, 317 and 89, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 90-92, respectively;

(b) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 11-13, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 1416, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 19-21, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 2224, respectively;

(d) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 19-21, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 2527, respectively;

(e) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 31-33, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 3436, respectively;

(f) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 39-41, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 4244, respectively;

(g) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 39-41, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 4547, respectively;

- 91 040561 (h) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 51-53, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 5456, respectively;

(i) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 59-61, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 6264, respectively;

(j) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 67-69, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 7072, respectively;

(k) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 75-77, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 7880, respectively;

(l) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 75-77, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 8183, respectively; or (m) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO: 87-89, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NO: 9092, respectively.

21. An isolated monoclonal antibody or antigen-binding portion thereof that binds to OX40, containing:

(a) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 87, 317 and 89, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 90-92, respectively;

(b) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 11-13, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 1416, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 19-21, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 2224, respectively;

(d) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 19-21, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 2527, respectively;

(e) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 31-33, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 3436, respectively;

(f) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 39-41, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 4244, respectively;

(g) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 39-41, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 4547, respectively;

(h) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 51-53, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NOs: 5456, respectively;

(i) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 59-61, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 6264, respectively;

(j) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 67-69, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 7072, respectively;

(k) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 75-77, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 7880, respectively;

(l) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 75-77, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 8183, respectively; or (ii i) heavy chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 87-89, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences consisting of SEQ ID NO: 9092, respectively.

22. An isolated monoclonal antibody, or an antigen-binding portion thereof, that binds to OX40 and contains heavy and light chain variable regions, wherein the heavy chain variable region contains an amino acid sequence that is at least 90% identical

- 92 040561 amino acid sequence selected from the group consisting of SEQ ID NO: 17, 28, 37, 48, 57,

65, 73, 84 and 93.

23. An isolated monoclonal antibody, or an antigen-binding portion thereof, that binds to OX40 and contains heavy and light chain variable regions, wherein the light chain variable region contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 29, 30, 38, 49, 50, 58, 66, 74, 85, 86 and 94.

24. An isolated monoclonal antibody, or antigen-binding portion thereof, that binds to OX40 and contains heavy and light chain variable region sequences that are at least 85% identical to amino acid sequences selected from the group consisting of:

(a) SEQ ID NOs: 318 and 94;

(b) SEQ ID NOs: 17 and 18;

(c) SEQ ID NOs: 28 and 29;

(d) SEQ ID NOs: 28 and 30;

(e) SEQ ID NOs: 37 and 38;

(f) SEQ ID NOs: 48 and 49;

(g) SEQ ID NOs: 48 and 50;

(h) SEQ ID NOs: 57 and 58;

(i) SEQ ID NOs: 65 and 66;

(j) SEQ ID NOs: 73 and 74;

(k) SEQ ID NOs: 84 and 85;

(l) SEQ ID NOs: 84 and 86; and (m) SEQ ID NOs: 93 and 94.

25. The antibody or antigen-binding portion thereof of embodiment 24, wherein the heavy and light chain variable regions comprise an amino acid sequence that is at least 90% identical to the heavy and light chain variable regions selected from the group consisting of (a)-( l) Embodiment 24.

26. The antibody or antigen-binding portion thereof according to embodiment 25, wherein the heavy and light chain variable region contains an amino acid sequence that is at least 95% identical to the heavy and light chain variable regions selected from the group consisting of (a)-( l) Embodiment 24.

27. The antibody or antigen-binding portion thereof of embodiment 26, wherein the heavy and light chain variable region comprises heavy and light chain variable regions selected from the group consisting of (a)-(l) of embodiment 24.

28. An isolated monoclonal antibody, or an antigen-binding portion thereof, that binds to OX40 and contains heavy chain and light chain sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acid sequences selected from group consisting of:

(a) SEQ ID NOs: 124 and 116, respectively;

(b) SEQ ID NOs: 95 and 96, respectively;

(c) SEQ ID NOs: 97 and 98, respectively;

(d) SEQ ID NOs: 99 and 100, respectively;

(e) SEQ ID NOs: 101 and 102, respectively;

(f) SEQ ID NOs: 103 and 104, respectively;

(g) SEQ ID NOs: 105 and 106, respectively;

(h) SEQ ID NOs: 107 and 108, respectively;

(i) SEQ ID NOs: 109 and 110, respectively;

(j) SEQ ID NOs: 111 and 112, respectively;

(k) SEQ ID NOs: 113 and 114, respectively;

(l) SEQ ID NOs: 115 and 116, respectively;

(m) SEQ ID NOs: 117 and 118, respectively;

(n) SEQ ID NOs: 119 and 120, respectively;

(o) SEQ ID NOs: 121 and 122, respectively;

(p) SEQ ID NOs: 123 and 116, respectively; and (q) SEQ ID NOs: 125 and 116, respectively.

29. An antibody or antigen-binding portion thereof according to embodiment 28, wherein the heavy and light chains comprise a heavy and light chain selected from the group consisting of (a)-(r) of embodiment 28.

30. An isolated monoclonal antibody, or antigen-binding portion thereof, that (a) binds to the same epitope on OX40 as the antibody of embodiment 27 and/or (b) inhibits the binding of the antibody of embodiment 27 to OX40 on activated T -cells by

- 93 040561 at least 95% as measured by FACS.

31. An antibody or antigen-binding portion thereof according to any one of embodiments 19-30, wherein the antibody binds to the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) of human OX40 (SEQ ID NO: 2).

32. An antibody or antigen-binding portion thereof according to any one of embodiments 19-30, wherein the antibody binds to the sequence DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK (SEQ ID NO: 179) of human OX40 (SEQ ID NO: 2).

33. The antibody, or antigen-binding portion thereof, of any one of embodiments 19-31, wherein the antibody binds to both human OX40 and cynomolgus monkey OX40.

34. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, or a variant thereof.

35. An antibody or antigen-binding portion thereof according to embodiment 34, wherein the antibody is an IgG1 antibody.

36. The antibody of embodiment 35, wherein the antibody, or antigen-binding portion thereof, contains an Fc having improved binding to an activating FcyR.

37. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein one or more methionine residues in the CDR regions are replaced with amino acid residues that do not undergo oxidation.

38. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody or antigen-binding portion thereof is a human or humanized antibody.

39. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody is non-immunogenic as defined in accordance with Example 21.

40. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the Asp-Gly amino acid sequence, if present in the heavy and/or light chain CDR sequences, is replaced with an amino acid sequence that does not undergo isomerization.

41. An antibody or antigen-binding portion thereof according to embodiment 40, wherein the antibody comprises the heavy chain variable region CDR2 sequence shown in SEQ ID NO: 76, but wherein the Asp-Gly sequence is replaced by an amino acid sequence that is not isomerized.

42. The antibody of embodiment 41 wherein Asp or Gly in the Asp-Gly sequence is replaced by Ser.

43. A bispecific molecule comprising an antibody of any of the preceding embodiments is linked to a molecule having a second binding specificity.

44. Nucleic acid encoding the heavy and/or light chain variable region of an antibody or antigen-binding portion thereof according to any one of embodiments 1-42.

45. An expression vector containing the nucleic acid molecule of embodiment 44.

46. Cell transformed with the expression vector of embodiment 45.

47. An immunoconjugate containing an antibody according to any of embodiments 1-42 associated with an agent.

48. A composition comprising an antibody or antigen-binding portion thereof, a bispecific molecule or an immunoconjugate according to any one of embodiments 1-43 and 47, and a carrier.

49. A kit containing the antibody or antigen-binding portion or bispecific molecule or immunoconjugate of any one of embodiments 1-43 and 47 and instructions for use.

50. A method for producing an OX40 antibody or antigen-binding portion thereof, comprising expressing the antibody or antigen-binding portion thereof in a cell according to embodiment 46 and isolating the antibody or antigen-binding portion thereof from the cell.

51. A method of stimulating an antigen-specific T cell response, comprising contacting a T cell with an antibody or antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate according to any one of embodiments 1-43 and 47, such that an antigen-specific T cell response is stimulated.

52. A method for activating or costimulating an effector T cell, comprising contacting the effector T cell with an anti-OX40 antibody or antigen-binding portion thereof, a bispecific molecule or immunoconjugate according to any one of embodiments 1-43 and 47, and CD3, wherein the effector T cell is activated or co-stimulated.

53. A method of increasing the production of IL-2 and/or IFN-γ in a T cell, comprising contacting the T cell with an effective amount of an antibody or antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate according to any one of embodiments 1-43 and 47.

54. A method for increasing T cell proliferation, comprising contacting a cell with

- 94 040561 an effective amount of the antibody or antigen-binding portion, bispecific molecule or immunoconjugate according to any of embodiments 1-43 and 47.

55. A method of increasing the production of IL-2 and/or IFN-γ in T cells in a subject, comprising administering an effective amount of an antibody or antigen-binding portion, bispecific molecule or immunoconjugate according to any one of embodiments 1-43 and 47, to increase the production of IL -2 and/or IFN-γ from T cells.

56. A method for reducing or depleting the number of regulatory T cells in a tumor in a subject in need thereof, comprising administering an effective amount of an antibody or an antigen-binding portion, a bispecific molecule, or an immunoconjugate according to any one of embodiments 1-43 and 47, wherein the antibody or antigen-binding portion thereof characterized by effector or improved effector function, to reduce the number of regulatory T cells in the tumor.

57. A method of stimulating an immune response in a subject, comprising administering an antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate according to any one of embodiments 1-43 and 47 to a subject such that an immune response is stimulated in the subject.

58. The method of embodiment 57 wherein the subject contains a tumor and an immune response against the tumor is stimulated.

59. A method for inhibiting tumor cell growth in a subject, comprising administering to the subject an antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate according to any one of embodiments 1-43 and 47 such that tumor growth is inhibited.

60. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding portion, bispecific molecule, or immunoconjugate of any one of embodiments 1-43 and 47 for treating cancer.

61. The method of Embodiment 60 wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer , gastrointestinal cancer, pancreatic cancer, colon and rectum cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, cancer bones, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasm, lymphoma, leukemia, myeloma, sarcoma, and virus-associated cancer.

62. The method of embodiment 60 or 61 wherein the cancer is a metastatic cancer, a refractory cancer, or a recurrent cancer.

63. The method of any one of embodiments 56-62, further comprising administering one or more additional therapeutic agents.

64. The method of embodiment 63 wherein the one or more additional therapeutic agents is an antibody or small molecule.

65. The method of embodiment 64, wherein the additional treatment is an anti-PD1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-PD-L1 antibody, or an anti-TGFe antibody.

66. A method of treating a solid tumor in a human, the method comprising administering to the subject an effective amount of an anti-OX40 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of the sequence shown in SEQ ID NO: ID NO: 318 and the CDR1, CDR2 domains and a light chain variable region CDR3 with the sequence shown in SEQ ID NO: 94, wherein the method comprises at least one cycle of administration, wherein the cycle is a period of two weeks, with one dose of anti-OX40 antibody for each of the at least one cycle administered at a dose of 1 mg/kg body weight; fixed dose of 20, 40, 80, 160 or 320 mg; a dose of approximately 1 mg/kg body weight or a fixed dose of approximately 20, 40, 80, 160 or 320 mg.

67. A method of treating a solid tumor in a human, the method comprising administering to the subject an effective amount of each of:

(a) an anti-OX40 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 318 and the light chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 94 ;

(b) an anti-PD-1 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 301 and the light chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: : 302, and the method provides for at least one cycle of administration, and the cycle represents

- 95 040561 is a period of two, three or four weeks, and for each of at least one cycle, one dose of antibodies to OX40 is administered at a dose of 1 mg/kg of body weight; fixed dose of 20, 40, 80, 160 or 320 mg;

dose of approximately 1 mg/kg body weight or a fixed dose of approximately 20, 40, 80, 160 or 320 mg, and a single dose of anti-PD-1 antibody is administered at a dose of 240, 360 or 480 mg or a dose of approximately 240, 360 or

480 mg.

68. The method of embodiment 67 wherein the anti-OX40 antibody and anti-PD-1 antibody are administered at the following doses:

(a) 1 mg/kg anti-OX40 antibody and 240 mg, 360 mg or 480 mg anti-PD-1 antibody;

(b) 20 mg anti-OX40 antibody and 240 mg, 360 mg or 480 mg anti-PD-1 antibody;

(c) 40 mg anti-OX40 antibody and 240 mg, 360 mg or 480 mg anti-PD-1 antibody;

(d) 80 mg anti-OX40 antibody and 240 mg, 360 mg or 480 mg anti-PD-1 antibody;

(e) 160 mg anti-OX40 antibody and 240 mg, 360 mg or 480 mg anti-PD-1 antibody; or (f) 320 mg anti-OX40 antibody and 240 mg, 360 mg or 480 mg anti-PD-1 antibody.

69. A method of treating a solid tumor in a human, the method comprising administering to the subject an effective amount of each of:

(a) an anti-OX40 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 318 and the light chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 94 ;

(b) an anti-CTLA-4 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 309 and the light chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO : 310, wherein the method comprises at least one cycle of administration, wherein the cycle is a period of three weeks, wherein for each of the at least one cycle, one dose of anti-OX40 antibody is administered at a dose of 1 mg/kg of body weight; fixed dose of 20, 40, 80, 160 or 320 mg; dose of approximately 1 mg/kg body weight or a fixed dose of approximately 20, 40, 80, 160 or 320 mg, and a single dose of anti-CTLA-4 antibody is administered at a dose of 1 mg/kg or a dose of approximately 1 mg/kg, and the antibody to OX40 co-administered with anti-CTLA-4 antibody for at least one cycle followed by monotherapy with anti-OX40 antibody for at least one cycle.

70. The method of embodiment 67 wherein the anti-OX40 antibody and anti-CTLA-4 antibody are administered at the following doses:

(a) 1 mg/kg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(b) 20 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(c) 40 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(d) 80 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody;

(e) 160 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody; or (f) 320 mg anti-OX40 antibody and 1 mg/kg anti-CTLA-4 antibody.

71. The method of any one of embodiments 66-70, wherein the anti-OX40 antibody or anti-OX40 antibody and the anti-PD-1 antibody or anti-CTLA-4 antibody are formulated for intravenous administration.

72. The method of any one of embodiments 67-71, wherein an anti-OX40 antibody and an anti-PD-1 or anti-CTLA-4 antibody are put together.

73. The method of any one of embodiments 67-71, wherein the anti-OX40 antibody and anti-PD-1 or anti-CTLA-4 antibody are separately formulated.

74. The method of any one of embodiments 66-68 and 71-73, wherein the treatment consists of 12 cycles.

75. The method of any one of embodiments 69-73 wherein the treatment consists of 8 cycles.

76. The method of embodiment 75 wherein the anti-OX40 antibody is co-administered with the anti-CTLA-4 antibody for the first 4 cycles followed by anti-OX40 antibody monotherapy for the last 4 cycles.

77. The method of any one of embodiments 66-76, wherein an anti-OX40 antibody or an anti-OX40 antibody and an anti-PD-1 or anti-CTLA-4 antibody are administered on the first day of each cycle.

78. The method of any one of embodiments 67-77 wherein the anti-OX40 antibody is administered prior to the administration of the anti-PD-1 or anti-CTLA-4 antibody.

79. The method of embodiment 78 wherein the anti-OX40 antibody is administered within about 30 minutes prior to the administration of the anti-PD-1 or anti-CTLA-4 antibody.

80. The method of any one of embodiments 67-77, wherein the anti-OX40 antibody is administered after administration of the anti-PD-1 or anti-CTLA-4 antibody.

81. The method of any one of embodiments 67-77 wherein the anti-OX40 antibody is administered simultaneously with the anti-PD-1 or anti-CTLA-4 antibody.

82. The method of any one of embodiments 66-81, wherein the treatment produces at least one therapeutic effect selected from reduction in tumor size, reduction in number of metastatic lesions over time, complete response, partial response, and stable disease.

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83. The method of any one of embodiments 66-82, wherein the solid tumor is associated with a cancer selected from the group consisting of cervical cancer, bladder cancer, colon cancer, and ovarian cancer.

84. The method of any one of embodiments 66-83, wherein the anti-OX40 antibody comprises heavy chain and light chain variable region CDRs comprising the amino acid sequences shown in SEQ ID NOs: 87, 317 and 89 and 90-92, respectively.

85. The method of any one of embodiments 66-84, wherein the anti-OX40 antibody comprises the heavy and light chain variable region sequences set forth in SEQ ID NOs: 318 and 94, respectively.

86. The method of any one of embodiments 66-85, wherein the anti-OX40 antibody comprises the heavy and light chain sequences set forth in SEQ ID NOs: 124 and 116, respectively.

87. The method of any one of embodiments 67, 68, and 71-86, wherein the anti-PD-1 antibody comprises heavy chain and light chain variable region CDRs comprising the amino acid sequences set forth in SEQ ID NOs: 303-305 and 306-308 , respectively.

88. The method of any one of embodiments 67, 68, and 71-87, wherein the anti-PD-1 antibody comprises the heavy and light chain variable region sequences set forth in SEQ ID NOs: 301 and 302, respectively.

89. The method of any one of embodiments 69-86, wherein the anti-CTLA-4 antibody comprises heavy chain and light chain variable region CDRs comprising the amino acid sequences shown in SEQ ID NOs: 311-313 and 314-316, respectively.

90. The method of any one of embodiments 69-86 and 89, wherein the anti-CTLA-4 antibody comprises the heavy and light chain variable region sequences set forth in SEQ ID NOs: 309 and 310, respectively.

91. A kit for the treatment of a human solid tumor, the kit comprising a dose of an anti-OX40 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 318 and the light variable region CDR1, CDR2 and CDR3 domains chains with the sequence shown in SEQ ID NO: 94, and instructions for use.

92. The kit of embodiment 91 further comprising (a) a dose of an anti-PD-1 antibody comprising the heavy chain variable region CDR1, CDR2 and CDR3 domains of SEQ ID NO: 301 and the variable heavy chain CDR1, CDR2 and CDR3 domains light chain region with the sequence shown in SEQ ID NO: 302, or (b) a dose of an anti-CTLA-4 antibody containing the heavy chain variable region CDR1, CDR2 and CDR3 domains with the sequence shown in SEQ ID NO: 309 and the CDR1 domains , CDR2 and CDR3 of the light chain variable region with the sequence shown in SEQ ID NO: 310.

93. A method for detecting the presence of OX40 in a sample, comprising contacting the sample with an antibody, or antigen-binding portion thereof, according to any one of embodiments 1-38 under conditions that allow formation of a complex between the antibody or binding of the antigen.

94. An antibody that binds to OX40 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 282-296.

95. The antibody of embodiment 94, wherein the antibody comprises a heavy chain consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 282-296.

96. Isolated monoclonal antibody that binds to OX40, containing the heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 87, 317 and 89, respectively, and the light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 90 -92, respectively.

97. An isolated monoclonal antibody that binds to OX40 containing heavy and light chain variable regions containing the amino acid sequences of SEQ ID NOs: 318 and 94, respectively.

98. An isolated monoclonal antibody that binds to OX40 containing heavy and light chains containing the amino acid sequences of SEQ ID NOs: 124 and 116, respectively.

99. An isolated monoclonal antibody that binds to OX40, wherein the antibody binds to all or part of the sequence DVVSSKPCKPCTWCNLR (SEQ ID NO: 178) of human OX40 (SEQ ID NO: 2).

100. A composition comprising an isolated monoclonal antibody according to any of embodiments 96-99 and a carrier.

101. Nucleic acid encoding the heavy and/or light chain variable region of an antibody of embodiment 96 or 97, or the heavy and/or light chain of embodiment 98.

102. A kit containing an antibody according to any one of embodiments 96-99.

103. A method of stimulating an antigen-specific T cell response, comprising contacting a T cell with an antibody according to any one of embodiments 96-99.

104. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody according to any one of the embodiments

- 97 040561 implementation 96-99 for the treatment of malignant tumors.

105. The method of embodiment 104, wherein the cancer or solid tumor is selected from the group consisting of cervical cancer, bladder cancer, colon cancer, ovarian cancer, non-small cell lung cancer, and head and neck squamous cell carcinoma.

Examples

Example 1. Obtaining antibodies to OX40.

Monoclonal antibodies to human OX40 were generated in Hco7, Hco12, Hco17 and Hco38 strains of HuMAb® transgenic mice (HuMAb is a trademark of Medarex, Inc., Princeton, NJ) and KM mice (KM Mouse® strain contains the SC20 transchromosome as described in PCT publication WO 02/43478) using the recombinant hexachistidine-OX40 antigen.

A total of 52 mice, including 5 transgenic mouse genotypes (KM, Hco7, Hco12, Hco17 and Hco38), were immunized with various immunization strategies. The immunogen was huOX40-6x, which was obtained locally and used at a dose of 2.0 mg/ml to give a total dose of 20 μg per mouse. Routes of administration included: tail base injection, Hock immunization, intraperitoneal (ip) and subcutaneous (sc) injection, and adjuvant (Ribi, Cat # S6322, Sigma). 27 fusions from 30 mice were performed and screened. Of these 27 fusions, 541 antigen-positive antibodies were identified by ELISA, and further characterization led to the isolation of antibodies of particular interest, including antibodies designated as 3F4, 14B6-1, 14B6-2, 23H3, 6E1-1, 6E1-2, 18E9, 8B11, 20B3 (also called OX40.17), 14A2-1, 14A2-2 and 20C1. Their variable region amino acid sequences and isotype are shown in FIG. 1-9. The 3F4 heavy and light chain variable regions consist of the amino acid sequences of SEQ ID NOs: 17 and 18. The 14B6-1 heavy and light chain variable regions consist of the amino acid sequences of SEQ ID NOs: 28 and 29. The 14B6-2 heavy and light chain variable regions consist of of the amino acid sequences of SEQ ID NOS: 28 and 30. The 23H3 heavy and light chain variable regions consist of the amino acid sequences of SEQ ID NOS: 37 and 38. The 6E1-1 heavy and light chain variable regions consist of the amino acid sequences of SEQ ID NOS: 48 and 49 The 6E1-2 heavy and light chain variable regions consist of the amino acid sequences of SEQ ID NOs: 48 and 50. The 18E9 heavy and light chain variable regions consist of the amino acid sequences of SEQ ID NOs: 57 and 58. The 8B11 heavy and light chain variable regions consist of amino acid sequences of SEQ ID NOs: 65 and 66. The variable regions of the heavy and light chains of 20B3 consist of amino acids of SEQ ID NOs: 73 and 74. The 14A2-1 heavy and light chain variable regions consist of the amino acid sequences of SEQ ID NOs: 84 and 85. The 14A2-2 heavy and light chain variable regions consist of the amino acid sequences of SEQ ID NOs: 84 and 86 The 20C1 heavy and light chain variable regions consist of the amino acid sequences of SEQ ID NOS: 93 and 94.

cDNA sequencing identified one heavy and one light chain for each of the antibodies 3F4, 23H3, 18E9, 8B11, 20B3 (also referred to as OX40.17) and 20C1, as well as one heavy chain and two light chains (light chain 1 or L1 and light chain 2 or L2) for each of the antibodies 14B6, 14A2 and 6E1. Protein analysis identified a single light chain for antibodies 14B6, 6E1 and 14A2, and N-terminal sequencing and molecular weight determination showed that it was the L1 light chain for 14B6 and 14A2 and the L2 light chain for 6E1. Antibodies 14B6-1 and 14B6-2 correspond to the light chain antibody 14B6 L1 and L2, respectively. Antibodies 14A2-1 and 14A2-2 correspond to the light chain antibody 14A2 L1 and L2, respectively. Antibodies 6E1-1 and 6E1-2 correspond to the light chain 6E1 antibody L1 and L2, respectively. Amino acid and nucleotide sequences of each of the light chains of 3 antibodies are presented in table. 23.

For some of the above antibodies in HCDR2, substitutions were made in the parent antibody to remove the presence of an isomerization site (DG) and framework substitutions (due to DP44 germline origin) were introduced to make the structure more similar to the normally expressed antibody. For 20C1-based antibodies, three additional unusual framework residues were returned to the germline (A2V, D24G and G82bS). Reversal of the G82bS framework also eliminates the deamidation (NG) site. A summary of the various substitutions introduced in the sequence of the original hybrid clone is given in table. 6.

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Table 6

Name The original hybridoma clone Isotype Variable region substitutions 0X40.6 23H3 gif 23H3 anti-OX40 with VH-H13Q/M87T 0X40.7 23H3 gif 23H3 anti-OX40 with VH-M87T/M95Y 0X40.8 14A2 gif 14A2 anti-OX40 with VH-G103W 0X40.9 14A2 gif 14A2 anti-OX40 with VH-M97Y/G103W 0X40.10 14A2 gif 14A2 anti-OX40 with VH-D53S 0X40.11 14A2 gif 14A2 anti-OX40 with VH-G54S 0X40.12 14A2 gif 14A2 anti-OX40 with VH-D53S/G103W 0X40.13 14A2 gif 14A2 anti-OX40 with VH-G54S/G103W 0X40.14 14A2 gif 14A2 anti-OX40 with VH-D53S/M97Y/G103W 0X40.15 14A2 gif 14A2 anti-OX40 with VH-G54S/M97Y/G103W 0X40.16 20С1 gif 20C1 anti-OX40 with VH- A2V/H13Q/D24G/M87T/G82bS 0X40.17 20VZ gif 20VZ anti-OH40 (no replacements) 0X40.18 3F4 gif 3F4 anti-OX40 with VH-N27Y/N72D/P102Y 0X40.19 14A2 gif 14A2 anti-OX40 with VH-M97L/G103W 0X40.20 20С1 gif 20C1 anti-OX40 with VH- A2V/H13Q/D24G/D54S/M87T/G82bS 0X40.21 20С1 gif 20C1 anti-OX40 with VH- A2 V/H13 Q/D24G/G5 5 A/M87T/G82b S 0X40.22 20С1 gif 20C1 anti-OX40 with VH- A2V/H13Q/D24G/D54S/G55T/M87T/G82bS

*Depending on the germline, D53 and G54 or D54 and G55 may be present as a potential isomerization site.

Example 2 Anti-OX40 Antibodies Binding to Activated Primary Human T Cells.

The anti-human OX40 monoclonal antibodies prepared in Example 1 were tested for their ability to bind to activated primary human T cells.

Cells were activated for several days prior to binding assay to induce OX40 expression. Briefly, PBMCs were cultured for three or four days with anti-human CD3 plus anti-human CD28 coated magnetic beads in the presence of recombinant human IL-2. On the day of analysis, the beads were removed and the cells were stained by titrating each with anti-OX40 antibody. Bound antibodies were detected with a fluorescently conjugated anti-human IgG polyclonal secondary antibody, and the cells were co-stained for CD4 and CD25 to detect activated CD4 T cells. Staining fluorescence intensity was measured using a FACSCanto II flow cytometer (Becton Dickinson). For the CD4+ CD25+ population (FACSDiva software), geometric mean fluorescence intensity (GMFI) or mean fluorescence intensity (MedFI) of anti-OX40 staining was calculated. EC ₅₀ for antibody binding was calculated using GraphPad Prism software.

As shown in FIG. 11A, anti-OX40 antibodies bind to activated primary human T cells with subnanomolar EC ₅₀ . It is noteworthy that OX40.5 showed low binding of the studied antibodies to OX40. The same experiment was performed with antibodies to OX40 with variations in the variable region. Initial experiments were performed using antibodies in the form of supernatants from host cell cultures transfected with recombinant antibody expression vectors. As shown in FIG. 11B, some substitutions caused a significant loss of binding, namely for antibodies OX40.7, OX40.9, OX40.14 and OX40.15. A set of antibodies with variable region substitutions was analyzed further using purified antibody material.

As shown in FIG. 11C and 11D, all tested antibodies bound subnanomolar EC ₅₀ to OX40, except for OX40.18, which showed lower binding than the original 3F4 hybridoma clone. OX40.5 showed the least binding to the panel of antibodies examined in FIG. 11B, while OX40.1 showed the lowest binding to the panel of antibodies examined in FIG. 11c. A summary of the values of EC ₅₀ is presented in table. 7 below.

Table 7. EC ₅₀ for binding of OX40 antibodies to activated _______ human primary T cells______

Name Human T cell binding EC50 (nM) (mean ± SD) η 3F4 0.19±0.15 3 8B11 0.14 ± 0.07 2 18E9 0.22 ± 0.12 2 20VZ 0.34 ± 0.23 3 20С1 0.10±0.06 3 23H3 0.15 ± 0.09 3 6E1 0.97±0.05 2 14A2 0.33±0.30 3 14V6 0.18 ± 0.16 2 0X40.6 0.13 ± 0.06 7 0X40.8 0.14 ± 0.07 7 0X40.16 0.06 ± 0.03 5 0X40.17 0.26 ± 0.17 3 0X40.18 3.15 ± 3.9 2 0X40.21 0.07 ± 0.02 5 0X40.1 0.35 ± 0.09 2 0X40.4 0.36 ± 0.06 2 0X40.5 3.20±0.00 2

Example 3 Anti-OX-40 Antibodies Binding to Activated Primary Cynomolgus T Cells.

The anti-human OX-40 monoclonal antibody that was tested for binding to activated primary human T cells in Example 1 was tested for its ability to bind to activated primary cynomolgus monkey T cells.

Briefly, the cells were activated for several days prior to the binding assay to induce OX40 expression. The total number of leukocytes was isolated from the peripheral blood of the cynomolgus monkey by lysis of erythrocytes using ammonium chloride buffer. The leukocytes were then cultured for four to five days in flasks pre-coated with anti-human CD3 plus anti-human CD28 antibodies that cross-react with cynomolgus monkey in the presence of recombinant human IL-2 to increase and activate T cells. On the day of analysis, cells were harvested and stained by titration of each anti-OX40 antibody. Bound antibodies were detected with a fluorescently conjugated anti-human IgG polyclonal secondary antibody and the cells were co-stained for CD4 and CD25 to detect activated CD4 T cells. Staining fluorescence intensity was measured using a FACSCanto II flow cytometer (Becton Dickinson). For the CD4+ CD25+ population (FACSDiva software), geometric mean fluorescence intensity (GMFI) or mean fluorescence intensity (MedFI) of anti-OX40 staining was calculated. EC ₅₀ for antibody binding was calculated using GraphPad Prism software. Dose-response curves were generated and EC ₅₀ for antibody binding was calculated using GraphPad Prism software.

As shown in FIG. 12A and 12B, the anti-OX40 antibodies tested bind with high activity to activated cynomolgus monkey CD4 T cells with an EC ₅₀ ranging from 0.068 nM (20C1) to 1.4 nM (20B3). 18E9 and 20B3 bound to EC _50s from 1 to 1.5 nM, while the rest of the antibodies bound to EC _50s below 1 nM. OX40.1 showed the least binding among the anti-OX40 antibodies examined in FIG. 12A and OX40.5 showed the least binding among the anti-OX40 antibodies examined in FIG. 12b. The same experiment was carried out with antibodies to OX40 with substitutions in the variable region. As shown in FIG. 12C, antibodies OX40.6, OX40.8 and OX40.21 showed the highest binding efficiency with an EC ₅₀ of 0.12 nM or lower. OX40.1 showed significantly lower binding to cynomolgus monkey CD4 T cells. No binding of the OX40.21 antibody was found on activated mouse or rat CD4+ T cells. A summary of the values of EC ₅₀ is presented in table. 8 below.

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Table 8

EC50 T cell binding cynomolgus macaque (nM) Name (mean ± SD) η 3F4 0.37 ± 0.13 4 8B11 0.20 ± 0.08 4 18E9 41.70±37.78 4 20VZ 1.18±0.27 4 20С1* 0.17±0.07 4 23H3 0.18±0.10 4 6E1 0.77±0.14 4 14A2 0.27 ± 0.02 4 14V6 0.31 1 0X40.6 0.11 ± 0.01 5 0X40.8 0.10±0.02 4 0X40.16 0.06 ± 0.01 3 0X40.17 0.52 ± 0.12 3 0X40.18 0X40.21 0.07±0.01 5 OH40.1 23.40 ± 37.75 4 0X40.4 1.3 1 0X40.5 ~2.2е+012 1

Example 4 Scatchard Analysis of Anti-OX40 Antibodies Binding to Activated Primary T Cells and Cells Overexpressing Human and Cynomolgus OX40.

Binding of OX40.21 (IgG1 isotype) to activated human T cells was further assessed using the Scatchard assay. Briefly, OX40.21 was labeled with radioactive iodine ¹²⁵ I-Na (1mCi, PerkinElmer Catalog NEZ033H001 MC) using IODO-GEN® solid phase iodination reagent (1,3,4,6-tetrachloro-3a-6a-diphenylglycoluril, Pierce Catalog 28601 ). Activated human CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMC), donor W326470, purchased from Stanford Blood Bank. CD4+ T cells were isolated by negative selection (RosetteSep™ human CD4+ T cell enrichment cocktail, StemCell Technologies Catalog 15062) and freezing. The isolated CD4+ T cells were activated for four days prior to the binding assay to induce OX40 expression as follows. Thawed cells were cultured for four days with magnetic beads coated with anti-human CD3 plus anti-human CD28 (human T-Expander CD3/CD28 Dynabeads, Invitrogen Catalog 111.41D) at a bead ratio of 1:1 in the presence of 200 IU/ml recombinant human IL-2 ( Peprotech Catalog 200-02).

Binding of radioiodine labeled OX40.21 IgG1 to activated human T cells was demonstrated by incubating activated human T cells with a titration of ¹²⁵ I-OX40.21 IgG1. Non-specific binding was determined by binding in the presence of titration with a 100-fold molar excess of unlabeled antibody and subtracted from the total CPM to calculate specific binding. A linear standard curve of ¹²⁵ I-OX40.21 IgG1 concentration versus CPM was used to extrapolate specific activity, maximum bound ¹²⁵ I-OX40.21 IgG1 in nM, and hence calculate the number of receptors per cell.

As shown in Table. 9 and in FIG. 13A, rich OX40.21 IgG1 binding was observed on activated human T cells endogenously expressing OX40 with a KD of 0.05 nM for each of the two T cell donors.

- 101 040561

Table 9 Binding of ¹²⁵ 1-OX40.21 to activated human T cells

Concentration- General binding non-specific specific tion labeled 125j binding binding antibodies labeled 125j labeled 125j antibodies (nM) (nM) antibodies (nM) antibodies (nM) 10 0.0317 0.0292 0.0120 0.0117 0.0197 0.0175 5 0.0283 0.0275 0.0073 0.0072 0.0210 0.0204 2.5 0.0230 0.0256 0.0036 0.0030 0.0195 0.0226 1.25 0.0205 0.0221 0.0029 0.0025 0.0176 0.0196 0.625 0.0197 0.0223 0.0014 0.0017 0.0183 0.0207 0.3125 0.0197 0.0229 0.0013 0.0012 0.0184 0.0217 0.15625 0.0177 0.0201 0.0012 0.0011 0.0165 0.0190 0.078125 0.0140 0.0152 0.0010 0.0011 0.0129 0.0141 0.039063 0.0083 0.0091 0.0007 0.0010 0.0076 0.0081 0.019531 0.0057 0.0057 0.0009 0.0010 0.0049 0.0047 0.009766 0.0024 0.0030 0.0007 0.0008 0.0017 0.0022

The same assay was performed using HEK293 cells overexpressing human 0X40 (hOX40-293). Briefly, binding of radioiodine labeled 0X40.21 to overexpressed human OX40 was demonstrated by incubating 110X40-293 cells with a titration of ¹²⁵ 1-OX40.21. Non-specific binding was determined by binding in the presence of titrating a 100-fold molar excess of unlabeled antibody and subtracting from the total CPM to calculate specific binding. A linear standard curve of ¹²⁵ I-OX40.21 IgGl concentration versus CPM was used to extrapolate specific activity, maximum bound ¹²⁵ I-OX40.21 IgGl in nM and thus calculate the number of receptors per cell. As shown in FIG. 13B and in table. 10, saturation binding of 0X40.21 IgGl was observed for binding to 0X40 expressed on 110X40-293 cells. The mean K _D value for binding from the two test conditions using different numbers of hOX40-293 cells per sample was 0.22 nM.

Table 10 Binding of 0X40.21 to 110X40-293 cells

Antibody concentration (nM) Total binding of labeled ¹²⁵ 1 antibody (nM) Non-specific binding of labeled ¹²⁵ 1 antibody (nM) Specific binding of labeled ¹²⁵ 1 antibody (nM) 10 0.1866 0.1712 0.0129 0.0137 0.1737 0.1575 5 0.1800 0.1710 0.0080 0.0099 0.1720 0.1611 2.5 0.1799 0.1643 0.0065 0.0065 0.1734 0.1578 1.25 0.1722 0.1628 0.0057 0.0054 0.1665 0.1574 0.625 0.1583 0.1436 0.0067 0.0048 0.1515 0.1388 0.3125 0.0986 0.0936 0.0038 0.0044 0.0948 0.0891 0.15625 0.0624 0.0501 0.0035 0.0048 0.0589 0.0453 0.078125 0.0351 0.0289 0.0035 0.0033 0.0316 0.0255 0.039063 0.0211 0.0162 0.0038 0.0027 0.0173 0.0136 0.019531 0.0117 0.0091 0.0029 0.0028 0.0088 0.0063 0.009766 0.0075 0.0056 0.0027 0.0028 0.0048 0.0028

The same assay was performed using CHO cells overexpressing cynomolgus monkey 0X40 (supoOX40-CHO). Briefly, binding of radioiodine labeled 0X40.21 to cynomolgus monkey 0X40 was demonstrated by incubating supoOX40-CHO cells with I-OX40.21 titration. Non-specific binding was determined by binding in the presence of titrating a 100-fold molar excess of unlabeled antibody and subtracting from the total CPM to calculate specific binding. A linear standard curve of I-OX40.21 IgGl concentration versus CPM was used to extrapolate the maximum bound ¹²⁵ I-OX40.21 IgGl in nM and thus calculate the number of receptors per cell. As shown in FIG. 13C and in table. 11, saturation binding of 0X40.21 IgGl was observed for binding to cynomolgus monkey 0X40 expressed on supoOX40-CHO cells.

- 102040561

The mean KD value for binding from the two test conditions using different numbers of cells per sample was 0.63 nM.

Table 11 Binding of OX40.21 to cynoOX40-CHO cells

Antibody concentration (nM) Total labeled antibodies th binding ¹²⁵ 1 a (nM) Non-specific binding Specific binding of labeled ¹²⁵ 1 antibody (nM) labeled antibodies (ι ¹ HgM) 20 0.1781 0.1814 0.0266 0.0414 0.1515 0.1400 10 0.1768 0.1651 0.0161 0.0197 0.1607 0.1454 5 0.1629 0.1820 0.0109 0.0171 0.1520 0.1649 2.5 0.1665 0.1659 0.0080 0.0092 0.1586 0.1567 1.25 0.1197 0.1839 0.0079 0.0084 0.1117 0.1755 0.625 0.0892 0.1197 0.0060 0.0074 0.0832 0.1123 0.3125 0.0630 0.0754 0.0057 0.0053 0.0573 0.0701 0.15625 0.0318 0.0437 0.0049 0.0050 0.0269 0.0386 0.078125 0.0158 0.0212 0.0030 0.0034 0.0128 0.0179 0.039063 0.0082 0.0110 0.0027 0.0029 0.0055 0.0082 0.019531 0.0058 0.0058 0.0026 0.0023 0.0032 0.0035 0.009766 0.0030 0.0032 0.0022 0.0021 0.0009 0.0011

Example 5 Specific binding of anti-OX40 antibodies to lymphocytes.

The specificity of various OX40 antibodies was examined in a panel of 22 types of normal human tissues, including spleen, tonsil, thymus, brain, cerebellum, heart, liver, lung, kidney, pancreas, pituitary gland, peripheral nerves, stomach, large intestine, small intestines, thyroid, skin, skeletal muscle, prostate, uterus, testicles, and placenta, via immunohistochemistry.

Fresh, frozen and/or OCT-embedded human tissues were purchased from several commercial tissue networks/suppliers (Asterand Inc. Detroit, MI; Cooperative Human Tissue Network, Philadelphia, PA; ProteoGenex Inc, Culver City, CA). To determine tissue binding, a number of anti-OX40 antibodies (OX40.6-FITC, OX40.8-FITC, 6E1-FITC, OX40.16-FITC, OX40.17-FITC, OX40.20-FITC and OX40.21FITC) were labeled with fluorescein and was applied to acetone-fixed cryostatic sections followed by an anti-FITC bridging antibody and imaging with the EnVision+ system. An isotype control antibody was used as non-specific fluorescein-labeled human IgG1. HT1080 cells stably expressing human OX40 (HT1080/huOX40) and hyperplastic sections of human tonsil tissue were used as positive control cells and tissues. To determine if FITC conjugation has any effect on binding properties, both FITC-conjugated and unconjugated anti-OX40 antibodies were compared in HT1080/huOX40 cells using anti-huIgG as a bridge antibody. Stained sections were evaluated under a light microscope.

Initial studies have shown that both unconjugated and FITC-conjugated anti-OX40 antibodies specifically stain the cytoplasm and membrane of transfected human OX40 cells, but not native HT1080 cells. There were no differences between unconjugated and FITC-conjugated anti-OX40 antibodies. These results indicate that the antibodies were suitable for immunohistochemistry analysis and that FITC conjugation did not affect tissue binding properties.

All tested anti-OX40 antibodies showed positive staining in a small subset of both scattered and small clusters of mononuclear cells (MNC) in lymphoid tissues (tonsil, spleen, and thymus) and lymphocyte-rich tissues (colon, stomach, and small intestine). as well as several scattered MNCs in several tissues (lung, skin, and thyroid). Based on morphology, these positive cells are primarily lymphocytes.

In addition to staining a subset of lymphocytes, the ligand-blocking antibody OX40.6 showed strong staining in subsets of the endothelial/subendothelial matrix and interstitial elements, more commonly associated with small arteries and adventitia of the vessel and its surrounding connective tissues, of virtually all tissues examined (Fig. 14A). , as well as specialized interstitial tissue elements, such as the sheath interstitium that surrounds the seminiferous tubule in the testis. Antibody OX40.8, a non-blocking ligand, positively methylated myofilament-like structures in cardiac muscle (Fig. 14A) and mesangial-like cells in glomeruli

- 103 040561 kidneys. Staining with another non-blocking ligand, ie, 6E1 antibody, also revealed staining in cardiac muscle cells, as well as in neurons and neuropils of the brain and cerebellum, and a subset of tubular epithelial cells in the kidney. In general, non-lymphocyte staining was only detected when antibodies were used at relatively high concentrations (3 or 5 µg/mL), but not at lower concentrations (1 µg/mL), indicating low binding affinity or potential off-target binding.

Further examination of other ligand-blocking antibodies, OX40.16 (FIG. 14A) and OX40.17, showed clear staining of a subset of lymphocytes without any specific staining of other tissue elements for all tissues examined. Antibody OX40.21, a variant of antibody OX40.16, had a similar binding profile as antibody OX40.16 (Fig. 14B). Immunohistochemistry in a similar panel of normal cynomolgus monkey tissues showed very similar staining in humans, demonstrating the usefulness of the cynomolgus monkey as a relevant preclinical species.

Example 6: Expression of OX40 in malignant tumors.

FFPE tumor tissue samples (formalin-fixed, paraffin-embedded) were purchased from commercial tissue dealers (n = 12-20 for each tumor type). To detect tissue binding, an automated IHC assay was developed with a commercial anti-human OX40 antibody using the Leica BondRX platform. Briefly, heat-induced antigen (HIER) preparation was performed in ER2 pH9 buffer (Leica) for 20 min at 95°C. Anti-human OX40 mouse monoclonal antibody clone ACT35 (BD Pharmingen) was incubated at 5 μg/mL for 60 min followed by NovolinkMax polymer (Leica) for 30 min. Finally, the sections were exposed to the substrate DAB-chromogenic solution for 6 min, counterstained with Mayer's hematoxylin, dehydrated, cleaned and plated with Permount. The Dako protein block was used as a diluent for the primary antibody.

For TIL profiling, commercially available monoclonal antibodies to CD3 (T-cell marker) and to FoxP3 (Treg marker) were used to stain adjacent sections. Commercial mouse IgG1 was used as a negative control and hyperplastic human tonsil tissue was used as a positive control. After immunostaining, sections were manually assessed under a light microscope.

In the four tumors studied, CD3+ TILs were present in all samples studied, with the amount of TILs varying within the samples and the distribution within the same tissue being heterogeneous. In some cases, TILs were more widely distributed across the tumor and host region, as expected. Most TILs were localized to the stromal tumor in most tissue samples. However, in many cases they were easily found in intratumor nests. Positive OX40 staining was observed in a small proportion of TILs and was mainly distributed in the tumor stroma. In general, the abundance of OX40+ TIL was proportional to that of CD3+ TIL. Among the four OX40+ tumor types studied, TILs were more numerous in HCC and CRC (FIGS. 15A-15C).

Example 7 Anti-human OX40 monoclonal antibodies that block the binding of OX-40L to OX-40.

Several anti-OX40 antibodies were tested for their ability to block the binding of recombinant soluble OX40L to OX40-transfected 293 human cells. Briefly, 293 cells stably transfected with human OX40 were first preincubated with various concentrations of anti-OX40 antibodies. A fixed concentration (0.2 μg/mL) of recombinant soluble his labeled human OX40L (OX40L-His, R&D Systems) was then added and the samples were further incubated. After cell washing, bound OX40L-His was detected using APC's own labeled His-tagged antibody. Staining fluorescence intensity was measured using a FACSCanto II flow cytometer (Becton Dickinson). The geometric mean fluorescence intensity (GMFI) of the APC-anti-His/OX40LHis antibody staining for the cell population was calculated (FACSDiva software). Dose response curves and EC ₅₀ for antibody blocking OX40L binding were generated using GraphPad Prism software; EC ₅₀ are presented in table. 12.

Table 12. EC50 values for blocking OX40L/OX40 interaction measured by FACS

Antibody clone EC50 (nM) 14В6.С5.С8 1.0

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3F4.G11.D2 0.54 8В11.Н9.С1 0.45 18E9.G5.H4 0.48 20B3.G12.A2 0.93 20C1.F2.D1 0.34 6E1.A12.A2 no blocking 23H3.C6 0.38 0X40.4 no blocking 0X40.5 ~ 1.2е+013

As shown in FIG. 16, most of the anti-OX40 antibodies tested completely blocked the binding of soluble human OX40L to human OX40 on the surface of transfected cells, with the exception of 6E1, OX40.4, and OX40.5. Inadequate blocking of OX40.5 may be due to lower binding efficiency to human OX40 than other tested antibodies, or binding to an overlapping but different epitope. In contrast, 6E1 and OX40.4 did not block the binding of human OX-40L to OX40. This lack of blocking is likely due to binding to a different epitope than the rest of the antibodies.

Example 8 Antibody Competition/Sorting.

Antibody sorting experiments were performed as follows. One or more anti-OX40 antibodies were coated directly onto the Biacore CM5 chip using amine coupling chemistry. Anti-OX40 antibodies serially diluted (1:3) from an initial concentration of 60 μg/ml were incubated with 20 nM of OX40-6X-His antigen for at least 1 hour. The incubated complex was passed through antibody-bound surfaces and observed for cross blocking. . The study was repeated with several antibodies on the surface to create an epitope map based on mutual cross-blocking of all antibodies. The surface of OX40L was also coated to identify and sort out antibodies that are capable of blocking the OX40-OX40L interaction. The experiments were carried out on SPR Biacore T200 or Biacore 3000 instruments.

As summarized in FIG. 17, antibodies 20C1, 20B3, 8B11, 23H3, 18E9, 14B6, OX40.1 and OX40.2 were ligand blockers; the 3F4 antibody was a partial ligand blocker; and 14A2, 6E1 and OX40.5 were non-ligand blockers.

Example 9 Biophysical properties of OX40 antibodies.

The affinity of several anti-OX40 antibodies to soluble human OX40 was examined by SPR analysis. Briefly, affinity measurements were performed by capturing 1-10 μg/ml of the appropriate antibody on a CM5 chip coated with anti-human CH1. The human OX40-6XHIS antigen was used either at a single concentration of 400 nM or in a 1:2 serial dilution from 400 nM. Experiments were performed on SPR BIACORE® T200 or BIACORE® 3000 instruments. Data were fitted to the model 1:1.

As shown in Table. 13, the studied antibodies to OX40 were characterized by dissociation constants (KD) in the range from 10 ^-8 to 10 ^-9 M.

Table 13. K _D values for OX-40 antibodies

Clone K _D (M) k-on (1/ms) k-off (1/s) 3F4 7.13 e-9 5.31 e4 3.79 e-4 8B11 1.05 e-8 4.8 e4 5.1 e-4 14V6 8.84 e-9 7.4 E4 6.54 e-4 6E1 1.1 e-8 1.28 e5 1.41 e-3 14A2 1.51 e-9 1.46 e5 2.2 e-4 18E9 2.04 e-9 6.83 E4 1.39 e-4 20VZ 3.71 e-9 5.42 e4 2.01 e-4 23H3 3.6 e-9 1.1 E5 3.95 e-4 20С1 3.22 e-9 6.48 E4 2.09 e-4 0X40.21 1.49 e-9 9.41e+5 0.0014

The thermal stability of the OX40.21 antibody was also investigated, the results are summarized in Table 1. 14. Thermal stability was determined using GE Healthcare CAP-DSC. Samples were run at a concentration of 250 μg/ml in PBS. The scanning frequency was 60°C/h. Data fit

- 105 040561 for a non-bi-state model. The OX40.21 antibody was determined to be one of the more stable antibodies tested when considered along with other features (eg, low off-target effects, immunogenicity, etc.).

Table 14

Clone Tsh1 Tsh2 TshZ % reversibility 3F4 68 83 8B11 72.7 82.9 14V6 66.3 70.5 18E9 65.8 71.2 23H3 72.3 82.7 20С1 68.0 83.0 0X40.21 72.2 79.5 48% at 80°C

The pharmacokinetics of the OX40.21 antibody was also studied after a single intravenous injection in cynomolgus monkeys. The OX40.21 antibody showed acceptable pharmacokinetic (PK) properties after a single intravenous (IV) administration to cynomolgus monkeys with a linear PK (0.4-4 mg/kg) and a long half-life (6 days).

Table 15. Pharmacokinetic parameters of OX40.1 after intravenous administration in cynomolgus monkeys (N = 3)

Dose (mg/kg) AUC(INF)* (mcg/ml day) tl/2 (day) CLT (ml/h/kg) Vss (ml/kg) 0.4 86+5 5.6 + 0.5 0.20 + 0.01 36+2 4 785+138 6.2±0.6 0.22 ± 0.04 49+12

PK parameters were calculated by the non-compartmental method. Values are mean ± SD.

* PK parameters were calculated using plasma concentration up to 10 days except monkey 2 at 0.4 mg/kg up to 7 days;

*% AUCextra ranged from 24 to 42%.

Human PK parameters for OX40.21 were projected from cynomolgus monkey PK data using allometric scaling (assuming power factor = 0.85 for CLT and 1 for Vss). The projected human t _1/2 was 10 days (Table 16) _. PK parameters were calculated by the two-compartment method.

Table 16. Projected pharmacokinetic parameters of human OX40.21

Dose AUC(INF) CLT Vss

Antibody (mg/kg) (mcg/ml xday) (day) (ml/h/kg) (ml/kg)

0X40.21 k 303 10 0L4 47

Example 10 FACS Cross-blocking of PE Labeled OX40 L106 Antibody Clone with a Panel of Unlabeled Anti-OX40 Antibodies.

Several anti-OX40 antibodies were tested for their ability to block the binding of PE labeled anti-OX40 antibody clone L106 to OX40-transfected 293 human cells. Briefly, 293 cells stably transfected with human OX40 were first incubated with various concentrations of unlabeled anti-OX40 antibodies. The cells were then washed and incubated with a fixed concentration of 2.5 μg/ml of PE labeled L106 antibody (BD Biosciences). Staining fluorescence intensity was measured using a FACS Canto II flow cytometer (Becton Dickinson). The geometric mean fluorescence intensity (GMFI) of PE-L106 antibody staining for the cell population was calculated (FACSDiva software). Dose-response curves for blocking L106 binding were generated using GraphPad Prism software.

As shown in FIG. 18, antibodies 18E9 and OX40.1 completely blocked the binding of L106 to cells transfected with human OX40, while 20B3 showed partial blocking. The rest of the antibodies were virtually unblocked, indicating that 18E9 and OX40.1 bind to a different epitope on OX40 than the other antibodies tested.

Example 11 Anti-OX40 Antibody Cross Blocking Assay.

This experiment was performed to test the cross-blocking properties of various anti-OX40 antibodies to assess binding specificity. Briefly, the OX40 antibody OX40.1 was conjugated with allocycocyanin (APC) and the human OX40 antibodies OX40.4 and OX40.5 were biotinylated. Panel

- 106 040561 unconjugated anti-human OX40 antibodies were dosed to engineered 293 or HT1080 cell lines that overexpress human OX40 protein on their surface and allowed to bind at 4°C for 30 minutes. Without washing off the unconjugated antibody, APC-OX40.1 (1 µg/mL), 6-iotin-OX40.4 (0.4 µg/mL) or 6-iotin-0X40.5 (0.4 µg/mL) was applied to the assay wells and allowed to bind at 4°C for 30 min. Cells were washed and, if necessary, additionally incubated in the presence of streptavidin-APC conjugate under the same conditions. After the final wash, the cells were analyzed on a FACSCanto flow cytometer (BD Bioscience, San Jose, CA). The mean fluorescence intensity (MFI) signal was proportional to the bound conjugated antibody.

As shown in FIG. 19A-19C, binding of APC-OX40.1 to cells overexpressing human OX-40 protein was blocked by OX40.2 and OX40.5, but only slightly, if at all, by OX40.4. APC-OX40.1 binding was blocked by 8B11.H9, 3F4.G11, 20B3.G2 and 14B6.C5, but not by 6E1.A12 and 14A2.B9. A diagram of observed binding relationships between the antibodies evaluated in FIG. 19A19C is shown in FIG. 19h.

In FIG. 19D-19E shows that biotin-OX40.4 binding was strongly blocked by 20B3.G2, moderately blocked by 20C1.F2, weakly, if at all, blocked by 3F4.G11 and 23H3.C6, and not blocked by 14A2.B9. Biotin-OX40.5 binding was strongly blocked by 20B3.G2, 23H3.C6, and 20C1.F2, moderately blocked by 3F4.G11, and weakly blocked by 14A2.B9.

In FIG. 19F-19G show that biotin-OX40.4 binding was not blocked by OX40.1 or OX40.8 and was weakly, if at all, blocked by OX40.5 or OX40.6. Biotin-OX40.5 binding was blocked by OX40.1, moderately blocked by OX40.6, and very weakly, if at all, blocked by OX40.4 or OX40.6.

The plot of observed binding relationships between antibodies, evaluated in FIG. 19D-19G is shown in FIG. 19I.

Example 12 Anti-OX40 antibodies bind to conformational epitope/epitope mapping.

This example shows that OX40.21 binds to undenatured human OX40 but not to denatured human OX40, and this binding is independent of N-glycosylation.

Binding of OX40.21 to native or denatured OX40, whether or not it contains N-linked glycosylation, was determined as follows. Samples of native (ie, undenatured) and denatured human OX40 were incubated with or without the enzyme PNGase F N-glycanase to remove N-glycosylation. Natural human OX40 samples with or without N-linked glycosylation were subjected to SDS gel electrophoresis, and denatured human OX40 samples with or without N-linked glycosylation were subjected to denaturing SDS gel electrophoresis.

As shown in FIG. 20A, OX40.21 only binds to natural OX40 and not to the denatured form, and the presence or absence of glycosylation does not affect binding to OX40. In FIG. 20B and 20C show that two N-glycopeptides were identified by peptide mapping after deglycosylation (60% occupancy for AspN118 and AspN12).

These data suggest that OX40.21 binds to an epitope that is conformational and independent of N-linked glycosylation.

Epitope mapping studies have also been conducted using mass spectrometry. Peptide fragments labeled with his OX40 human (hOX40) were obtained by enzymatic cleavage with endoproteinases. LC-MS was performed using an AB Sciex 5600 Triple-TOF.

As shown in FIG. 20D and 20E, binding experiments to native hOX40 by limited proteolysis showed that OX40.16 and OX40.21 bind preferentially to the DVVSSKPCKPCTWCNLR peptide (SEQ ID NO: 178), which corresponds to amino acids 46-62 of the extracellular portion of mature human OX-40. (SEQ ID NO: 2). The OX40.8 antibody bound to the DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK peptide (SEQ ID NO: 179), which corresponds to amino acids 89-124 of the extracellular portion of mature human OX40 (SEQ ID NO: 2). The location of the OX40.21-bound epitope overlaps a portion of the OX40 ligand binding site as determined by the crystal structure of the human OX40/OX40L complex (Protein Structure Bank (PDB) identification code 2HEV). Additional peptides identified by mass spectrometry for OX40.21 are shown in the top panel of FIG. 20E and include QLCTATQDTVCR (SEQ ID NO: 184), SQNTVCRPCGPGFYN (SEQ ID NO: 185), SQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLR (SEQ ID NO: 182), and PCKPCTWCNLR (SEQ ID NO: 183).

Example 13 Antibodies to OX-40 promote T cell proliferation and induce secretion of IFN-γ and IL-2 from T cells.

Anti-OX-40 antibodies were tested for their ability to induce T cell activity in vitro by measuring the proliferation and amount of IL-2 and IFN-γ released by T cells incubated with the antibodies.

- 107 040561

The transfected CHO cell line was prepared for use as artificial antigen presenting cells in the primary T cell activation assay. The CHO-CD3CD32A cell line expresses the anti-human CD3 antibody in single chain Fv format together with the human CD32A Fc receptor to present the anti-OX40 antibody on the surface of CHO cells. Briefly, primary human CD4 T cells were isolated by negative selection (RosetteSepTM, StemCell Technologies) and co-cultured with irradiated CHO-CD3-CD32A cells at a T:CHO ratio of 8:1 in the presence of graduated doses of anti-OX40 antibodies or an isotype control antibody. After 3-4 days in culture at 37° C., supernatants were harvested to assess T-cell activation by measuring secreted human IFNy either by ELISA (BD Biosciences) or HTRF (Cisbio) following manufacturers' recommendations. Tritiated thymidine was then added during the final approximately 18 hours of culture to measure T cell proliferation by including tritiated thymidine as an additional measure of T cell activation.

As shown in FIG. 21A-21D and 22A-22D (and summarized in Table 17 below), most of the anti-OX40 antibodies tested strongly potentiated the activation of human CD4 T cells stimulated by CHO-CD3-CD32 cells in a dose-dependent manner, as measured by IFNy proliferation and secretion. The panel of antibodies tested in this assay co-stimulated T cell activation at least as well as or even better than OX40.1, OX40.4 and OX40.5.

Table 17

Name ECso (nM) of proliferation (mean ± SD) η IFNy EC ₅ o (nM) (mean ± SD) η 3F4 0.016±0.008 5 8B11 0.022 ± 0.027 3 18E9 0.010±0.005 3 20VZ 0.008 ± 0.003 3 20С1 0.008 ± 0.006 4 23H3 0.028 ± 0.017 3 6E1 0.014±0.008 2 14A2 0.037 ± 0.044 4 14V6 0.012±0.008 3 0X40.6 0.032 ± 0.028 2 0.033 ± 0.004 2 0X40.8 0.043 ± 0.037 2 0.024 1 0X40.16 0.017 1 0.044 1 0X40.17 0.009 ± 1 0.044 1 0X40.18 0.230± 1 0.490 1 0X40.21 0.011±0.006 9 0.043 ± 0.023 9 OH40.1 0.024±0.012 4 0X40.4 0.094 1 ~ 2.3 e+009 1 0X40.5 1,900 1 ~ 37 1

Antibodies to human OX40 have also been investigated for their effect on primary T cell stimulation in cultures activated with enterotoxin B (SEB) human peripheral blood mononuclear cells (PBMC). Whole human blood was obtained from AllCells, Inc. (Berkeley, CA) or from donors at Bristol-Myers Squibb, Redwood City, CA through their own phlebotomy program. PBMCs were isolated by ficoll gradient purification and cultured for 3 days in culture medium supplemented with fixed suboptimal (85 ng/ml) Staphylococcus enterotoxin B superantigen (SEB; Toxin Technologies, Sarasota, FL) in the presence of graduated doses of OX40 antibodies or isotype controls. antibodies together with 2-5 μg/ml soluble cross-linking antibody, goat F(ab') ₂ to human Fcy. After culturing for 3 days at 37° C., supernatants were harvested to assess T cell activation by human secreted IL-2 ELISA assay.

Briefly, culture supernatants were diluted 1:10 in sample diluent and tested for human IL-2 by ELISA (BD Bioscience) according to the manufacturer's recommended protocol. After addition of TBM substrate, the assay plates were read on a Spectramax 340PC reader using Softmax software at 650 nm. The measured optical densities of the chromogenic substrate were proportional to the bound detection antibody.

Data from PBMCs isolated from different donors are shown in FIG. 23A-23F. In general, dissolve

- 108 040561 My cross-linked clone 20C1.F2 caused a more stable cytokine response (EC50 1.3 - 2.0 nM) compared to soluble cross-linked clones 23H3.C6, 8B11.H9, 3F4.G11, 18E9.G5, 6E1 .A12 and 20B3.G2 (FIGS. 23A-23C). For antibodies with variable region mutations, OX-40.21 generally elicited a more robust cytokine response compared to soluble cross-linked antibodies OX-40.17, OX40.18, OX40.6, and OX40.8 (Fig. 23D-23F) . Data from these donors, together with data from 8 additional donors in which additional anti-OX40 antibodies were tested, collectively demonstrate that, on average, OX40.21 exhibits superior efficacy in increasing T cell responses compared to OX40.1, OX40.2, OX40.4, OX40.5, OX40.17 and OX40.18. These results again show that OX40.21 elicits responses that are comparable to those elicited by OX40.6 and OX40.8 (Table 18).

Table 18

Donor number 0X40.21 0X40.17 0X40.18 0X40.6 0X40.8 BMS-009 EC50 (nM) 0.34 0.97 3.21 0.45 0.50 BMS-O12 EC50 (nM) 0.29 1.74 -1.67 0.66 0.09 BMS-016 EC50 (nM) 0.04 1.40 >100 0.29 0.09 Donor number 0X40.21 0X40.17 0X40.18 0X40.6 0X40.8 OX40.1 0X40.2 0X40.4 0X40.5 WB10024 ^EC5i (uM) 0.82 1.06 -3.17 Ό.42 Ό.44 1.87 2.53 1.25 -1.83 WB10025 EC50 (nM) 1.31 1.35 - 1.14 Ό.85 0.79 2.08 '3.26 1.08 3.07 WB10026 EC50 (nM) 0.79 1.68 3.02 0.28 0.63 2.24 3.16 1.14 2.59 WB10027 ^E C50 (nM) 1.08 1.15 '3.27 0.45 Ό.46 1.26 2.26 0.93 '3.09 Donor number 0X40.21 0X40.17 0X40.18 0X40.6 0X40.8 OX40.1 0X40.2 0X40.4 0X40.5 WB10137 EC50 (nM) 0.56 0.92 >100 0.56 0.42 Ί.824 >100 1.37 >100 BMS-OO1 EC50 (nM) 0.41 0.41 '84.76 0.49 ' 0.42 0.64 4.95 ~ 0.89 ~ 0.43 BMS-004 ^EC5 °( ^4m ) Ό.84 1.03 2.43 0.48 0.55 2.52 13.89 0.84 1.94 BMS-015 EC3U (nM) 0.76 1.16 >100 Ό.46 Ό.42 2.41 >100 0.91 1.92 Mean EC50 (nM) 0.64 1.17 2.89 0.46 0.44 1.86 5.36 1.07 2.38

* Each set of experiments was performed on different days.

Example 14 Promotion of anti-OX40 antibody to NK92 mediated cell lysis using cell lines.

Several anti-human OX40 antibodies have been investigated for their ability to promote NK92-mediated lysis of activated CD4+ T cells using calcein release as a readout. Briefly, CD4+ T cells for use as target cells were separated by negative selection using magnetic beads and activated for 72 hours with anti-CD3 and anti-CD28 coated beads. Three days later, NK92 cells were plated with calcein-labeled AM activated CD4+ cells at a ratio of 5 to 1. A titration of each anti-OX40 antibody was then added and the cells were incubated for two hours. Calcein release was measured by reading the fluorescence intensity of the medium using an Envision plate reader (Perkin Elmer). The percentage of antibody-dependent cell lysis was calculated based on the mean fluorescence intensity (MFI) using the following formula:

(explored MFI - average background) / (average maximum - average background)] x 100.

As shown in FIG. 24, OX40.8 and OX40.16 caused the highest amount of specific lysis of target cells (60 and 30%, respectively). The EC ₅₀ of OX40.8 was 16 ng/mL and for OX40.16 it was 4 ng/mL. All other antibodies tested elicited ADCC at levels too low to accurately quantify.

Example 15 Promotion of anti-OX40 antibody to NK-mediated cell lysis of primary human CD4+ T cells.

Several anti-human OX40 antibodies have been investigated for their ability to promote primary NK-mediated lysis of activated CD4 ⁺ T cells. Briefly, CD4 ⁺ T cells for use as target cells were separated from PBMCs from two donors by magnetic selection and activated for 72 h with anti-CD3 and anti-CD28 coated beads. NK cells for use as effectors were separated from a single donor by negative selection using magnetic beads and activated with IL-2 for 24 h. After the activation period, NK effector cells were mixed with calcein-labeled target T cells in a ratio of 20:1, 10: 1 or 5:1 in the presence of antibody at a concentration of 1 μg/ml for 2 h. The level of calcein secreted by lysed target cells was measured by reading the fluorescence intensity of the medium using an Envision plate reader (Perkin Elmer).

The percentage of antibody-dependent cell lysis was calculated based on the mean fluorescence intensity (MFI) using the following formula:

[(tested MFI - mean background)/(mean maximum - mean background)] x 100.

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As shown in FIG. 25A and 25B, activated target CD4+ T cells from two donors were most efficiently lysed with OX40.8. Lower levels of ADCC activity were observed with both OX40.21 and OX40.1.

Example 16 Promotion of anti-OX40 antibody to macrophage-mediated cellular phagocytosis of OX40-expressing HEK293 cells.

To determine the antibody-mediated phagocytic activity of several anti-OX40 antibodies, primary human macrophages were cultured for four hours with CellTrace Violet labeled HEK293/OX40 cells and titrated with anti-OX40 antibodies. Four hours later, cells were harvested, stained with anti-CD64-APC and run on a flow cytometer. Cells that were double positive for CD64 and CellTrace Violet were considered phagocytosed. The percentage of phagocytosed target cells was calculated using the formula: 100 x (double positive cells/total CellTrace Violet positive cells).

As shown in FIG. 26, all anti-OX40 antibodies tested induced phagocytosis of OX40-expressing target cells in a dose-dependent manner. OX40.8 was characterized by the highest overall level of phagocytosis and the lowest concentration of EC ₅₀ 6.2 ng/ml. This demonstrates that anti-OX40 human IgG1 antibodies induce FcR-mediated phagocytosis in a dose-dependent manner.

Example 17 Antibodies to OX40 bind the C1q component of human complement.

A colorimetric ELISA was developed to assess whether the C1q component of human serum complement binds to the OX40.21 antibody. A high binding immunoassay plate was coated with all test antibodies at a concentration of 10 μg/ml. After blocking unoccupied protein binding sites, graduated doses of human C1q (3.125-200 μM) were added to the wells, including blocked blank wells, which served as a control group for non-specific background C1q binding to the assay plate. C1q binding to immobilized antibodies was detected using a combination of biotinylated mouse anti-C1q antibody and streptavidin-poly-HRP together with tetramethylbenzidine substrate. The results are presented as optical density read at 450-630 nm.

As shown in FIG. 27, C1q bound to OX40.21 (solid squares) and human IgG1 isotype control (open circles) in a dose dependent manner. However, the level of C1q binding to OX40.21 was lower than with the IgG1 isotype control antibody. As expected, there was little background signal (gray circles) and no clear binding of C1q to the IgG1 isotype control. 1 (solid black circles). The IgG1.1 antibody contains five mutations in the Fc portion designed to eliminate C1q binding and FcR interaction. This result demonstrates that the C1q component of human serum complement can bind to OX40.21 and indicates that OX40.21 can induce complement-mediated lysis of OX40-expressing cells in vivo.

Example 18 OX40 is expressed in tumor infiltrating lymphocytes.

OX-40 is expressed in tumor-infiltrating lymphocytes with a profile that is generally limited to CD4+ cells (Fig. 28A) with minimal expression on CD8+ T cells (Fig. 28B) in colon and rectal, lung, and ovarian cancer (Fig. .28C).

Similarly, OX40 is expressed by CD4+ T cells and Treg in Sa1N mouse tumors (FIG. 28D) and MC38 mouse tumors (FIG. 28E). To test mouse OX-40 expression in tumors, 2x10 ⁶ SA1N sarcoma cells or 2x10 ⁶ MC38 cells were implanted subcutaneously in AJ or B6 mice, respectively. On day 15 post-implantation, tumors were harvested, dissociated into single cell suspensions, and stained for flow cytometry. T cell populations were identified based on their expression of CD8, CD4 and Foxp3. For Sa1N tumors, CD4+ Foxp3+ cells from the tumor are shown in the red histogram, CD4+ Foxp4- cells are shown in the blue histogram, and CD8+ cells are shown in the orange histogram (FIG. 28D). Isotype control-stained cells are shown in the green histogram. For MC38 tumors, Tregs are shown in the blue histogram, CD4+ cells are shown in the green histogram, and CD8+ cells are shown in the red histogram (FIG. 28E).

Example 19 Anti-OX40 Reversal of Treg Cell-Mediated Suppression.

Several anti-human OX40 antibodies have been investigated for their ability to abolish regulatory T cell (Treg) mediated suppression of human CD4+ T cell proliferation. Briefly, Treg cells and immunoreactive T cells (Tresp) were isolated by enriching PBMC with CD4+ cells by magnetic bead separation and then sorting CD4 ⁺ CD25 ^hl CD127 ^l o Treg and CD4 ⁺ CD25 ^1o CD127 ^hl CD45RO ⁺ Tresp cells. Tresp cells were then labeled with proliferation dyes and coated with titrated amounts of Treg cells starting at a 1:1 ratio. Cultures were stimulated with 3 μg/ml plate-bound anti-CD3 antibody, 1 μg/ml soluble anti-CD28 antibody, and 2 μg/ml plate-bound OX40 antibody or isotype control. After 96 hours, Tresp cell proliferation was measured by assessing dye dilution using flow cytometry.

As shown in FIG. 29, in both the presence and absence of Treg cells, anti-OX40 antibodies increased Tresp cell proliferation compared to isotype control. This suggests that

- 110 040561 Investigational Anti-OX40 Antibodies Change the Suppressive Effects of Treg Cells on Cell Proliferation

Tresp.

Example 20 Toxicity studies

OX40.6 (2 mg/kg) was administered intravenously to the monkeys on days 1 (FIG. 30A) and 29 (FIG. 30B) to evaluate any associated toxic effects. There were no signs of tolerability or clinical pathology. OX40.6 stimulated an enhanced immune response to KLH, which tended to upregulate CD69 expression in CD4+ T cells in an ex vivo KLH secondary immune response assay. Two of the four monkeys showed accelerated clearance, which correlated with the formation of antibodies to drugs.

The concentration of 0X40.6 in the cynomolgus monkey serum samples for the experiment above was analyzed by chemiluminescence (CL) enzyme-linked immunosorbent assay. The OX40.6 antibody was used to generate calibrators and quality control (QC) samples. Biotinylated human OX40-his was immobilized on streptavidin-coated microplates (Greiner Bio-one) as a capture molecule for OX40.6. Samples, standards and quality control samples adjusted to a final matrix of 10% cynomolgus serum were incubated on plates. Samples were analyzed at the minimum required dilution of 10% in 1% BSA/PBS/0.05% Tween 20 (PTB) containing 2% mouse serum. The unbound material was washed away and the fixed anti-OX40.6 antibody was detected using an HRP-labeled mouse anti-human IgG monoclonal antibody as the detection molecule. After addition of SuperSignal ELISA Pico (Thermo Scientific) chemiluminescent substrate, OX40.6 concentration in cynomolgus monkey serum samples was calculated from luminescence intensity measured with an M5 plate reader using a 4-parameter logistic (4-PL) calibration curve obtained from OX40.6 antibody calibrators . The range of the calibration curve of the OX40.6 antibody ranged from 5 to 5000 ng/ml in cynomolgus monkey serum. The upper and lower limits of quantitation were 5000 and 10 ng/mL, respectively (ie ULOQ 5000 ng/mL, LLOQ 10 ng/mL). Quality control samples were prepared at 3750, 400 and 20 ng/ml in cynomolgus monkey serum and analyzed on each plate to ensure acceptable assay results. Calibrators, QCs and samples were diluted 5-fold in PTB containing 2% mouse serum. Four streptavidin plates were used for sample analysis. Assay performance was within an acceptable range: %CV between standard plates was below 25% and QC recovery was ±30% of nominal.

The presence of drug antibodies to OX40.6 in the cynomolgus monkey serum in the experiment described above was determined using a bridged electrochemiluminescence (ECL) immunoassay. In particular, a mouse anti-human Fc IgG monoclonal antibody was used to obtain a positive control. A biotinylated anti-OX40:k hOX40-his antibody was used at a concentration of 25 ng/ml as a capture molecule, and a ruthenylated anti-OX40:k hOX40-his antibody was used at a concentration of 25 ng/ml as a detection molecule. Samples were analyzed at 100-fold dilution in 1% BSA/PBS/0.05% Tween 20 (PTB) containing capture and detection molecules. After 2 h incubation in polypropylene plates, the sample mixture was transferred to streptavidin-coated MSD plates. After a one hour incubation, unbound material was washed away, MSD read buffer was added and ECL was measured using an MSD SI6000 plate reader. A positive control (mouse anti-human Fc IgG) was prepared at 1000 (HPC), 100 (MPC) and 10 ng/ml (LPC) in cynomolgus monkey serum. Pooled cynomolgus monkey serum was used as a negative control (NC). The signal ratio for HPC, MPC, and LPC versus NC was 102, 10, and 2, respectively. A single streptavidin plate was used for sample analysis. The performance of the assay was within the acceptable range: % CV PC was below 10%, and the raw signal for the negative control (54 RLU) was comparable to the raw signal for pre-exposure samples (48-55 RLU).

Example 21 Immunogenicity risk assessment study.

In vitro T cell proliferation assays were performed for several anti-human OX40 antibodies to evaluate their potential immunogenicity in humans. Briefly, peripheral blood mononuclear cells (PBMC) from healthy volunteers were isolated by Ficoll (GE Healthcare) and gradient centrifugation, and class II human lymphocytes (HLA) were characterized by polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes (ProImmune).

The analysis used a panel of 40 PBMC donors with HLA class II types that were close to the frequencies of world populations. PBMC were labeled with CFSE (Invitrogen) to control proliferation and seeded in 96-well plates in 6 replicates at 200,000 cells per well in RPMI (Lonzo) containing 10% human AB (Bioreclamation), essential amino acids (Gibco) penicillin-steptomycin (Gibco). Anti-human OX40 antibodies, control proteins, control antibodies, and ConA were cultured with PBMC at 1 μM for 7 days, after which the medium was washed off and the cells were labeled with APC anti-human CD4 monoclonal antibody (BD science). After removal of unbound anti-CD4 antibody with a washing step, the cells were fixed with formalin at a concentration of

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3.7% (Sigma) in PBS and analyzed by flow cytometry to determine the percentage of proliferating CD4+ cells.

The percentage of 40 donors that showed a positive response (defined as a significant increase in proliferating CD4+ T cells compared to those incubated in PBMC medium) for various anti-human OX40 antibodies is shown in FIG. 31. All anti-human OX40 antibody variants showed low potential to activate CD4+ cells in this assay, comparable to low QC protein, with the exception of OX40.16 and OX40.21, which did not respond positively to CD4 proliferation in any of the 40 donors. These results indicate that these anti-human OX40 antibodies have a low potential to induce an anti-drug response in humans.

Example 22 Binding to Activated Fc Receptors Enhances Anti-mOX40 Activity in a Colon Carcinoma Model.

To investigate the role of FcR binding in the activity of anti-mouse OX40 antibodies in mouse tumor models, anti-OX40 antibodies of different isotypes were investigated. C57BL/6 mice were injected subcutaneously with 2 million MC38 tumor cells. After 7 days, tumor volumes were determined and mice were randomized into treatment groups to have comparable mean tumor volumes. Antibodies formulated in PBS were administered intraperitoneally on days 7, 10 and 14 at 200 μg per 200 μl dose.

In synergistic mouse tumor models, anti-mouse OX40 monoclonal antibodies (eg, OX86, rat IgG1) exhibit antitumor activity. Because variation in the isotype of many antibodies specific for T cell surface receptors (both co-stimulatory and co-inhibitory) can alter the antitumor activity of these antibodies, OX86 mouse Fc isotype variants produced an antibody that did not block the OX40/OX40L interaction. As shown in FIG. 32A-32C, OX86 formatted as mouse IgG2a Fc (FIG. 32C) provided superior antitumor activity compared to OX86 formatted as mouse IgG1 (FIG. 32B). This is likely due to both the depletion of Treg cells at the tumor site and the increase in effector T cells from antibody-mediated OX40 agonism.

To confirm the effect of different antibody isotypes on tumor-infiltrating T cell populations, tumors from MC38 mice that had been treated with different isotypes were evaluated by flow cytometry. Selected mice were sacrificed, and tumors and spleens were collected for analysis on the 15th day after tumor implantation. Single cell suspensions were prepared by dissociating the tumor and lymph node with the back of a syringe in a 24-well plate. Cell suspensions were passed through 70 µm filters, pelletized, resuspended and counted. Cells were then plated in 96-well plates with 1x106 cells per staining well. The samples were then analyzed on a FACS Canto (BD) flow cytometer. Analysis of spleens and tumor-bearing mice treated with anti-OX40 antibodies shows that the IgG2a isotype can deplete CD4+ Treg in tumors (Fig. 33A) and that the IgG1 and IgG2a isotypes can activate an increase in T cells in the periphery (Fig. 33B) and lead to an increase in the number of cells in the spleen (Fig. 33C). These results indicate that OX40 agonism (but not necessarily blocking the OX40/OX40L interaction) and Fc receptor binding of the OX86 antibody contributes to antitumor activity.

The role of human Fc and FcR was investigated using mice, where mouse FcR was knocked down and replaced with human FcR. These experiments were performed using a bone marrow chimera system in which host congenic CD45.1 cells were irradiated, then reconstituted with FcR transgenic human bone marrow cells. These mice were then allowed to recover for 8 weeks prior to inoculation with 2x106 MC38 tumor cells. After 7 days, tumor volumes were determined and mice were randomized into treatment groups to have comparable mean tumor volumes. Antibodies formulated in PBS were administered intraperitoneally on days 7, 10 and 14 at 200 μg per 200 μl dose. Mice were treated with either control human IgG1 (FIG. 34A) or human G1 chimeric fusion antibody OX-86 (FIG. 34B), or human G1 OX-86 chimeric fusion antibody S267E (FIG. 34C). The results were similar to those found using mouse isotypes, ie. the human IgG1 antibody had a significant antitumor effect because it can bind to activate FcRs, while the S267E mutation, which increases binding to both CD32B and CD32A, has higher activity (FIGS. 34A-34C). This higher level of activity is likely due to increased agonism on effector T cells as well as increased Treg depletion at the tumor site.

T cell populations in the tumor site and spleens of tumor-bearing mice were examined as previously described. Tregs were less prevalent in mice treated with either the G1 or G1 S267E antibody, with a greater effect seen with the S267E isotype (FIG. 35A). An increase in the percentage of CD8+ T cells (FIG. 35B) and CD4+ effector (FIG. 35C) and tumor site was also evident, and these increases were higher with the S267E G1 antibody. Increased cellularity was also noted in the spleens of mice treated with antibodies to OX-40 (Fig. 35D). These results show that the OX86-hIgG1 antibody exhibited potent antitumor activity (FIGS. 34A-34C) and

- 112 040561 measured Treg depletion (FIGS. 35A-35D).

Example 23 An anti-OX40 blocking antibody exhibits antitumor activity in a mouse tumor model.

Conducted the following experiment to determine whether the antibody, which blocks the interaction between OX40/OX40L, strong antitumor activity. For this purpose, a hamster anti-mouse OX40 antibody that blocks the OX40/OX40L interaction (hamster IgG1 8E5 antibody) was prepared and tested for its antitumor activity in the CT26 mouse subcutaneous tumor model. CT-26 is a mouse colon adenocarcinoma tumor cell line whose solid tumor growth can be controlled in BALB/c mice when the cells are transplanted subcutaneously.

Female BALB/c mice (Charles River Laboratories, Hollister, CA) were acclimatized for at least three days prior to the study. Mice were housed 5 animals per cage, and the cages were placed in a ventilated microisolator. Animals were kept at a temperature in the range of 18-26°C and a relative humidity of 50 + 20% with at least twelve airings per hour. A 12-hour day/night cycle was maintained. Animals received a sanitized laboratory rodent diet and municipal water ad libitum.

CT-26 cells were maintained in RPMI-1640 medium (Hyclone, cat. no. SH30096.01) supplemented with 10% fetal bovine serum (FBS, Hyclone, cat. no. SH30071.03). Approximately twice a week, the cells contained in one T175 flask were divided and diluted to four T175 flasks at a 1:5 dilution until sufficient cells were obtained for tumor implantation. Cells were harvested at approximately 80% confluency, washed and resuspended in PBS.

On day 0, mice were implanted with 1 x 106 CT-26 cells using a 1 ^cc syringe (Becton Dickinson, Franklin Lakes, NJ) and a 5/8 inch 27 gauge needle. Tumors were then measured twice a week in three dimensions with an electronic caliper (Mitutoyo, Aurora, Illinois) and recorded. Tumor volumes (mm ³ ) were calculated using the formula: width x length x height x 0.5. After measuring the tumor volume on the 6th day after implantation, the mice were classified into stages according to the tumor volume. Mice with an average tumor volume of 26 mm ³ randomized into groups and treated as shown in table. 19.

The hamster isotype control antibody is an inert anti-GST Armenian hamster IgG monoclonal antibody (mAb) (clone PIP, catalog # BE0260, BioXcell, West Lebanon, NH). It was prepared in PBS immediately prior to administration to provide a dose of 10 mg/kg per mouse via intraperitoneal (IP) injection on days 6, 10 and 14, as shown in Table 1. 19.

An anti-mouse OX40 monoclonal antibody (clone 8E5) was prepared in PBS immediately prior to administration to give doses of 10, 3, 1, or 0.3 mg/kg per mouse by intraperitoneal injection on days 6, 10, and 13, as shown in Table 1. 19.

Table 19

Impact Dose Number Path Schedule of introductions Isotype control hamster IgG monoclonal antibody 10 mg/kg 12 IP Days 6, 10, 14 Hamster monoclonal antibody to mouse OX40 (clone 8E5) 10 mg/kg 12 IP Days 6, 10, 14 Hamster monoclonal antibody to mouse OX40 (clone 8E5) 3 mg/kg 12 IP Days 6, 10, 14 Hamster monoclonal antibody to mouse OX40 (clone 8E5) 1 mg/kg 12 IP Days 6, 10, 14 Hamster monoclonal antibody to mouse OX40 (clone 8E5) 0.3 mg/kg 12 IP Days 6, 10, 14

Animals were checked daily for postural, hygienic and respiratory changes, as well as for lethargy. Animals were weighed twice a week and sacrificed if the weight loss was >20%. Mice were checked for the presence and size of tumors twice a week until death or euthanasia. Tumors were measured in three dimensions with an electronic caliper (Mitutoyo, Aurora, Illinois) and recorded. The response to the compounds for treatment was measured depending on the growth of the tumor. If the tumor reached a volume > 1500 mm ³ or ulceration appeared, the animals were euthanized.

As shown in FIG. 36A-36E, a hamster anti-mouse OX40 monoclonal antibody (clone 8E5, FIGS. 36B-36E showing exposure to various doses of 8E5) demonstrated potent antitumor activity in the subcutaneous CT-26 model compared to a hamster IgG isotype control group (Fig. 36A). ). The 8E5 antibody was administered at doses ranging from 0.3 to 10 mg/kg and even at the most

- 113 040561 low dose (0.3 mg/kg) 10 of 12 mice were tumor-free (TF) at the end of the study period (day 72). Although the number of tumor-free mice did not differ significantly in each dose group, with 9 or 10 tumor-free mice in each group at the end of the study period, mice receiving one of the two highest doses (3 or 10 mg/kg) showed a greater delay in tumor growth compared to mice treated with the lowest two doses (0.3 or 1 mg/kg). There were no tumor-free mice in the isotype control group; all mice in this group were sacrificed by day 39 as a result of ulceration or tumor burden (>1500 mm ³ ).

These data indicate that an anti-OX40 antibody that blocks the interaction between OX40 and OX40-L exhibits potent antitumor activity in the CT-26 subcutaneous tumor mouse model when administered to mice with established tumors.

Example 24 OX40 agonism acts synergistically with PD-1 blockade in the MC38 mouse model of colon carcinoma.

To test the synergy between the effects of an anti-OX-40 antibody and an anti-PD1 antibody, combinations of these antibodies were tested in the MC38 mouse tumor model. C57BL/6 mice were injected subcutaneously with 2 million MC38 tumor cells. After 7 days, tumor volumes were determined and mice were randomized into treatment groups to have comparable mean tumor volumes. Antibodies formulated in PBS were administered intraperitoneally on days 7, 10 and 14 at 200 μg per 200 μl dose.

As shown in FIG. 37A-37D, both the anti-PD-1 antibody (FIG. 37B) and the anti-OX40 antibody (FIG. 37C) showed minimal activity when used alone, but had significant antitumor activity when combined (FIG. 37D), with 5 of 8 mice were tumor free.

Example 25 OX40 agonism enhances vaccine response in cynomolgus monkeys.

The enhancement of immune responses to vaccines was measured to assess the ability of the OX40.6 antibody to stimulate immune responses in cynomolgus monkeys. This approach was chosen because the desired effect, ie. enhancement of immune responses to tumors cannot be assessed in healthy non-human primates because they do not contain a tumor.

Monkeys were immunized with fisurella hemocyanin (KLH) on day 1 (10 mg, IM) and hepatitis B surface antigen (HBsAg) (ENGERIX-B) (20 μg IM on days 1 and 29). Following vaccine administration, monkeys were injected intravenously with 0 or 2 mg/kg of OX40.6 antibody on days 1 and 29. Immune responses were measured on days 22 and 41 by ex vivo T cell response to KLH and T cell dependent antibody responses to KLH and HBsAg. As shown in FIG. 38A and 38B, OX40.6-related results at 2 mg/kg on days 22 (FIG. 38A) and 41 (FIG. 38B) included an increase in the ex vivo secondary response to KLH, characterized by an increase in the mean percentage of CD69+, IFN- γ + and TNF-a+ expressing CD4+ CD8- T cells.

Example 26 Fc Receptor Binding for Antibodies with Constructed Constant Domains

This example demonstrates that antibodies with modified heavy chain constant regions containing CH1 and an IgG2 hinge bind to FcyR when they contain IgG1 CH2 and CH3 domains.

In addition to antigen binding via variable domains, antibodies can interact with Fc-γ receptors (FcgR) through interaction with constant domains. These interactions mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The efficiency of functional activity is high for the IgG1 isotype, but very low or absent for IgG2 and IgG4 due to these isotypes having low affinity for FcgR. In addition, the effector function of IgG1 can be modified by mutation of amino acid residues in the constant regions to change the affinity and selectivity of FcgR.

The binding of antibodies to Fc-γ receptors (FcyR or FcgR) was studied using biosensor technologies including Biacore Surface Plasmon Resonance (SPR) and Fortebio Biolayer Interferometry (BLI). SPR studies were performed on a Biacore T100 instrument (GE Healthcare) at 25°C. A Fab fragment from a mouse anti-6xHis antibody was immobilized on a CM5 sensor chip using EDC/NHS at ~3000 RU. Various labeled his FcgRs (7 μg/ml) were captured with the his C-terminal tag using a contact time of 30 s at 10 μl/min and binding of 1.0 μM antibody was evaluated in running buffer 10 mM NaPO ₄ , 130 mM NaCl, 0.05% p20 (PBS-T), pH 7.1. The FcgRs used for these experiments included CD64 (FcgRI), CD32a-H131 (FcgRIIaH131), CD32a-R131 (FcgRIIa-R131), CD32b (FcgRIIb), CD16a-V158 (FcgRIIIa-V158), CD16b-NAl (FcgRIIIb-NA1 ) and CD16B-NA2 (FcgRIIIb-NA2). BLI experiments were performed on a RED Fortebio Octet instrument (Pall, Fortebio) at 25° C. in 10 mM NaPO ₄ , 130 mM NaCl, 0.05% p20 (PBS-T), pH 7.1. Antibodies were captured from undiluted expression supernatants on protein A coated sensors followed by binding of 1 μM hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158 or 0.1 μM hCD64 analytes.

First, antibodies were obtained that contained modified IgG1 Fc domains containing

- 114 040561 substitutions S267E (SE) and S267E/L328F (SELF), as well as various combinations of mutations P238D, P271G, H268D, A330R, G237D, E233D, called V4, V7, V8, V9 and V12. The binding of these antibodies was studied using SPR Biacore compared with IgG1f, IgG2.3 (IgG2-C219S) and IgG4.1 (IgG4-S228P) antibodies, as well as with an IgG1.1f antibody that was designed to reduce binding to all FcgRs. The results shown in FIG. 70 demonstrate expected FcgR binding properties for IgG1f, IgG2.3, and IgG4.1 and mutated IgG1 antibodies, including increased binding of CD32a-H131, CD32a-R131, and CD32b for SE and SELF, and increased selectivity for V4, V7 mutants, V8, V9 and V12 for CD32b versus CD32a-H131 and CD32a-R131 (Figure 39).

The following set of constructs was used to generate effector function in the effector function-negative IgG2 isotype. For this study, the mutations described above were introduced in the context of an IgG2.3 constant region or a hybrid IgG2.3/IgG1f termed IgG2.3G1AY (Table 20). Antibodies were expressed on a small scale as supernatants and tested for FcgR binding using Fortebio Octet BioLayer Interferometry biosensor technology. Since antibodies were present at low concentrations in the supernatants, the experiment was performed by capturing antibodies from the supernatants using protein A coated sensors, followed by binding of the analyzed FcgRs in solution. Purified and supernatant IgG1f controls were also included for comparison, including wild-type IgG1, SE, P238D, V4 and V12 antibodies, and each of these control antibodies showed the expected FcgR binding properties (FIG. 40). The IgG2.3 antibody also showed the expected binding profile with notable binding only to CD32a-H131. However, all mutations to introduce the S267E, L328F, P238D, P271G, H268D, A330R, G237D, or E233D mutations in IgG2.3 failed to replicate the FcgR affinity of the corresponding engineered IgG1 monoclonal antibodies (FIG. 40). In contrast, the IgG2.3G1-AY construct was able to fully retain the binding properties of wild-type IgG1 FcgRs while retaining CH1 and IgG2.3 hinge regions. In addition, all IgG2.3G1-AY mutants containing S267E, L328F, P238D, P271G, H268D, A330R, G237D and E233D showed FcgR binding properties comparable to IgG1 version monoclonal antibodies containing the same mutations (Fig. 40) . This demonstrates the successful construction of antibodies with CH1 and IgG2 hinge regions in combination with wild-type or mutant IgG1 IgG1 effector function.

Table 20 Engineered IgG2 Constructs

Kit ID Design SeqID# 1 IgG23 hHC-IgG2-C219S 258 IgG2.3-V13 hHC-IgG2-C219S-P238D 267 IgG2.3-V14 hHC-IgG2-C219S - P238D,P271G 268 IgG2.3-V15 hHC-IgG2-C219S - P238D,H268D,P271G 269 IgG2.3-V16 hHC-IgG2-C219S - P238D,P271G,A330R 270 IgG2.3-V17 hHC-IgG2-C219S - P23 8D,H268D,P271G, A3 3 OR 271 IgG2.3-V18 hHC-IgG2-C219S-S267E 272 IgG2.3-V19 hHC-IgG2-C219S - S267E,L328F 273 2 IgG2.3Gl hHC-IgG2-C219S/hHC-IgGl f 262 IgG2.3Gl-AY- V20 hHC-IgG2-C219S/hHC-IgGlf-P238D 274 IgG2.3Gl-AY- V21 hHC-IgG2-C219S/hHC-IgGlf - P238D,P271G 275 IgG2.3Gl-AY- V22 hHC-IgG2-C219S/hHC-IgGlf - P238D,H268D,P271G 276 IgG2.3Gl-AY- V23 hHC-IgG2-C219S/hHC-IgGlf - P238D,P271G,A330R 277 IgG2.3Gl-AY- V24 hHC-IgG2-C219S/hHC-IgGlf - P23 8D,H268D,P271G, A3 3 OR 278

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IgG2.3Gl-AY- V25 hHC-IgG2-C219S/hHC-IgGlf - G23 7D,P23 8D,H268D,P271G, A3 3 OR 279 IgG2.3Gl-AY- V26 hHC-IgG2-C219S/hHC-IgGlf - E233D,G237D,P238D,H268D,P271G,A330R 280 IgG2.3Gl-AY- V27 hHC-IgG2-C219S/hHC-IgGlf-S267E 266 IgG2.3Gl-AY- V28 hHC-IgG2-C219S/hHC-IgGlf - S267E,L328F 281

This construction strategy has been further explored by producing other antibodies, with the specified IgG2.3G1-AY, IgG2.3G1-AY-S267E (IgG2.3G1-AY-V27) format, and IgG2-B form variants (IgG2.5G1-AY and IgG2.5G1-AY-V27), and other hybrid antibodies containing various combinations of IgG1 and IgG2 constant domains, and examining the binding of these antibodies to captured anti-his Fab labeled with his FcgR using SPR Biacore technology. Consistent with Octet supernatant data, SPR data showed that IgG2.3G1-AY and IgG2.3G1-AY-V27 antibodies have comparable FcgR binding properties to IgG1f and IgG1f-S267E, respectively, despite containing CH1 and hinge regions. IgG2 A-form antibodies (IgG2.3) (Table 21). Similar data were generated using IgG2.5G1-AY and IgG2.5G1-AY-V27 antibodies, demonstrating the successful construction of IgG2 B-form antibodies (containing the C131S mutation termed IgG2.5) containing IgG1f-like or modified IgG1f effector functions. Data for several other antibodies with IgG2.3G1-AY, IgG2.3G1-AY-V27, IgG2.5G1-AY, or IgG2.5G1-AY-V27 constant regions but different variable regions show that this design strategy is broadly applicable to other antibodies, regardless of variable domains (Table 21).

Table 21. %Rmax values for 1 μM antibodies binding to captured anti-his Fab FcgR-his proteins

monoclonal antibody hCD64 hCD32a- H131 hCD32a- R131 hCD32b hCD16a- V158 hCD16B- NA2 mAb8-IgGlf 80% 82% 51% 27% 51% 21% mAb9-IgGlf 70% 33% 19% 4% 28% 10%

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mAbll-IgG2.3 2% 44% 17% 5% 1% 0% mAb6-IgG2.3 3% 66% 14% 3% 1% 0% mAb4-IgG2.3 1% 39% 6% 1% 1% 0% mAb5-IgG2.3 6% 100% thirty% 4% 3% 0% mAb!2-IgG2.3 2% 39% 7% 1% 1% 0% mAbl3-IgG2.3 2% 40% 7% 1% 1% 0% mAbll-IgG2.5 0% 40% 13% 3% 0% -1% mAb7-IgG2.5 4% 72% 19% 2% 2% 0% mAb8-IgG2.5 3% 59% 14% 3% 2% 0% mAblO-IgG2.5 1% 29% 5% 1% 1% 0% mAb6-IgG2.5 3% 75% 17% 4% 2% 0% mAb4-IgG2.5 2% 46% 8% 1% 1% 0% mAb5-IgG2.5 6% 89% 26% 5% 4% 1% mAbl2-IgG2.5 1% 36% 6% 1% 1% 0% mAbl3-IgG2.5 -2% 39% 4% -2% 0% -2% mAb8-IgG2.3Gl-AY 77% 61% 38% 10% 38% 13% mAblO-IgG2.3Gl-AY 67% 23% 14% 4% 24% 8% mAb7-IgG2.5Gl-AY 80% 73% 45% 12% 47% 19% mAb8-IgG2.5Gl-AY 77% 70% 45% 17% 48% 22% mAb7-IgG2.3Gl-AY-V27 84% 68% 92% 76% 26% 7% mAb8-IgG2.3Gl-AY-V27 78% 67% 80% 67% 24% 7% mAblO-IgG2.3Gl-AY- V27 69% 24% 57% 40% 12% 3% mAb7-IgG2.5Gl-AY-V27 81% 74% 89% 84% 32% 9% mAb8-IgG2.5Gl-AY-V27 77% 76% 79% 77% 33% 10%

Example 27 Effect of anti-OX40 antibodies with modified heavy chain constant regions on T cell proliferation and secretion of IFN-γ and IL-2 from T cells with or without crosslinks.

Anti-OX40 antibodies with modified IgG2 CH1/hinge regions may have the ability to stimulate T cell activation in the absence of cross-linking, and thus may stimulate T cell activation in vivo in the absence or low expression of FcyR-expressing cell types, and, possibly stimulate antitumor activity in a wider range of tumor types than antibodies of the IgG1 isotype.

Alternatively, antibodies with modified CH1/hinge regions may still need to be cross-linked to promote T cell activation, but may have increased FcyR binding agonist activity relative to IgG1 isotype antibodies and thus be more effective in stimulating T-cell activation and antitumor activity.

Anti-OX40 antibodies with modified heavy chain constant regions containing the sequences shown in Table 1. 22 are produced and examined for their effect on T cell proliferation and secretion of IFN-γ and IL-2 from T cells with or without crosslinking using the assays described below. The light chain sequences for antibodies OX40.6, 40.8, 40.16 and 40.21 correspond to SEQ ID NOS: 96, 110 and 116 (for both OX40.16 and 40.21), respectively.

- 117 040561

Table 22

Constructions SEQID NO OX40.6-Vh-hHC-IgG2.3 282 OX40.8-Vh-hHC-IgG2.3 283 0X40.16-Vh-hHC-IgG2.3 284 OX40.6-Vh-hHC-IgG2.3Gl 285 OX40.8-Vh-hHC-IgG2.3Gl 286 OX40.16-Vh-hHC-IgG2.3Gl 287 OX40.6-Vh-hHC-IgG2.3Gl-V27 288 OX40.8-Vh-hHC-IgG2.3Gl-V27 289 OX40.16-Vh-hHC-IgG2.3Gl-V27 290 OX40.6-Vh-hHC-IgG2.5 291 OX40.8-Vh-hHC-IgG2.5 292 OX40.16-Vh-hHC-IgG2.5 293 0X40.2 l-Vh-hHC-IgG2.5 294 0X40.2 l-Vh-hHC-IgG2.5Gl 295 0X40.2 l-Vh-hHC-IgG2.5Gl-V27 296

CHO-CD3 +/- CD32 analysis.

Antibodies to OX-40 with the sequences shown in table. 22 were examined for their ability to induce T cell activity in vitro by measuring the proliferation and the amount of IL-2 and IFN-γ secreted by T cells incubated with antibodies.

Transfected CHO cell lines are prepared for use as artificial antigen presenting cells in the primary T cell activation assay. The CHO-CD3CD32A cell line expresses the anti-human CD3 antibody in single chain Fv format together with the human CD32A Fc receptor (FcR) to present anti-OX40 antibodies on the surface of CHO cells. The CHO-CD3 cell line expresses the anti-human CD3 antibody in single chain Fv format without FcR.

Briefly, primary human CD4 T cells are isolated by negative selection (RosetteSep™, StemCell Technologies) and co-cultured with either irradiated CHOCD3-CD32A cells or irradiated CHO-CD3 cells at a T:CHO ratio of 8:1 in the presence of graduated doses of antibodies. to OX40 or isotype control antibodies. After 3-4 days in culture at 37° C., supernatants are harvested to assess T cell activation by measuring secreted human IFN-γ using either ELISA (BD Biosciences) or HTRF (Cisbio) assay, following manufacturer's recommendations. Tritiated thymidine is then added for a final culture of approximately 18 hours to measure T cell proliferation by including tritiated thymidine as an additional measure of T cell activation.

SEB PBMC analysis.

Antibodies to OX-40 with the sequences shown in table. 22 were investigated for their effect on primary T cell stimulation in cultures of enterotoxin B-activated staphylococcus (SEB) human peripheral blood mononuclear cells (PBMC). Human whole blood samples are obtained from AllCells, Inc. (Berkeley, CA) or from donors at Bristol-Myers Squibb, Redwood City, CA through their own phlebotomy program. PBMCs are isolated by Ficoll-Hypaque density gradient purification and cultured for 3 days in culture medium supplemented with fixed suboptimal (85 ng/mL) Staphylococcus enterotoxin B superantigen (SEB; Toxin Technologies, Sarasota, FL) in the presence of graduated doses of OX40 antibodies or isotype control antibodies. In some cases, 2-5 μg/ml of a soluble cross-linking antibody, goat F(ab') ₂ to human Fcy, is also added to the cultures. After culturing for 3 days at 37° C., supernatants are harvested for evaluation of T cell activation by human secreted IL-2 ELISA assay. Briefly, culture supernatants are diluted 1:10 in sample diluent and assayed for human IL-2 by ELISA (BD Bioscience) according to the manufacturer's recommended protocol. After adding the TMB substrate, the assay plates are read on a Spectramax 340PC reader using Softmax software at 650 nm. The measured optical densities of the chromogenic substrate were proportional to the bound detection antibody.

MLR analysis.

Antibodies to OX-40 with the sequences shown in table. 22, investigated in relation to their

- 118 040561 ability to potentiate the primary proliferation of human T cells and secretion of IFN-γ in the reaction of allogeneic mixed lymphocyte T cells: dendritic cells (T: DC AlloMLR). All T cells are isolated from peripheral blood from healthy human donors by negative selection (RosetteSep, Stemcell Technologies). Monocytes are isolated from peripheral blood from healthy human donors using CD14 microbeads (Miltenyi) and cultured for six days in the presence of GMCSF and IL-4 to obtain immature dendritic cells (DC). DC and T cells are co-cultured in the presence of graduated doses of OX40 antibodies or isotype control antibodies. In some cases, cultures are also supplemented with 2-5 μg/ml of a soluble cross-linking antibody such as goat F(ab') ₂ to human Fcy. The supernatant from each sample is collected between days 4 and 7 for measurement of secreted IFN-γ by ELISA assay (BD Biosciences) or HTRF (Cisbio) following the manufacturers' recommendations. After collecting the supernatant, cell cultures are pulsed with 1 μCi/well with ³ [H]-thymidine during the last 16-18 hours of culture. Cells are harvested onto filter plates and counts of ³ [H] per minute of ³ [H]thymidine injected into the cell are read as a measure of T cell proliferation.

Example 28 Antitumor Activity of OX40 Agonist Monoclonal Antibody with CTLA-4 Blocker in CT26 Model.

To test for synergy between an anti-OX-40 antibody and an anti-CTLA-4 antibody, combinations of these antibodies were tested in the CT26 mouse tumor model. Mice were inoculated with CT26 tumors and monoclonal antibody administration was started on day 3 post-inoculation (dosed on days 3, 7 and 10) at 200 μg/mouse of the antibody shown in FIG. 41A-41D.

As shown in FIG. 41A-41D, both the anti-CTLA-4 antibody (FIG. 41B) and the anti-OX40 antibody (FIG. 41C) showed minimal activity when applied alone (1 of 8 tumor-free mice for both treatments), but showed significant antitumor activity when combination (Fig. 41D), with 4 of 8 mice were characterized by the absence of a tumor.

Example 29 Phase 1/2a clinical trial in a subject with solid tumors.

A phase 1/2a study for OX40.21 administered alone or in combination with nivolumab or ipilimumab is being conducted in patients with advanced solid tumors to demonstrate the efficacy of OX40.21 alone or in combination with nivolumab or ipilimumab.

1. Purpose.

The primary aim of the study is to evaluate the safety, tolerability, dose-limiting toxicity (DLT) and maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D) of OX40.21 administered alone or in combination with nivolumab or ipilimumab in subjects with advanced cancers.

Secondary goals include investigating the preliminary antitumor activity of OX40.21, administered alone or in combination with nivolumab or ipilimumab, in subjects with advanced cancers; characterization of PK OX40.21 administered alone and in combination with nivolumab or ipilimumab; and characterization of the immunogenicity of OX40.21 administered alone or in combination with nivolumab or ipilimumab and the immunogenicity of nivolumab or ipilimumab administered with OX40.21. Additional research objectives include investigating potential associations between antitumor activity and selective biomarker measurements in tumor biopsy and peripheral blood specimens before and after OX40.21 administration alone or in combination with nivolumab or ipilimumab; evaluation of the potential impact of monotherapy and combination therapy of OX40.21 on the QTc interval; characterization of the PK of nivolumab in subjects receiving the combination of nivolumab and 0X40.21; characterization of the PK of ipilimumab in subjects receiving the combination of ipilimumab and OX40.21; assessment of overall survival (OC) in patients treated with OKC40.21 alone and in combination with nivolumab or ipilimumab; and investigating potential relationships between dose/exposure and antitumor activity, pharmacodynamic (PD) effects (selected biomarkers in peripheral blood samples and tumor biopsies), and key safety measures in patients treated with OX40.21 alone and in combination with nivolumab or ipilimumab.

2. Design and duration of the study.

This is a phase 1/2a open label study of OX40.21 in patients with advanced solid tumors that combines initial OX40.21 monotherapy followed by combination therapy with nivolumab or ipilimumab.

The sections of the study (dose escalation and dose escalation to maximum tolerated) undergo a stepwise approach based on safety data, PK and PD. The first section of the study begins with dose escalation of OX40.21 monotherapy in cohorts. Clinical data from the first 3 monotherapy dose cohorts serve as the basis for initiating dose escalation of OX40.21 in combination with nivolumab. Clinical data from the first three monotherapy dose cohorts, in addition to clinical data from the first cohort of OX40.21 in combination with nivolumab, provide the basis for initiating dose escalation of OX40.21 in combination with ipilimumab. Once a tolerable and pharmacologically active RP2D OX40.21 is established in the dose escalation section, dose escalation to the maximum tolerated in certain tumor cohorts begins.

3. Increasing the dose.

The study design scheme for Part 1A is shown in FIG. 42.

The dose escalation phase of the study is evaluating the safety and tolerability of OX40.21, alone or in combination with nivolumab or ipilimumab, in patients with advanced solid tumors.

The starting dose level for OX40.21 is 20 mg. Dose escalation decisions for subsequent doses are based on DLT using the BLRM model (for OX40.21 monotherapy) or the BLRM (-Copula) model (for OX40.21 in combination with nivolumab or ipilimumab). The DLT period is 28 days for dose escalation portions for both monotherapy and combination therapy. The frequency of DLT is determined based on the frequency, severity, and duration of AEs that occur during the DLT period and for which no alternative cause can be identified. Dose selection for the next monotherapy cohort/dose level takes into account the BLRM (-Copula) recommendation in conjunction with all available data on PK, PD, and clinical and laboratory safety in all subjects. The choice of starting dose of OX40.21 for Part 2A is determined using the data presented in Part 1A, including clinical and laboratory safety assessments, PK/PD data, and modeling recommendations from Bayesian Hierarchical Network Modeling by including single agent toxicity profiles as OX40 .21 (part 1A) and nivolumab (CA209 003). The choice of starting dose of OX40.21 for Part 3A is determined using the data presented in Parts 1A and 2A, including clinical and laboratory safety assessments, PK/PD data, and Bayesian network modeling recommendations by including single agent toxicity profiles as for OX40.21 (part 1A) and ipilimumab (CA184-022). Actual doses may be modified for BLRM (-Copula) but not to exceed doubling the previously studied dose.

During dose escalation for all dose cohorts, the original subject (signal subject) is observed for 5 days before additional subjects in that cohort are treated with investigational drug.

Approximately 30 subjects are included in each dose escalation portion. The number of subjects in each dose escalation cohort varies depending on the BLRM (-Copula) recommendations. Initially, approximately 3 subjects are treated at initial dose levels of OX40.21 or OX40.21 in combination with nivolumab or ipilimumab. Additional cohorts of approximately 3 evaluable subjects are treated at the recommended doses for BLRM (-Copula) during the dose escalation phase. At least 6 subjects evaluated for DLT are treated with MTD.

Part 1A. Enrollment begins in Part 1A, dose escalation of OX40.21 monotherapy. The starting dose of OX40.21 for Part 1A is 20 mg, with subsequent doses expected of 40, 80, 160, and 320 mg. Actual doses are subject to change for BLRM, but do not exceed doubling the previously studied dose.

Part 2A. Part 2A is a combination treatment arm for OX40.21 with nivolumab that begins after at least 3 dose levels of monotherapy dose escalation have been found to be tolerable or an MTD has been determined with monotherapy dose escalation (Part 1A). The starting dose of OX40.21 in Part 2A is at least 1 dose level lower than the dose shown to be tolerated in Part 1A to provide additional safety for the combination. In no case should the dose for OX40.21 in Part 2A exceed the maximum allowable dose in Part 1A. Nivolumab is administered at a constant dose of 240 mg. Each treatment cycle lasts 2 weeks, and study drugs are administered every 2 weeks starting on day 1 of each cycle for up to 12 cycles.

Part 3A. Part 3A is a combination of the OX40.21 treatment arm with ipilimumab that is initiated only after at least 3 dose levels in monotherapy dose escalation have been found to be tolerated or MTD is determined with monotherapy dose escalation (Part 1A), and at least one dose cohort was tolerable in terms of dose escalation of OX40.21 with nivolumab. The starting dose of OX40.21 in Part 3A is at least 1 dose level below the dose shown to be tolerated in Part 1A. In no case should the dose for OX40.21 in Part 3A exceed the maximum allowable dose in Part 1A to further ensure the safety of combined doses in treated patients. Ipilimumab is administered at a dose of 1 mg/kg. Each treatment cycle lasts 3 weeks. OX40.21 is administered every 3 weeks starting on day 1 of cycle 1 for up to 8 cycles, and ipilimumab is administered every 3 weeks starting on day 1 for 4 cycles. In the last 4 cycles, only OX40.21 is injected.

Increase the dose to the maximum tolerated.

Treatment in escalation cohorts to the maximum tolerated dose begins when the MTD/RP2D has been determined based on the available clinical safety population (DLT, significant AE occurring after the DLT period), PK, PD, and modeling data from the escalation part.

- 120 040561 doses (parts 1A, 2A and 3A). Approximately 110 subjects are treated across all escalation cohorts to the maximum tolerated dose.

Part 1B is a dose escalation cohort to maximally tolerated OX40.21 monotherapy in cervical cancer patients with MTD/RP2D as defined in Part 1A. Dosing of OX40.21 begins on day 1 of each cycle and is administered every 2 weeks for 12 cycles. Approximately 12 subjects are treated in this escalation cohort to the maximum tolerated dose.

Parts 2B are part of the dose escalation to maximum tolerated combination therapy (OX40.21 with nivolumab) for subjects with CRC in MTD/RP2D as defined in Part 2A. Nivolumab is administered at a constant dose of 240 mg. Each treatment cycle lasts 2 weeks and study drugs are administered every 2 weeks starting on day 1 of each cycle for up to 12 cycles. Approximately 35 subjects are treated in this escalation cohort to the maximum tolerated dose.

Part 2C is part of the dose escalation to maximum tolerated combination therapy (OX40.21 with nivolumab) for subjects with BC in MTD/RP2D as defined in Part 2A. Each treatment cycle lasts 2 weeks and study drugs are administered every 2 weeks starting on day 1 of each cycle for up to 12 cycles. Approximately 27 subjects are treated in this escalation cohort to the maximum tolerated dose.

Part 3B is part of the dose escalation to maximally tolerated combination therapy (OX40.21 with ipilimumab) in OS subjects for MTD/RP2D as defined in Part 3A. Each treatment cycle lasts 3 weeks. Ipilimumab is given for the initial 4 cycles in combination with OX40.21. The subject then continues OX40.21 monotherapy for up to 4 additional cycles for a total of 24 weeks (8 cycles) of treatment. Approximately 35 OC subjects are treated in this escalation cohort to the maximum tolerated dose.

Brief description of the study periods.

Subjects go through up to 5 study periods: screening (up to 28 days), treatment (up to 24 weeks), safety follow-up (minimum 100 days), response follow-up, and long-term survival follow-up (up to approximately 2 years). from the first dose) as described below. The study visit scheme is shown in Fig. 43.

selection period.

The selection period lasts up to 28 days. The screening period begins with establishing initial eligibility for the subject and signing the informed consent form. Subjects are enabled using interactive communication technology (IRT).

Treatment period.

The treatment period includes up to 24 weeks of dosed administration. After each treatment cycle, the decision to treat the subject in the next cycle of study therapy, up to 24 weeks of treatment, is based on an assessment of risk/benefit and tumor. Tumor assessments are performed every 8 weeks for each 2-week (q2w) dosing regimen and every 9 weeks for each 3-week (q3w) dosing regimen. Partial response (PR) and complete response (CR) scores are confirmed at least 4 weeks after the initial score. Endpoints of tumor progression or response are assessed using the Solid Tumor Response Evaluation Criteria (RECIST) v1.1.

Subjects with a stable disease response (SD), PR, or CR at the end of a given cycle continue on to the next treatment cycle. Subjects are generally allowed to continue investigational therapy until the first occurrence of one of the following: 1) completion of the maximum number of cycles; 2) disease progression; 3) clinical deterioration, suggesting that there will be no additional benefit from the treatment; 4) intolerance to therapy; or 5) meeting the criteria for discontinuation of study therapy.

Follow-up to assess safety.

Upon completion of study therapy, subjects enter a follow-up period to assess safety. Following an end-of-treatment (EOT) visit, subjects are evaluated for any new adverse events (AE) for at least 100 days after the last dose of therapy. Follow-up visits occur on days 30, 60, and 100 after the last dose or date of discontinuation. Subjects (excluding those with early withdrawal of consent to participate in the study) complete 3 clinical follow-up visits to assess safety, regardless of whether they initiate new anticancer therapy.

Follow-up to assess survival.

After completion of the safety follow-up period, subjects enter the survival follow-up period. Subjects are seen approximately every 3 months (12 weeks) until death, withdrawal from follow-up, withdrawal of consent, or completion of the study, whichever comes first. This phase lasts up to 2 years after the first dose of study drug.

Follow-up to assess response.

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After the completion of the safety follow-up period, all subjects with current SD, PR, or CR at the EOT visit enter a response follow-up period that occurs concurrently with the survival follow-up period. These subjects continue to receive radiographic and clinical tumor assessments every 3 months (12 weeks) during the follow-up period to assess response or until disease progression or withdrawal of consent to the study. Tumor radiological evaluations for subjects that are characterized by the presence of ongoing clinical benefit continue to be collected after completion of the study survival phase. Subjects who experience disease progression after an initial course of investigational therapy are not assessed for response after the EOT visit and are allowed to receive other tumor-targeted therapy.

Study duration.

The total length of study time for any individual subject is approximately 2 years. The study ends when the last subject completes their last study visit, which occurs approximately 4 years after the start of the study.

Number of subjects.

Approximately 225 subjects will be enrolled and approximately 200 subjects will be treated in the study.

Study sample.

Subjects at least 18 years of age and characterized by the presence of histological or cytological evidence of malignancy that is advanced (metastatic, recurrent, refractory, and/or inoperable) with measurable disease according to RECIST v1.1.

Rules for increasing the dose and stopping.

In Parts 1A, 2A, and 3A, the BLRM and BLRM (-Copula) models are used to recommend dose escalation once DLT information is available for each cohort of subjects. Dose selection of OX40.21 for the next cohort/dose level considers the BLRM (-Copula) recommendation in conjunction with the clinical recommendation and all available PK, PD, and clinical and laboratory safety data for all subjects.

Dose-limiting toxicities.

For direction of dose escalation, DLT is determined based on the frequency, intensity, and duration of AEs for which no clear alternative cause has been identified. The DLT period is 28 days after the start of the investigational drug(s). For the management of subjects, an AE that meets DLT criteria, regardless of the cycle in which it occurs, results in discontinuation of study drug use. Subjects who withdraw from the study during the DLT evaluation interval for reasons other than DLT may be replaced with a new subject at the same dose level. The frequency of DLT during the DLT assessment period is used in dose escalation decisions and in determining the MTD. AEs occurring after the DLT period are considered for MTD purposes unless they have a clear alternative cause and are not associated with disease progression. Subjects experiencing DLT do not deviate from the investigational drug and enter an observation period to assess safety. AE is assessed according to the National Cancer Institute (NCI) Common Criteria for Terminology of Adverse Events (CTCAE) v4.03.

Non-hematological DLT.

A. Liver-acting DLT.

Any > grade 3 increase in AST, ALT, or total bilirubin;

AST, ALT or total bilirubin;

grade of AST or ALT with symptomatic liver inflammation (eg, right upper quadrant tenderness, jaundice, pruritus);

AST or ALT > 3 x ULN and concurrent total bilirubin > 2 x ULN without baseline evidence of cholestasis (elevated serum alkaline phosphatase [ALP]) (eg, results consistent with High's law or FDA definition of potential drug-induced liver injury or pDILI).

B. Non-liver DLT.

Uveitis, episcleritis, or iritis grade 2 or more.

Any other grade 2 eye pain or blurred vision that does not respond to topical treatment and does not improve to grade 1 within 2 weeks or requires systemic treatment.

Pneumonitis, bronchospasm, neurological toxicity, hypersensitivity reaction or infusion reaction grade 3 or higher.

Non-dermatological, non-hepatic toxicity grade 3 or higher will be considered DLT with the following specific exceptions.

Grade 3 or 4 electrolyte abnormalities that are not complicated by clinical adverse experience, last less than 72 hours, and either resolve spontaneously or respond to routine medical intervention.

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Grade 3 nausea, vomiting, or diarrhea that lasts less than 72 hours and either resolves spontaneously or responds to conventional medical intervention.

Grade 3 or 4 amylase or lipase elevation not associated with clinical or radiographic evidence of pancreatitis.

Separate grade 3 fever not associated with hemodynamic disturbances (eg, hypotension, clinical or laboratory evidence of worsening end organ perfusion).

Grade 3 endocrinopathy, which is well controlled by hormone replacement therapy.

Transient worsening of the clinical manifestations of a grade 3 tumor (defined as pain, irritation, or rash that is localized to areas of a known or suspected tumor).

Grade 3 fatigue for less than 7 days.

Grade 3 infusion reaction that returns to grade 1 in less than 6 hours.

Dermatological DLT.

Rash of the 4th degree.

Grade 3 rash if not improved (eg, resolution to < Grade 1) after infusion delay 1 to 2 weeks. Subjects who have not experienced Grade 3 cutaneous AE may resume treatment in the presence of Grade 2 cutaneous toxicity.

Hematological DLT.

Grade 4 neutropenia > 5 days.

Grade 4 thrombocytopenia or grade 3 thrombocytopenia with clinically significant bleeding or any requirement for platelet transfusion.

Anemia of the 4th degree, not explained by the underlying disease.

Febrile neutropenia of the 4th degree.

Grade 3 febrile neutropenia that lasts > 48 hours.

Grade > 3 hemolysis (i.e. requiring a transfusion or medical intervention such as steroids).

Treatment with additional cycles over 24 weeks.

Subjects are treated for 24 weeks if the study drug withdrawal criteria have not been previously met. Subjects completing approximately 24 weeks of treatment with ongoing disease control (CR, PR, or SD) are eligible for an additional 24 weeks of study therapy in monotherapy (Part 1) and combination therapy (Parts 2 and 3) after the first 24 weeks when the risk assessment /benefit promotes continuation of therapy. Upon completion of the additional 24 weeks of study therapy, subjects enter a safety follow-up period.

Treatment without progression.

Treatment beyond progression is permitted in selected subjects with initial progressive disease based on RECIST v1.1, after determining that a benefit/risk assessment is conducive to continuing study therapy (e.g., subjects continue to experience clinical benefit, tolerate treatment, and comply with other criteria).

Re-treatment.

Retreatment is permitted if there is confirmed disease progression during the follow-up period to assess response. Subjects completing approximately 24 weeks (or additional treatment cycles if necessary) of therapy who enter an observation period to assess response with persistent disease control (CR, PR, or SD) without any significant toxicity are eligible for retreatment . Such subjects are eligible for re-treatment on a case-by-case basis after evaluating and determining whether the risk/benefit ratio supports the introduction of further investigational therapy and whether the subject continues to meet the eligibility criteria for treatment with investigational therapy. Subjects eligible for retreatment are treated with the originally prescribed monotherapy or combination therapy regimen (e.g., same dose and dose schedule as administered during the first 24 weeks), unless that dose and schedule is subsequently exceeded. MTD, in which case the subject is treated with the next lower dose considered acceptable/safe.

Inclusion Criteria.

1) Signed written informed consent.

a) The subject must sign an informed consent form prior to performing any study-related procedures that are not considered part of the SOC.

b) Consent for tumor biopsy specimens (mandatory pre- and post-treatment biopsies are required for escalation cohorts up to the maximum tolerated dose as well as additional subjects added to any previously completed escalation cohort and optionally for a dose escalation cohort).

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2) Target sample.

Subjects must be at least 18 years of age and have histological or cytological evidence of a malignancy that is advanced (metastatic, recurrent, resistant, and/or inoperable) with measurable disease according to RECIST v1.1.

A. Dose escalation.

Subjects must have received and then progressed to or been resistant to or intolerant of at least 1 standard treatment regimen in advanced or metastatic cancer therapy, if such therapy exists. Subjects not eligible for any standard therapy are allowed to register if their non-compliance is documented in medical records. The following tumor histologies are permitted, except for subjects with primary central nervous system (CNS) tumors or CNS metastases as the only site of active disease.

(i) Melanoma: BRAF mutational status should be documented if known.

(ii) NSCLC mutational status: EGFR, ALK, KRAS and ROS1 should be documented if known.

(iii) Head and neck malignancy limited to squamous cell carcinoma. HPV status should be documented if known.

(iv) Transient cell carcinoma of the genitourinary tract.

(v) Renal cell carcinoma.

(vi) Adenocarcinoma of the pancreas.

(vii) CRC: MSI, KRAS and BRAF status should be documented if known.

(viii) Cervical cancer: HPV status should be documented if known.

(ix) Triple negative breast cancer. HER2, ER and PR status should be documented.

(x) Endometrial adenocarcinoma.

(xi) Malignant tumor of the ovaries.

(xii) Adenocarcinoma of the prostate.

(xiii) Hepatocellular malignancy - Child-Pugh class only.

(xiv) Small cell malignant tumor of the lung.

(xv) Gastrointestinal and gastric cancer: HER2 status should be documented if known.

B. Increasing the dose to the maximum allowable: parts 1B, 2B, 2C and 3B.

The following types of tumors will be allowed:

(a) Cervical cancer - part 1B.

(i) Histologically confirmed cervical cancer that is inoperable, metastatic, or recurrent with documented disease progression.

(ii) Documented HPV tumor status, if known. If unknown, subjects must agree to have their submitted archival tumor tissue sample (block or unstained sections) examined.

(iii) Prior need for therapy.

1. Must have received and then progressed or not tolerated or been resistant to at least 1 standard systemic therapy for metastatic and/or inoperable disease (eg, paclitaxel/cisplatin, paclitaxel/cisplatin/bevacizumab). Concurrent chemotherapy given with primary radiation and additional chemotherapy received after completion of radiation therapy is not considered a systemic chemotherapy regimen.

(b) Colon cancer - part 2B.

(i) Histologically confirmed CRC that is metastatic or recurrent with documented disease progression.

(ii) Documented status of MSI, MMR, KRAS and BRAF, if known. If unknown, subjects must agree to have their submitted archival tumor tissue sample (block or unstained sections) examined.

(iii) Prior need for therapy.

Subjects were required to receive and then progress to or not tolerate or be resistant to at least one standard systemic therapy for metastatic and/or inoperable disease (or progress within 6 months of additional therapy).

(c) Bladder cancer - part 2C.

(i) Histologically or cytologically confirmed urothelial carcinoma (including mixed histologies of urothelial carcinoma with elements of other subtypes) of the renal pelvis, ureter, bladder, or urethra with progressive or persistent disease.

- 124 040561 (ii) Prior need for therapy.

Subjects were required to receive and then progress to or not tolerate or be resistant to at least 1 standard systemic treatment regimen (eg, platinum-based chemotherapy) for the treatment of metastatic (stage IV) or locally advanced inoperable disease.

(d) Malignant tumor of the ovary - Part 3B.

(i) Histologically or cytologically confirmed ovarian carcinoma (including epithelial OC, primary peritoneal or fallopian carcinoma) with documented disease progression.

(ii) Registered germline BRCA mutation status, if known. If unknown, subjects must agree to have their submitted archival tumor tissue sample (block or unstained sections) examined.

(iii) Prior need for therapy.

Subjects were required to receive and then progress to or not tolerate or be resistant to at least 1 standard systemic therapy (eg, platinum-based chemotherapy) for the treatment of metastatic or inoperable disease.

3) The general condition of the cancer patient according to the scale of the Eastern United Oncology Group (ECOG) < 1.

4) Presence of at least 1 lesion with measurable disease, as determined by RECIST v1.1 for response assessment. Subjects with lesions in a previously irradiated field as the only site of measurable disease are allowed to be recorded if the lesion(s) has shown a clear progression and can be accurately measured.

5) For subjects requiring a new tumor biopsy, subjects must have at least one lesion available for biopsy before and after treatment, in addition to the minimum measured RECIST v1.1 lesion required for response assessment. This lesion must be distinguishable from the lesion index(es) that is being assessed for radiological response.

6) Subjects who have received prior therapy with any agent specifically targeted at checkpoint pathway inhibition (e.g., anti-PD-1, anti-PD-L1, anti-PD-L2, anti-LAG-3, and anti-CTLA-4 antibodies) are allowed to participate. after a washout period at any time more than 4 weeks after the last treatment.

Note: (i) Subjects who have experienced prior therapy-related checkpoint grade 1 and 2 immune-mediated AE must confirm recovery from these events at study entry, except for endocrinopathies treated with replenishment as described in the authorization. all associated clinical symptoms, abnormal physical examination findings, and/or associated laboratory abnormalities. Where applicable, these subjects must also have completed steroids for the treatment of these AEs at least 14 days prior to initiation of study therapy, (ii) Eligibility of subjects with prior therapy-related immune-mediated AE checkpoints > Grade 3, will be considered on a case-by-case basis after discussion with a medical supervisor (e.g., inclusion with asymptomatic isolated grade 3 lipase elevation without clinical or radiological evidence of pancreatitis is permitted).

7) Subjects with prior therapy with any agent specifically targeting T-cell costimulation pathways, excluding anti-OX40, anti-CD137, anti-GITR, and anti-CD27 antibodies, are permitted after a washout period at any time greater than 4 weeks after last exposure.

8) Prior palliative radiotherapy must be completed at least 2 weeks before the first dose of study drug. Subjects with symptomatic tumor lesions at baseline who may require palliative radiotherapy within 4 weeks of the first dose of study drug are strongly encouraged to receive palliative radiotherapy prior to enrollment in the study.

9) Subjects included in the escalation and escalation to maximum tolerated dose cohorts must agree to receive existing formalin-fixed, paraffin-embedded (FFPE) tumor tissue, either a block, or a minimum of 15 unstained sections (preferably 25 sections) to perform a correlation study . If an archival sample is not available, the subject must consent to a biopsy of the tumor prior to treatment. Subjects who are unable to provide an archived tumor sample and who either do not consent to pre-treatment tumor biopsy or do not have available lesions do not qualify. (However, subjects whose pre-treatment biopsy results in insufficient tissue quantity or quality will not be excluded on that basis alone). For any additional subjects added to any of the previously completed dose escalation cohorts, mandatory pre- and post-treatment biopsies are required.

10) Subjects included in the dose escalation group to the maximum tolerated dose or added to any previously completed dose escalation cohort should undergo mandatory biopsy prior to and

- 125 040561 after treatment with acceptable clinical risk.

(a) The tissue sample from a solid tumor must be a punch, excision, or incisional biopsy. Fine needle biopsy, drainage of pleural effusions with cytocentrifuges, or punch biopsy are not considered adequate for reviewing biomarkers. Biopsies of bone lesions that do not have soft tissue components or tumor specimens of decalcified bone are also unacceptable.

(b) Lesion biopsies must be distinct from the index lesion(s) being evaluated for radiological response.

11) The adequate function of an organ for subjects is defined as follows.

(a) Neutrophils >1500/µl (stable to any growth factor within 4 weeks of first administration of study drug).

(b) Platelets >80 x 10 ³ /µl (transfusion to reach this level is not allowed within 2 weeks after the first administration of study drug).

(c) Hemoglobin >8 g/dl (transfusion to reach this level is not allowed within 2 weeks after the first administration of study drug).

(d) ALT and AST <3 x upper limit of normal (ULN).

(e) Total bilirubin <1.5 x ULN (except subjects with Gilbert's syndrome, who should have normal direct bilirubin).

(f) Normal thyroid function or stable hormonal supplement as judged by the investigator.

(g) Albumin > 2 mg/dl.

(h) Serum creatinine < 1.5 x ULN or creatinine clearance (CrCl) >40 ml/min (measured using the Cockcroft-Gault formula below):

Female CrCl = (140 - age in years) x weight in kg x 0.85 x serum creatinine in mg/dl;

Male CrCl = (140 - age in years) x weight in kg x 1.00 x serum creatinine in mg/dL.

12) Ability to comply with treatment conditions, collection of PK and PD samples and follow-up. Age and reproductive status.

a) Men and women, age > 18 years at the time of informed consent.

b) Women of reproductive potential (WOCBP) must have a negative serum or urine pregnancy test (minimum sensitivity of 25 IU/L or equivalent units of human chorionic gonadotropin [hCG]) within 24 hours prior to initiation of drug study.

c) Women should not breastfeed.

d) WOCBPs must agree to follow instructions on the contraceptive method(s) for the duration of study drug OX40.21 treatment plus 5 study drug half-lives plus 30 days.

This duration should be 12 weeks for Part 1 and 3 subjects (50 days plus 30 days) or 23 weeks for Part 2 subjects (130 days plus 30 days [ovulatory cycle duration]), up to a total of 160 days after completion of treatment.

e) Men who are sexually active with WOCBP must agree to follow instructions on their contraceptive method(s) for the duration of study drug OX40.21 treatment plus 5 study drug half-lives plus 90 days. The duration must be 20 weeks for Part 1 and 3 subjects (50 days plus 90 days) or 31 weeks for Part 2 subjects (130 days after completion). In addition, men must be prepared to refrain from donating sperm during this time.

f) Men with azoospermia are exempt from requirements for contraception. WOCBPs that have not been heterosexually active for a long time are also exempt from contraceptive requirements, but still get tested for pregnancy.

exclusion criteria.

1) Exceptions for the target disease.

a) Subjects with known or suspected CNS metastases or untreated CNS metastases or with the CNS as the sole site of disease are excluded. However, subjects with controlled brain metastases may be included in the study. Controlled brain metastases are defined as no radiographic progression for at least 4 weeks after radiation and/or surgery (or 4 weeks of follow-up if no clinical intervention), and from steroids for at least 2 weeks, and no new or progressive neurological signs and symptoms.

b) Subjects with carcinomatous meningitis.

c) For a malignant tumor of the ovaries.

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i) Subjects with ovarian cancer with a history of bowel obstruction within the previous 6 months or with a Tenckhoff catheter are excluded.

ii) Up to 4 previous anticancer treatments (ie chemotherapy, radiotherapy, hormonal or immunotherapy) are allowed. Restarting the same treatment regimen after a drug interruption may be considered one treatment regimen; however, it will count as two treatment regimens if any other treatment regimen existed between them.

2) Medical history and comorbidities.

a) Subjects with a previous malignancy other than that used for inclusion in this study, diagnosed less than 2 years prior to study entry, are excluded (except for non-melanoma skin cancers and in situ cancers such as bladder, colon, cervix/dysplasia, melanoma, or breast). In addition, patients with other secondary malignancies diagnosed more than 2 years ago who received curative therapy without evidence of disease during this interval and who are at low risk of recurrence are eligible.

b) Other active malignancy requiring concurrent intervention.

c) Previous organ allograft.

d) Previous treatment.

i) Prior cancer treatment (ie chemotherapy, radiation therapy, hormonal or immunotherapy) is acceptable.

ii) Toxicity (other than alopecia) associated with prior anticancer therapy and/or surgery must either resolve or return to baseline or to Grade 1 or be considered irreversible.

iii) For cytotoxic agents, at least 4 weeks must elapse between the last dose prior to anticancer treatment and the start of study treatment.

iv) For non-cytotoxic agents, at least 4 weeks or 5 half-lives (whichever is less) must elapse from the last dose of prior anticancer therapy and the start of investigational therapy.

e) Prior therapy with anti-OX40 antibody.

f) Subjects with active, known or suspected autoimmune disease are excluded. Subjects with vitiligo, type 1 diabetes mellitus, residual hypothyroidism due to an autoimmune condition requiring only hormone replacement therapy, subjects with normal thyroid function with a history of Graves' disease (subjects with suspected autoimmune thyroid disorders must be negative in thyroglobulin and thyroid peroxidase antibodies and thyroid-stimulating immunoglobulin prior to the first dose of study drug), psoriasis not requiring systemic treatment, or conditions that are not expected to recur in the absence of an external trigger, study enrollment is permitted. Subjects with well-controlled asthma and/or mild allergic rhinitis (seasonal allergies) are eligible.

g) Subjects with a history of life-threatening toxicity associated with prior immune therapy (eg, treatment with an anti-CTLA-4 or anti-PD-1/PD-L1 antibody or any other antibody or drug specifically designed to stimulate T cells or immune checkpoint pathways), except for those that are unlikely to be repeated with standard responses (eg, hormone replacement therapy after an adrenal crisis).

h) Subjects with interstitial lung disease that is symptomatic or may interfere with detection or treatment of expected drug-related pulmonary toxicity.

i) Chronic obstructive pulmonary disease requiring repeated pulse therapy with steroids or chronic steroids at doses greater than 10 mg/day of prednisone or equivalent.

j) Subjects with a condition requiring systemic treatment with either corticosteroids (> 10 mg daily prednisone equivalents) or other immunosuppressive drugs within 14 days of study drug administration, excluding adrenal steroid replacement doses > 10 mg daily prednisone equivalents in the absence of active autoimmune disease. Note: Treatment with a short course of steroids (<5 days) up to 7 days prior to the start of the drug study is permitted.

k) Uncontrolled or significant cardiovascular disease, including, without limitation, one of the following:

i) myocardial infarction or stroke/transient ischemic attack within the last 6 months;

ii) uncontrolled sore throat within the last 3 months;

iii) any history of clinically significant arrhythmia (such as ventricular tachycardia, ventricular fibrillation, or bidirectional tachycardia);

iv) history of other clinically significant heart disease (eg, cardiomyopathy,

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v) cardiovascular disease-related requirement for daily supplemental oxygen therapy;

vi) QT interval corrected for heart rate using the Friedrichia formula (QTcF) > 480 ms.

l) Any history of chronic hepatitis as evidenced by the following:

i) a positive hepatitis B surface antigen test;

ii) a positive test for qualitative hepatitis C viral load (by PCR).

Note. Subjects with positive hepatitis C antibody and negative quantitative hepatitis C by PCR are eligible. A history of resolved hepatitis A virus infection is not an exclusion criterion. Additional testing or replacement testing is permitted under institutional guidance to rule out infection.

m) Evidence of active infection requiring systemic antibiotic, antiviral, or antifungal therapy < 7 days prior to initiation of study drug therapy (does not apply to viral infections suspected to be associated with the underlying tumor type required for study enrollment).

n) Known positive HIV test or history of acquired immunodeficiency syndrome.

o) Evidence of active or latent tuberculosis infection, including PPD recently converted to positive; chest x-ray with signs of infectious infiltrate; recent unexplained changes in fever/chill profiles or a history of them.

p) Any major surgery within 4 weeks of study drug administration. Subjects must have recovered from the effects of major surgery or significant traumatic injury at least 14 days prior to the first dose of study drug.

q) Use of non-cancer vaccines containing live virus for the prevention of infectious diseases within 4 weeks prior to the start of the drug study. The use of inactivated seasonal flu vaccines such as Fluzone® is permitted.

r) Use of pRBC or platelet transfusion within 2 weeks prior to the first dose of study drug.

s) A known or underlying medical or psychiatric condition and/or social reason that may cause the administration of the investigational medicinal product to be dangerous to subjects or may adversely affect the subject's ability to comply with or tolerate the study.

3) Allergies and adverse drug reactions.

a) History of allergy to nivolumab or ipilimumab (parts 2 and 3 only, respectively).

b) History of any significant drug allergy (such as anaphylaxis or hepatotoxicity) prior to prior anticancer immunomodulatory therapy (eg, checkpoint inhibitors, costimulatory T-cell antibodies).

Research estimates.

Physical examinations, vital signs measurements, 12-lead electrocardiograms (ECGs), and clinical laboratory evaluations are performed at selected time points during the dosing interval. Subjects are closely monitored for the presence of AE throughout the study.

Safety Assessments: AE is assessed during the study and up to 100 days after the last treatment. AE scores are scored according to NCI CTCAE v4.03. Subjects are monitored until all treatment-related AEs have recovered to baseline or are considered irreversible.

Efficacy Evaluations: Disease assessment by CT and/or MRI as needed is done at baseline and every 8 weeks (± 1 week) for the q2w dosing regimen and every 9 weeks (± 1 week) for the q3w dosing regimen, then every 12 weeks for throughout the treatment and response observation phase until treatment is discontinued or the study is withdrawn. Tumor evaluations at other time points are performed if there are concerns about tumor progression. Evaluation of tumor response is performed according to RECIST v1.1 for subjects with malignant tumors.

Pharmacokinetic and immunogenicity assessment: Samples for PK and immunogenicity assessment are collected from subjects receiving OX40.21 alone or in combination with nivolumab or ipilimumab. PK OX40.21 is characterized by a non-selective analysis (NCA) method. Immunogenicity samples are analyzed for anti-OX40.21 antibodies and/or anti-nivolumab antibodies and/or anti-ipilimumab antibodies using validated immunoassays.

Experimental assessments of biomarkers. To investigate potential predictive markers for clinical response to OX40.21 in relation to dose and PK, 3 types of specimens were obtained from

- 128 040561 all subjects for biomarker testing: (i) whole blood, (ii) serum/plasma, and (iii) tumor tissue.

Statistical aspects.

Sample size determination.

Dose increase.

As a phase 1 dose escalation clinical trial, the sample size for each escalation cohort depends on the observed toxicity and the inference of the posterior distribution. Approximately 30 subjects are treated during each dose escalation portion (OX40.21 monotherapy [Part 1A], OX40.21 in combination with nivolumab [Part 2A], and OX40.21 in combination with ipilimumab [Part 3A]) for a total of approximately 90 subjects in parts 1A, 2A and 3A. Initially, approximately 3 subjects are treated at initial dose levels of OX40.21 or OX40.21 in combination with nivolumab or ipilimumab. Additional cohorts of approximately 3 evaluable subjects are treated at the recommended doses as recommended by BLRM (-Copula) during the dose escalation phase. At least 6 subjects with assessed DLT are treated at MTD.

Increasing the dose to the maximum allowable.

In general terms, the size of the escalation phase to the maximum tolerable dose is based on the target response rate (target overall response rate) and the ability to identify a signal for such a clinical response that exceeds the standard of care (historical overall response rate).

Approximately 12 subjects are treated in Part 1B of the dose escalation cohort to the maximum tolerated dose. Approximately 35 subjects are treated in Part 2B of the dose escalation cohort to the maximum tolerated dose. Approximately 27 subjects are treated in Part 2C of the escalation cohort to the maximum tolerated dose. Approximately 35 subjects are treated in Part 3B of the escalation cohort to the maximum tolerated dose.

end points.

primary endpoints.

The safety score is based on prevalence of AE, severe AE, AE leading to termination and death. In addition, anomalies in clinical laboratory tests are investigated.

secondary endpoints.

Efficacy: The antitumor activity of OX40.21 and OX40.21 alone in combination with nivolumab or ipilimumab is measured by ORR, duration of response, and progression-free survival (PFSR) at 24 weeks based on RECIST v1.1. The above definition is based on measurements of tumors occurring at baseline every 8 weeks (± 1 week) for the q2w dosing regimen and every 9 weeks (± 1 week) for the q3w dosing regimen during the treatment period and every 3 months (12 weeks) during the time of the observation period for assessing survival.

The Best Overall Response (BOR) is scored according to RECIST 1.1 criteria.

ORR is the proportion of all treated subjects whose BOR is CR or PR.

Duration of response, calculated for all treated subjects with BOR CR or PR, is the time between the date of first response and the date of disease progression or death, whichever comes first.

PFSR at 24 weeks is defined as the proportion of treated subjects without progression and survivors at 24 weeks. The share is calculated from the Kaplan-Meier estimate, which takes into account the censored data.

Pharmacokinetics.

Selected parameters such as Cmax, Tmax, AUC(O-t) and AUC(TAU) are evaluated in 2 cycles depending on the schedule for monotherapy or in combination with nivolumab or ipilimumab. Parameters such as Ctau, CLT, Css-avg, accumulation index (AI) and effective half-life (T-HALFeff) are evaluated in the second cycle with intensive PK collected.

Immunogenicity.

The second immunogenicity target is assessed by the frequency of positive ADA to OX40.21 or nivolumab or ipilimumab.

Search endpoints.

OS-related search targets are evaluated by the frequency of OS at a given point in time (eg, 2 years). OS frequency is the proportion of subjects living at a given time. OS for a subject is defined as the time from the date of the first dose of study drug to the date of death from any cause. Biomarker-related search targets are assessed by changes from baseline in measurements of biomarkers in peripheral blood (eg, soluble factors, including but not limited to cytokines and chemokines) or tumor tissue (eg, tumor-infiltrating lymphocytes).

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For subjects with multiple ECG measurements, the following parameters are additionally assessed:

changes in ECG intervals QT, QTc, QRS and P-R interval from the initial values.

Analysis.

Safety Analysis: All reported AEs are listed and tabulated by system organ class, preferred timing, and treatment. Vital signs and clinical laboratory test results are listed and summarized by treatment. Also note any important results of the study of physical indicators and the results of clinical laboratory studies. Assess the ECG results and note abnormalities, if present.

Performance analysis. A list of tumor measurements is provided by subject and day of study at each treatment group and dose level. The subject's individual BOR is indicated based on RECIST 1.1. To describe the antitumor activity of OX40.21 alone or in combination with nivolumab or ipilimumab, the ORR is calculated. ORR and the corresponding Klopper-Pearson 2-tailed 95% CI are provided by treatment and/or dose level as well as tumor type. The median duration of response and the corresponding 2-tailed 95% CI are reported by treatment and/or dose and tumor type. The duration of the response is analyzed using the Kaplan-Meier method. In addition, PFSR, the probability that the subject will not progress or survive after 24 weeks, is assessed by the Kaplan-Meier method for treatment, tumor type and dose level. The corresponding 95% CI is obtained based on the Greenwood formula. OS is plotted on a graph using the Kaplan-Meier method. Median OS and corresponding 2-sided 95% CI are reported.

Pharmacokinetic analyses: All individual PK parameters are listed for each analyte, including any exclusions and reasons for exclusion from the generalization. Summary statistics are given for each treatment PK parameter. Geometric means and coefficients of variation are presented for Cmax, AUC(O-t), AUC(TAU), Ctau, CLT, Cssavg, and AI. Medians and ranges are for Tmax. Values and standard deviations are given for all other PK parameters (eg T-HALFeff).

Dose dependence of OX40.21 is evaluated in dose escalation monotherapy. To describe the dose dependence of OX40.21, scatter plots of Cmax, AUC(O-t) and AUC(TAU) versus dose are provided for each day measured. An exploratory estimate of dose proportionality is made based on the power model and CI around the power factor. The end of nivolumab and ipilimumab infusion and residual concentrations (Ctrough) and residual concentration of OX40.21 are tabulated according to treatment and study day using summary statistics. These data can also be combined with other datasets to analyze the PK of the sample.

Immunogenicity Analysis: All available immunogenicity data are provided in relation to treatment, dose and immunogenicity. The frequency of subjects with a positive score for ADA OX40.21, nivolumab, and ipilimumab is determined.

Exploratory analysis of biomarkers: summary statistics for biomarkers and their respective changes (or percentage changes) from baseline are shown in the table depending on the planned study day and dose in each group. The temporal dynamics of biomarker measures is presented graphically. If there is an indication of significant structure over time, additional analysis (eg, a linear mixed model) is performed to characterize the relationship. Methods such as, without limitation, logistic regression are used to explore possible associations between biomarker measures from peripheral blood or tumor biopsy and clinical outcomes.

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Table 23. Summary table of sequences

SEQ ID Description Subsequence 1 Human 0X40 Predecessor MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTY PSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYN DVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCR AGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTN CTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPA RPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLG LVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFR TPIQEEQADAHSTLAKI 2 Extracellular domain mature 0x40 human AVAA 3 OX40 cynomolgus monkey MCVGARRLGRGPCAALLLLGLGLSTTAKLHCVGDTY PSNDRCCQECRPGNGMVSRCNRSQNTVCRPCGPGFY NDVVSAKPCKACTWCNLRSGSERKQPCTATQDTVCR CRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWT NCTLAGKHTLQPASNSSDAICEDRDPPPTQPQETQGPP ARPTTVQPTEAWPRTSQRPSTRPVEVPRGPAVAAILGL GLALGLLGPLAMLLALLLLRRDQRLPPDAPKAPGGGS FRTPIQEEQADAHSALAKI 4 OX40-L human MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLbLCF TYICLHFSTL QVSHRYPRIQ SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF YLISLKGYFS QEVSISbBYQ KDEEPbFQUK KVRSVNSIHV ASLTYKDKVY UHAWTIMTSL DDFBVHGGEL II.IHQNPGEF CVX 5 Human IgGl constant domain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV

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EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 6 Human IgGl constant domain (allotype) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG 7 Human IgGl kappa light chain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLS SP VTKSFNRGEC 8 Alternate C-terminus of constant region heavy chain LSPGK 9 Alternate C-terminus of constant region heavy chain LSPG 10 Constant domain kappa light chain (CL) human IgGl RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLS SP VTKSFNRGEC AND CDR1 VH 3F4 SYDVN 12 CDR2 VH 3F4 WMNPNSGNTGYAPKFQG 13 CDR3 VH 3F4 IYSSSYNWFDP 14 CDR1 VL 3F4 RASQSVSSYLA 15 CDR2 VL 3F4 DASNRAT 16 CDR3 VL 3F4 QQRSNWPLT 17 VH3F4 QVQLVQSGAEVKKPGASVKVSCKASGNTFTSYDVNW VRQATGQGLEWMGWMNPNSGNTGYAPKFQGRVTM TRNTSISTAYMELS SLRSEDTAVYYC ARIYS S S YNWFD PWGQGTLVTVSS

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18 VL 3F4 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPLTFGGGTKVEIK 19 CDR1 VH 14B6 SNWIG 20 CDR2 VH 14B6 FIYPGDSDTRYSPSFQG 21 CDR3 VH 14B6 YGDDWYFDL 22 CDR1 VL1 14B6 RASQSVSSYLA 23 CDR2 VL1 14B6 DASNRAT 24 CDR3 VL1 14B6 QQRGDWPIT 25 CDR1 VL2 14B6 RASQGISSWLA 26 CDR2 VL2 14B6 AASSLQS 27 CDR3 VL2 14B6 QQYNSYPRIT 28 VH 14B6 EVQLEQSGAEVKKPGESLKISCKGSGYSFTSNWIGWV RQMPGKGLEWMGFIYPGDSDTRYSPSFQGQVTISADK SISTAYLQWSSLKASDIAMYYCARYGDDWYFDLWGR GTLVTVSS 29 VL1 14B6 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWFQQ RPGQAPRLLIYDASNRATGIPARFSGSGSGTDFSLTISS LEPEDFAVYYCQQRGDWPITFGQGTRLEIK thirty VL2 14B6 DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQ KPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQYNSYPRITFGQGTRLEIK 31 CDR1 VH 23H3 NYAMY 32 CDR2 VH 23H3 AIGIGGDTFYTDSVKG 33 CDR3 VH 23H3 MGTGYFFDY 34 CDR1 VL 23H3 RASQSVSSYLA 35 CDR2 VL 23H3 DASNRAT 36 CDR3 VL 23H3 QQRSNWPLT 37 VH 23H3 EVQLVQ SGGGLVHPGGSLRLSC AGSGFTF SNYAMYW VRQAPGKGLEWVSAIGIGGDTFYTDSVKGRFTISRDN AKNSLSLQMNSLRAEDMAVYYCARMGTGGYFFDYWG QGTLVTVSS

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38 VL 23H3 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPLTFGPGTKVDIK 39 CDR1 VH 6E1 SFAMH 40 CDR2 VH 6E1 VISYDGSIKYYTDSVKG 41 CDR3 VH 6E1 DGNYGSARYFQH 42 CDR1 VL1 6E1 RASQGISSWLA 43 CDR2 VL1 6E1 AASSLQS 44 CDR3 VL1 6E1 QQYNSYPRT 45 CDR1 VL2 6E1 RASQSVSSYLA 46 CDR2 VL2 6E1 DASNRAT 47 CDR3 VL2 6E1 QQRSNWPYT 48 VH 6E1 Q VQLVESGGGVVQPGRSLRLSC AASGFTF S SF AMHW VRQAPGKGLEWVTVISYDGSIKYYTDSVKGRFTFSRD NSKNTLYLQMNSLRAEDTAVYYCTRDGNYGSARYFQ HWGQGTLVTVSS 49 VLI 6E1 DIQMTQ SP S SLS AS VGDRVTITCRASQGIS S WL AW YQQ KPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQYNSYPRTFGQGTKVEIK 50 VL2 6E1 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPYTFGQGTKLEIK 51 CDR1 VH 18E9 SSAMH 52 CDR2 VH 18E9 AIGTGGDTYYADSVKG 53 CDR3 VH 18E9 DFYDILTGIFDY 54 CDR1 VL 18E9 RASQGISSWLA 55 CDR2 VL 18E9 AASSLQS 56 CDR3 VL 18E9 QQANSFST 57 VH 18E9 EVQLVQSGGGLVHPGGSLRLSCAHSGFTFTSSAMHW VRQAPGKGLEWISAIGTGGDTYYADSVKGRFTISRDN AKNSLYLQINSLRAEDMAVYYCARDFYDILTGIFDYW GQGTLVTVSS

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58 VL 18E9 DIQMTQ SP S S VS AS VGDRVTITCRASQGIS S WL AW YQ HKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQANSFPSTFGQGTKVEIK 59 CDR1 VH8B11 SDAMY 60 CDR2 VH8B11 AIGIGGDTYYTDSVMG 61 CDR3 VH8B11 LGMGYYFDY 62 CDR1 VL8B11 RASQSVSSYLA 63 CDR2 VL8B11 DASNRAT 64 CDR3 VL8B11 QQRSNWPPT 65 VH8B11 MEFVLSWVFLVAILKGVQCEIQLVQSGGGLVHPGGSL RLSCAGSGFTFSSDAMYWVRQAPGKGLEWVSAIGIGG DT YYTD S VMGRFTISRDNAKNSLYLQMNSLRAEDMA VYYC ARLGMGYYFD YWGQGTL VT VS S 66 VL8B11 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPPTFGQGTKVEIK 67 CDR1 VH 20B3 SYDMH 68 CDR2 VH 20B3 VIGTAGDTYYPGSVKG 69 CDR3 VH 20B3 GGMGNYFDY 70 CDR1 VL 20B3 RASQSVSSYLA 71 CDR2 VL 20B3 DASNRAT 72 CDR3 VL 20B3 QQRSNWPLT 73 VH20B3 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHW VRQTTGKGLEWVSVIGTAGDTYYPGSVKGRFTISREN AKNSLYLQMNSLRAGDTAVYYCARGGMGNYFDYW GQGTLVTVSS 74 VL 20B3 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPLTFGGGTKVEIK 75 CDR1 VH 14A2 NYALH 76 CDR2 VH 14A2 LISYDGSRKHYADSVKG 77 CDR3 VH 14A2 LTMVREGG 78 CDR1 VL1 14A2 RASQSVSSSYLA

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79 CDR2 VL1 14A2 GASSRAT 80 CDR3 VL1 14A2 QQYGSSPFT 81 CDR1 VL2 14A2 RVSQGISSYLN 82 CDR2 VL2 14A2 SASNLQS 83 CDR3 VL2 14A2 QRTYNAPYT 84 VH 14A2 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGI<GLEWVALISYDGSRI<HYADSVI<GRF SISRD NSKNTLYLQMNSLRAEDTAVYYCASLTMVREGGQGT LVTVSS 85 VL1 14A2 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQ APRLLIYGAS SRATGIPDRF SGSGSGTDFTLTIS RLEPEDF AVYYCQQ YGS SPFTFGPGTKVDIK 86 VL2 14A2 DIQLTQSPSSLSASVGDRVTITCRVSQGISSYLNWYRQ KPGKVPKLLIYSASNLQSGVPSRFSGSGSGTDFTLTISS LQPEDVATYYGQRTYNAPYTFGGGTKVEIK 87 CDR1 VH20C1 SYAMY 88 CDR2 VH20C1 AIDTDGGTF YAD S VRG 89 CDR3 VH20C1 LGEGYFFDY 90 CDR1 VL20C1 RASQSVSSYLA 91 CDR2 VL20C1 DASNRAT 92 CDR3 VL20C1 QQRSNWPPT 93 VH20C1 EAQLVQSGGGLVHPGGSLRLSCADSGFTFSSYAMYW VRQAPGKGLEWVSAIDTDGGTFYADSVRGRFTISRDN AKNSLYLQMNGLRAEDMAVYFCARLGEGYFFDYWG QGTLVTVSS 94 VL20C1 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPPTFGGGTKVEIK 95 Heavy chain 0X40.6 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYW VRQAPGKGLEWVSAIGIGGDTFYTDSVKGRFTISRDN AKNSLSLQMNSLRAEDTAVYYCARMGTGYFFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

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WTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 96 Light chain 0X40.6 EIVLTQSPATLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPLTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEK HKVYACEVQACEV 97 Heavy chain 0X40.7 EVQLVQSGGGLVHPGGSLRLSCAGSGFTFSNYAMYW VRQAPGKGLEWVSAIGIGGDTFYTDSVKGRFTISRDN AKNSLSLQMNSLRAEDTAVYYCARYGTGYFFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 98 Light chain 0X40.7 EIVLTQSPATLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPLTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEK HKVYACEVQACEV

- 137 040561

99 Heavy chain 0X40.8 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGKGLEWVALISYDGSRKHYADSVKGRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 100 Light chain 0X40.8 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGAS SRATGIPDRF SGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSRGTYSLSSTLTLSKADYEKVEKN HKVYACE 101 Heavy chain 0X40.9 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGKGLEWVALISYDGSRKHYADSVKGRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTYVREWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 102 Light chain 0X40.9 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGAS SRATGIPDRF SGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS

- 138 040561

VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLS SP VTKSFNRGEC 103 Heavy chain 0X40.10 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGKGLEWVALISYSGSRKHYADSVKGRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTMVREGGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 104 Light chain 0X40.10 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQ APRLLIYGAS SRATGIPDRF SGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESSPTEQDSKDSKDSSLSSTLTLSKADYGLECTH HKVY 105 Heavy chain 0X40.11 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGKGLEWVALISYDSSRKHYADSVKGRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTMVREGGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

- 139 040561

106 Light chain 0X40.11 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQ APRLLIYGAS SRATGIPDRF SGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESSPTEQDSKDSKDSSLSSTLTLSKADYGLECTH HKVY 107 Heavy chain 0X40.12 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGKGLEWVALISYSGSRKHYADSVKGRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 108 Light chain 0X40.12 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQ APRLLIYGAS SRATGIPDRF SGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESSPTEQDSKDSKDSSLSSTLTLSKADYGLECTH HKVY 109 Heavy chain 0X40.13 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGI<GLEWVALISYDSSRI<HYADSVI<GRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK

- 140 040561

TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 110 Light chain 0X40.13 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGAS SRATGIPDRF SGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSRGTYSLSSTLTLSKADYEKVEKN HKVYACE 111 Heavy chain 0X40.14 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGKGLEWVALISYSGSRKHYADSVKGRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTYVREWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 112 Light chain 0X40.14 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGAS SRATGIPDRF SGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSRGTYSLSSTLTLSKADYEKVEKN HKVYACE FROM Heavy chain 0X40.15 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGKGLEWVALISYDSSRKHYADSVKGRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTYVREWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVSVTVSWNSGALTSGVHTFPAVLQSSGKSLYSLPSTHKNTNVSLGTPDKNTVCDLYSLPSTHKNTNVSLGEPDNKNHK

- 141 040561

TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYGNTHHYSKLT VDKKSVLDGSFFLYGNTHQLSVDKSRWQQQQLSV 114 Light chain 0X40.15 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQAPRLLIYGAS SRATGIPDRF SGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSRGTYSLSSTLTLSKADYEKVEKN HKVYACE 115 Heavy chain 0X40.16 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYW VRQAPGKGLEWVSAIDTDGGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG 116 Light chain 0X40.16 (contained in 0X40.20, 0X40.21, 0X40.22) EIVLTQSPATLSLPGERAATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPPTFGGGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEK HKVYACEVQACEV 117 Heavy chain 0X40.17 EVQLVESGGGLVQPGGSLRLSC AASGFTF S S YDMHW VRQTTGKGLEWVSVIGTAGDTYYPGSVKGRFTISREN

- 142 040561

AKNSLYLQMNSLRAGDTAVYYCARGGMGNYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKD YFPEP VTVSWNSGALTSGVHTFP AVLQ S SGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 118 Light chain 0X40.17 EIVLTQSPATLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEK HKVYACEVQACEV 119 Heavy chain 0X40.18 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVNW VRQATGQGLEWMGWMNPNSGNTGYAPKFQGRVTM TRDT SIS Т AYMELS SLRSEDT AVYYC ARIYS S S YNWFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLS S VVT VP S S SLGTQT YICNVNHKP SNTKVDKRVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 120 Light chain 0X40.18 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRTVAAPS

- 143 040561

VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLS SP VTKSFNRGEC 121 Heavy chain 0X40.19 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGKGLEWVALISYDGSRKHYADSVKGRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTLVREWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 122 Light chain 0X40.19 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQ QKPGQ APRLLIYGAS SRATGIPDRF SGSGSGTDFTLTIS RLEPEDFAVYYCQQYGSSPFTFGPGTKVDIKRTVAAPS VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESSPTEQDSKDSKDSSLSSTLTLSKADYGLECTH HKVY 123 Heavy chain 0X40.20 EVQLVQ SGGGLVQPGGSLRLSC AGSGFTFS S YAMYW VRQAPGKGLEWVSAIDTSGGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC WVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

- 144 040561

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG — Light chain 0X40.20 SEQID NO: 116 124 Heavy chain 0X40.21 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYW VRQAPGKGLEWVSAIDTDAGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC WVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG — Light chain 0X40.21 SEQID NO: 116 125 Heavy chain 0X40.22 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYW VRQAPGKGLEWVSAIDTSTGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG — Light chain 0X40.22 SEQ ID NO: 116 126 VH (nucleotide sequence)3F4 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAA GAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGG

- 145 040561

CTTCTGGAAACACCTTCACCAGTTATGATGTCAACT GGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGG ATGGGATGGATGAACCCTAACAGTGGTAACACAGG CTATGCACCGAAGTTCCAGGGCAGAGTCACCATGA CCAGGAACACCTCCATAAGCACAGCCTACATGGAG CTGAGCAGCCTGAGATCTGAGGACACGGCCGTTTAT TACTGTGCGAGAATATATAGCAGCTCGTACAACTGG TTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTC TCCTCA 127 VL 3F4 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTAC CAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTC ACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 128 VH 14B6 GAGGTGCAGCTGGAGCAGTCTGGAGCAGAGGTGAA AAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGG GTTCTGGATACAGCTTTACCAGCAACTGGATCGGCT GGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGG ATGGGGTTCATCTATCCTGGTGACTCTGATACCAGG TACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCA GCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGG AGCAGCCTCAAGGCCTCGGACATCGCCATGTATTAC TGTGCGAGATATGGGGATGACTGGTACTTCGATCTC TGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA 129 VL1 14B6 GAAATTGTGTTGACACAAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTTC CAACAGAGACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC

- 146 040561

AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCTCT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTGGCGACTGGCCCATC ACTTCGGCCAAGGGACACGACTGGAGATTAAA 130 VL2 14B6 GACATCCAGATGACCCAGTCTCCATCCTCACTGTCT GCATCTGTAGGAGACAGAGTCACCATCACTTGTCGG GCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTAT CAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGAT CTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATC AAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCA CTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTG CAACTTATTACTGCCAACAGTATAATAGTTACCCTC GGATCACCTTCGGCCAAGGGACACGACTGGAGATT AAA 131 VH 23H3 GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGT ACATCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGG CTCTGGATTCACCTTCAGTAACTATGCTATGTACTG GGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGG TATCAGCCATTGGTATTGGTGGTGACACATTCTATA CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAG ACAATGCCAAGAACTCCTTGTCTCTTCAAATGAACA GCCTGAGAGCCGAGGACATGGCTGTGTATTACTGTG CAAGAATGGGAACTGGGTACTTCTTTGACTACTGGG GCCAGGGAACCCTGGTCACCGTCTCCTCA 132 VL 23H3 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTAC CAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTC ACTTTCGGCCCTGGGACCAAAGTGGATATCAAA

- 147 040561

133 VH 6E1 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAGCTTTGCTATGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGACAGTTATTTCATATGATGGAAGCATTAAATACT ACACAGACTCCGTGAAGGGCCGATTCACCTTCTCCA GAGACAATTCCAAGAACACTCTGTATCTGCAAATGA ACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACT GTACGAGAGATGGAAACTATGGTTCGGCGAGATAC TTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTC TCCTCA 134 VLI 6E1 GACATCCAGATGACCCAGTCTCCATCCTCACTGTCT GCATCTGTAGGAGACAGAGTCACCATCACTTGTCGG GCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTAT CAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGAT CTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATC AAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCA CTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTG CAACTTATTACTGCCAACAGTATAATAGTTACCCTC GGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 135 VL2 6E1 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTAC CAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCGTAC ACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 136 VH 18E9 GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTT CATCCTGGGGGGTCCCTGAGACTCTCCTGTGCAACAC TCTGGATTCACCTTCACTAGCTCTGCTATGCACTGG GTTCGCCAGGCTCCAGGAAAAGGTCTGGAATGGAT

- 148 040561

ATCAGCTATTGGTACTGGTGGTGACACATACTATGC AGACTCCGTGAAGGGCCGATTCACCATCTCCAGAG ACAATGCCAAGAACTCCTTGTATCTTCAAATAAACA GCCTGAGAGCCGAGGACATGGCTGTATATTACTGTG CAAGAGACTTTTACGATATTTTGACTGGTATCTTTG ACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCT CA 137 VL 18E9 GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCT GCATCTGTAGGAGACAGAGTCACCATCACTTGTCGG GCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTAT CAGCATAAACCAGGGAAAGCCCCTAAGCTCCTGAT CTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATC AAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCA CTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTG CAACTTACTATTGTCAACAGGCTAATAGTTTCCCTT CGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 138 VH8B11 ATGGAGTTTGTGCTGAGCTGGGTTTTCCTTGTTGCTA TATTAAAAGGTGTCCAGTGTGAAATTCAGCTGGTGC AGTCTGGGGGAGGCTTGGTACATCCTGGGGGGTCCC TGAGACTCTCCTGTGCAGGCTCTGGATTCACCTTCA GTAGCGATGCTATGTACTGGGTTCGCCAGGCTCCAG GAAAAGGTCTGGAGTGGGTATCAGCTATTGGTATTG GTGGTGACACATACTATACAGACTCCGTGATGGGCC GATTCACCATCTCCAGAGACAATGCCAAGAACTCCT TGTATCTTCAAATGAACAGCCTGAGAGCCGAGGAC ATGGCTGTGTATTACTGTGCAAGGCTGGGGATGGGG TACTACTTTGACTACTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCA 139 VL8B11 GAAATTGTGTTGACACAAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTAC CAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC

- 149 040561

AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCG ACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 140 VH20B3 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAGCTACGACATGCACTG GGTCCGCCAAACTACAGGAAAAGGTCTGGAGTGGG TCTCAGTTATTGGTACTGCTGGTGACACATACTATC CAGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAG AAAATGCCAAGAACTCCTTGTATCTTCAAATGAACA GCCTGAGAGCCGGGGACACGGCTGTGTATTACTGTG CAAGAGGGGGGATGGGGAACTACTTTGACTACTGG GGCCAGGGAACCCTGGTCACCGTCTCCTCA 141 VL 20B3 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTAC CAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCGCTC ACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 142 VH 14A2 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAACTATGCTCTGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCACTTATATCATATGATGGAAGCAGGAAACACT ACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCA GAGACAATTCCAAGAACACACTGTATCTGCAAATG AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTA CTGTGCGAGTCTTACTATGGTTCGGGAGGGGGGCCA GGGAACCCTGGTCACCGTCTCCTCA

- 150 040561

143 VL1 14A2 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC ATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTT TGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACC ATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAA A 144 VL2 14A2 GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCT GCATCTGTAGGAGACAGAGTCACCATCACTTGCCGG GTGAGTCAGGGCATTAGCAGTTATTTAAATTGGTAT CGGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGAT CTATAGTGCATCCAATTTGCAATCTGGAGTCCCATC TCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCAC TCTCACTATCAGCAGCCTGCAGCCTGAAGATGTTGC AACTTATTACGGTCAACGGACTTACAATGCCCCTTA CACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 145 VH20C1 GAGGCTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTT CATCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGAC TCTGGATTCACCTTCAGTAGCTATGCTATGTACTGG GTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGT ATCAGCTATTGATACTGATGGTGGCACATTCTATGC AGACTCCGTGCGGGGCCGATTCACCATCTCCAGAGA CAATGCCAAGAACTCCTTGTATCTTCAAATGAACGG CCTGAGAGCCGAGGACATGGCTGTGTATTTCTGTGC AAGACTTGGGGAAGGGTACTTCTTTGACTACTGGGG CCAGGGAACCCTGGTCACCGTCTCCTCA 146 VL20C1 GAAATTGTGTTGACACAAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTAC CAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC

- 151 040561

TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCC ACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 147 Heavy chain 0X40.6 GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGT ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGG CTCTGGATTCACCTTCAGTAACTATGCTATGTACTG GGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGG TATCAGCCATTGGTATTGGTGGTGACACATTCTATA CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAG ACAATGCCAAGAACTCCTTGTCTCTTCAAATGAACA GCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTG CAAGAATGGGAACTGGGTACTTCTTTGACTACTGGG GCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCA CCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGC TGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACT CTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG CAGCTTGGGCACCCAGACCTACATCTGCAACGTGAA TCACAAGCCCAGCAACACCAAGGTGGACAAGAGAG TTGAGCCCAAATCTTGTGACAAAACTCACACATGCC CACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAG CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA AGGTCTCCA ACAAAGCCCTCCCAGCCCCCATCGAGA

- 152 040561

AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA CCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGG TCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCTCTCCCTGTCCCCGGGT 148 Light chain 0X40.6 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTAC CAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTC ACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGT ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATC GGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACA GCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTC GCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 149 Heavy chain 0X40.7 GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGT ACATCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGG CTCTGGATTACCTTCAGTAACTATGCTATGTACTG GGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGG TATCAGCCATTGGTATTGGTGGTGACACATTCTATA

- 153 040561

CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAG ACAATGCCAAGAACTCCTTGTCTCTTCAAATGAACA GCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTG CAAGATATGGAACTGGGTACTTCTTTGACTACTGGG GCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCA CCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGC TGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACT CTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG CAGCTTGGGCACCCAGACCTACATCTGCAACGTGAA TCACAAGCCCAGCAACACCAAGGTGGACAAGAGAG TTGAGCCCAAATCTTGTGACAAAACTCACACATGCC CACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCC TCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAG CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA AGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGA AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA CCACAGGTGTACACCCTGCCCCCATCCCGGGAGGA GATGACCAAGAACCAGGTCAGCCTGACCTGCCTGG TCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT GGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG

- 154 040561

150 Light chain 0X40.7 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTAC CAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTC ACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGT ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATC GGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACA GCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGT CTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTC GCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 151 Heavy chain 0X40.8 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAACTATGCTCTGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCACTTATATCATATGATGGAAGCAGGAAACACT ACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCA GAGACAATTCCAAGAACACACTGTATCTGCAAATG AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTA CTGTGCGAGTCTTACTATGGTTCGGGAGTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAA GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC

- 155 040561

TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA GCCCAAATCTTGTGACAAAACTCACACATGCCCACC GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCCCCGGGT 152 Light chain 0X40.8 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC ATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTT TGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACC ATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAA ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC

- 156 040561

GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT 153 Heavy chain 0X40.9 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAACTATGCTCTGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCACTTATATCATATGATGGAAGCAGGAAACACT ACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCA GAGACAATTCCAAGAACACACTGTATCTGCAAATG AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTA CTGTGCGAGTCTTACTTACGTTCGGGAGTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAA GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA GCCCAAATCTTGTGACAAAACTCACACATGCCCACC GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC

- 157 040561

AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCCCCGGGT 154 Light chain 0X40.9 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC ATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTT TGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACC ATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAA ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT

- 158 040561

155 Heavy chain 0X40.10 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAACTATGCTCTGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCACTTATATCATATAGTGGAAGCAGGAAACACT ACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCA GAGACAATTCCAAGAACACACTGTATCTGCAAATG AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTA CTGTGCGAGTCTTACTATGGTTCGGGAGGGGGGCCA GGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAA GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA GCCCAAATCTTGTGACAAAACTCACACATGCCCACC GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG TCTCCAACAAAG CCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT GACCAAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGGCAATGGGCAGCCGGAGAACAACTACAAGACC

- 159 040561

ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCCCCGGGT 156 Light chain 0X40.10 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC ATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTT TGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACC ATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAA ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT 157 Heavy chain 0X40.11 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAACTATGCTCTGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCACTTATATCATATGATAGTAGCAGGAAACACT ACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCA GAGACAATTCCAAGAACACACTGTATCTGCAAATG AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTA CTGTGCGAGTCTTACTATGGTTCGGGAGGGGGGCCA

- 160 040561

GGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAA GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA GCCCAAATCTTGTGACAAAACTCACACATGCCCACC GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCAC AACCACTACACCGCAGAAGAGCC TCTCCCTGTCCCCGGGT 158 Light chain 0X40.11 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC

- 161 040561

ATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTT TGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACC ATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAA ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT 159 Heavy chain 0X40.12 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAACTATGCTCTGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCACTTATATCATATAGTGGAAGCAGGAAACACT ACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCA GAGACAATTCCAAGAACACACTGTATCTGCAAATG AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTA CTGTGCGAGTCTTACTATGGTTCGGGAGTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAA GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA

- 162 040561

GCCCAAATCTTGTGACAAAACTCACACATGCCCACC GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCCCCGGGT 160 Light chain 0X40.12 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC ATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTT TGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACC ATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAA ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC

- 163 040561

AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT 161 Heavy chain 0X40.13 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAACTATGCTCTGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCACTTATATCATATGATAGTAGCAGGAAACACT ACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCA GAGACAATTCCAAGAACACACTGTATCTGCAAATG AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTA CTGTGCGAGTCTTACTATGGTTCGGGAGTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAA GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA GCCCAAATCTTGTGACAAAACTCACACATGCCCACC GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG

- 164 040561

TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCCCCGGGT 162 Light chain 0X40.13 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC ATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTT TGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACC ATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAA ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT 163 Heavy chain 0X40.14 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTACCTTCAGTAACTATGCCTCTGCACTG

- 165 040561

GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCACTTATATCATATAGTGGAAGCAGGAAACACT ACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCA GAGACAATTCCAAGAACACACTGTATCTGCAAATG AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTA CTGTGCGAGTCTTACTTACGTTCGGGAGTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAA GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA GCCCAAATCTTGTGACAAAACTCACACATGCCCACC GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT GACCAAGAACCAG GTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA

- 166 040561

TGAGGCCTCTGCACAACCACTACACCGCAGAAGAGCC TCTCCCTGTCCCCGGGT 164 Light chain 0X40.14 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC ATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTT TGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACC ATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAA ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT 165 Heavy chain 0X40.15 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAACTATGCTCTGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCACTTATATCATATGATAGTAGCAGGAAACACT ACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCA GAGACAATTCCAAGAACACACTGTATCTGCAAATG AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTA CTGTGCGAGTCTTACTTACGTTCGGGAGTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAA GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC

- 167 040561

TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA GCCCAAATCTTGTGACAAAACTCACACATGCCCACC GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCCCCGGGT 166 Light chain 0X40 L 5 GAAATTGTGTTGACGCAGTCTCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC ATTCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGACTGTCTGGGACAGACTT CACTCATCAGCTACT

- 168 040561

TGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACC ATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAA ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT 167 Heavy chain 0X40.16 GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTT CAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGGC TCTGGATTCACCTTCAGTAGCTATGCTATGTACTGG GTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGT ATCAGCTATTGATACTGATGGTGGCACATTCTATGC AGACTCCGTGCGGGGCCGATTCACCATCTCCAGAGA CAATGCCAAGAACTCCTTGTATCTTCAAATGAACAG CCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGC AAGACTTGGGGAAGGGTACTTCTTTGACTACTGGGG CCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCAC CAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA GCTTGGGCACCCAGACCTACATCTGCAACGTGAATC ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTT GAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC

- 169 040561

ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATG CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT CCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCCTGTCCCCGGGTTGA 168 Light chain 0X40.16 (contained in 0X40.20, 0X40.21, 0X40.22) GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTAC CAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCC ACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACG TACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCC ATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGC CAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAG

- 170 040561

TCTAGCCTGCGAAGTCACCCATCAGGGCCTGAGCT CGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TAG 169 Heavy chain 0X40.17 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGT ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAGCTACGACATGCACTG GGTCCGCCAAACTACAGGAAAAGGTCTGGAGTGGG TCTCAGTTATTGGTACTGCTGGTGACACATACTATC CAGGCTCCGTGAAGGGCCGATTCACCATCTCCAGAG AAAATGCCAAGAACTCCTTGTATCTTCAAATGAACA GCCTGAGAGCCGGGGACACGGCTGTGTATTACTGTG CAAGAGGGGGGATGGGGAACTACTTTGACTACTGG GGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGC ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGG CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGAC GGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCG TGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGAC TCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA ATCACAAGCCCAGCAACACCAAGGTGGACAAGAGA GTTGAGCCCAAATCTTGTGACAAAACTCACACATGC CCACCGTGCCCAGCACCTGAACTCCTGGGGGGACC GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACAC CCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCA AGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGT GCAAGGTCTCCA ACAAAGCCCTCCCAGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGA GAACCACAGGTGTACACCCTGCCCCCATCCCGGGA

- 171 040561

GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACGCAGAA GAGCCTCTCCCTGTCCCCGGGTTGA 170 Light chain 0X40 L 7 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTAC CAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCGCTC ACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACG TACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCC ATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGC CAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCT CGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TAG 171 Heavy chain 0X40 L 8 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAA GAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGG CTTCTGGATACACCTTCACCAGTTATGATGTCAACT GGGTGCGACAGGCCACTGGACAAGGGCTTGAGTGG ATGGGATGGATGAACCCTAACAGTGGTAACACAGG CTATGCACCGAAGTTCCAGGGCAGAGTCACCATGA

- 172 040561

CCAGGGACACCTCCATAAGCACAGCCTACATGGAG CTGAGCAGCCTGAGATCTGAGGACACGGCCGTTTAT TACTGTGCGAGAATATATAGCAGCTCGTACAACTGG TTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTC TCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACA GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCT GACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGAC CGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT CTGCAACGTGAATCACAAGCCCAGCAACACCAAGG TGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAACTC CTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGA CCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTC CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCC AGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG GGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC CCATCCCGGGAGGAGATGACCAAGAACCAGGTCAG CCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG AGAACAACTACAA GACCACGCCTCCCGTGCTGGAC TCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACC GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT

- 173 040561

172 Light chain 0X40.18 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTAC CAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGATGCATCCAACAGGGCCACTGGCATCCCAGCC AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACT CTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTC ACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACG TACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCC ATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGC CAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAG TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCT CGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TAG 173 Heavy chain 0X40.19 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGT CCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGC CTCTGGATTCACCTTCAGTAACTATGCTCTGCACTG GGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGG TGGCACTTATATCATATGATGGAAGCAGGAAACACT ACGCAGACTCCGTGAAGGGCCGATTCAGTATCTCCA GAGACAATTCCAAGAACACACTGTATCTGCAAATG AACAGCCTGAGAGCTGAGGACACGGCTGTGTATTA CTGTGCGAGTCTTACTCTGGTTCGGGAGTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAA GGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC

- 174 040561

ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC TTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA GCCCAAATCTTGTGACAAAACTCACACATGCCCACC GTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGAT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTATAGCAAGCTCACCGTGGACAAGAGTAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCC TCTCCCTGTCCCCGGGT 174 Light chain 0X40.19 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCT TTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGG GCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGG TACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC ATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTT CACTCTCACCATCAGCAGACTGGAGCCTGAAGATTT TGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACC ATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAA

- 175 040561

ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCC GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGA GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGT 175 Heavy chain 0X40.20 GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTT CAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGGC TCTGGATTCACCTTCAGTAGCTATGCTATGTACTGG GTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGT ATCAGCTATTGATACTAGTGGTGGCACATTCTATGC AGACTCCGTGCGGGGCCGATTCACCATCTCCAGAGA CAATGCCAAGAACTCCTTGTATCTTCAAATGAACAG CCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGC AAGACTTGGGGAAGGGTACTTCTTTGACTACTGGGG CCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCAC CAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA GCTTGGGCACCCAGACCTACATCTGCAACGTGAATC ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTT GAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT

- 176 040561

CAACTGGTACGTGGACGGCGTGGAGGTGCATAATG CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT CCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCCTGTCCCCGGGTTGA — Light chain 0X40.20 SEQTDNO: 168 176 Heavy chain 0X40.21 GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTT CAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGGC TCTGGATTCACCTTCAGTAGCTATGCTATGTACTGG GTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGT ATCAGCTATTGATACTGATGCTGGCACATTCTATGC AGACTCCGTGCGGGGCCGATTCACCATCTCCAGAGA CAATGCCAAGAACTCCTTGTATCTTCAAATGAACAG CCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGC AAGACTTGGGGAAGGGTACTTCTTTGACTACTGGGG CCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCAC CAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA GCTTGGGCACCCAGACCTACATCTGCAACGTGAATC

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ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTT GAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATG CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT CCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCCTGTCCCCGGGTTGA — Light chain 0X40.21 SEQID NO: 168 177 Heavy chain 0X40.22 GAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTT CAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGGC TCTGGATTCACCTTCAGTAGCTATGCTATGTACTGG GTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGT ATCAGCTATTGATACTAGTACTGGCACATTCTATGC AGACTCCGTGCGGGGCCGATTCACCATCTCCAGAGA CAATGCCAAGAACTCCTTGTATCTTCAAATGAACAG CCTGAGAGCCGAGGACACGGCTGTGTATTTCTGTGC AAGACTTGGGGAAGGGTACTTCTTTGACTACTGGGG CCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCAC CAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTC

- 178 040561

CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA GCTTGGGCACCCAGACCTACATCTGCAACGTGAATC ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTT GAGCCCAAATCTTGTGACAAAACTCACACATGCCCA CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATG CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAA AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC CACAGGTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT CCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGC CTCTCCCTGTCCCCGGGTTGA — Light chain 0X40.22 SEQID NO: 168 178 hOX40 epitope D VVS SKPCKPCTWCNLR 179 hOX40 epitope DSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGK 180 Peptide linker PVGVV 181 Sortase A recognition motif LPXTG where X is any amino acid

- 179 040561

182 hOX40 epitope QNT VCRPCGPGF YND VVS SKPCKPCTWCNLR 183 hOX40 epitope PCKPCTWCNLR 184 hOX40 epitope QLCTATQDTVCR 185 hOX40 epitope SQNTVCRPCGPGFYN 186 C-terminal CH1 of IgGl (same for IgG3 (17-15-1515), igG3 (17-15-15), IgG3 (17-15), IgG3 (15-15-15), IgG3 (15) and IgG4 VDKRV 187 CH1 C-terminus of IgG2 VDKTV 188 Upper Hinge IgGl EPKSCDKTHT 189 Upper hinge IgG3 (17-15-15-15) (same for IgG3 (17-15-15) and IgG3 (17-15)) ELKTPLGDTTHT 190 Upper hinge IgG3 (15-15-15) (same for IgG3(15)) EPKS 191 Upper hinge IgG4 ESKYGPP 192 Middle hinge IgGl CPPCP 193 Middle hinge IgG2 CCVECPPCP 194 Middle hinge IgG3 (17-15-15-15) CPRCP(EPKSCDTPPPCPRCP) ₃ 195 Middle hinge IgG3 (17-15-15) CPRCP(EPKSCDTPPPCPRCP) ₂ 196 Middle hinge IgG3 (P-15) CPRCP(EPKSCDTPPPCPRCP)i 197 Middle hinge IgG3 (15-15-15) CDTPPPPCPRCP(EPKSCDTPPPCPRCP) ₂ 198 Middle Hinge IgG3 (15) CDTPPPPCPRCP 199 Middle hinge IgG4 CPSCP

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200 IgGl lower hinge (same for IgG3 (1715-15-15), IgG3 (17-1515), IgG3 (17-15), IgG3 (15-15-15), IgG3 (15) and IgG4) APELLGG 201 Inferior hinge IgG2 APPVAG 202 CHI human wild-type IgGl ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKV 203 CHI human wild-type IgG2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTV 204 CH2 human wild-type IgGl PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAK 205 CH2 human wild-type IgG2 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTK 206 CH3 human wild-type IgGl GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 207 SNZ human wild-type IgG2 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 208 Alternative Hinge ERKCCVECPPCP APPVAG 209 Alternative Hinge ERKSCVECPPCP APPVAG 210 Alternative Hinge ERKC SVECPPCP APPVAG 211 Alternative Hinge ERKXCVECPPCP APPVAG 212 Alternative Hinge ERKCXVECPPCP APPVAG 213 Alternative Hinge ERKCCVECPPCP APPVAGX 214 Alternative Hinge ERKSCVECPPCP APPVAGX 215 Alternative Hinge ERKC SVECPPCP APPVAGX

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216 Alternative Hinge ERKXCVECPPCPAPPVAGX 217 Alternative Hinge ERKCXVECPPCPAPPVAGX 218 Alternative Hinge ERKCCVECCPPCPAPELLGG 219 Alternative Hinge ERKSCVECPPCPAPELLGG 220 Alternative Hinge ERKCCSVECPPCPAPELLGG 221 Alternative Hinge ERKXCVECPPCPAPELLGG 222 Alternative Hinge ERKCXVECPPCPAPELLGG 223 Alternative Hinge ERKCCVECCPPCPAPELLG 224 Alternative Hinge ERKSCVECPPCPAPELLG 225 Alternative Hinge ERKCCSVECPPCPAPELLG 226 Alternative Hinge ERKXCVECPPCPAPELLG 227 Alternative Hinge ERKCXVECPPCPAPELLG 228 Alternative Hinge ERKCCVECPPCPAP 229 Alternative Hinge ERKSCVECPPCPAP 230 Alternative Hinge ERKCSVECPPCPAP 231 Alternative Hinge ERKXCVECPPCPAP 232 Alternative Hinge ERKCXVECPPCPAP 233 Hinge part PVAG 234 Hinge part ELLG 235 Hinge part ELLGG 236 Hinge part SCDKTHT 237 Hinge part CCVE 238 Human wild-type IgG2 hinge ERKCCVECPPCPAPPVAG 239 Hinge with C219S human wild-type IgG2 ERKSCVECPPCPAPPVAG 240 IgG2/IgGl hinge ERKCCVECCPPCPAPELLGG 241 IgG2 hinge (C219S)/IgGl ERKSCVECPPCPAPELLGG 242 Human wild-type IgGl hinge EPKSCDKTHTCPPCPAPELLGG

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243 CH2 c A330S/P331S human IgGl PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPSSIEKTISKAK 244 IgGl-IgG2-IgGlf ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQT YICNVNHKP SNTKVDKKVERKCCVECPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 245 IgGl-IgG2-lgGlf2 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQT YICNVNHKP SNTKVDKKVERKCCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG 246 IgGl-IgG2CS-IgGlf ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVERKSCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG 247 IgGl-IgG2CS-IgGlf2 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVERKSCVECPPCPAPP

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VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYCSLTVDKLSSRWQQ 248 IgG2-IgGlf ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 249 IgG2-IgGlf2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG 250 IgG2CS-IgGlf ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG

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251 IgG2CS-IgGlf2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG 252 IgGl-IgG2-IgGl.lf ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPSRREMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG 253 IgGl-IgG2CS-IgGl.lf ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS S SL GTQTYICNVNHKPSNTKVDKKVERKSCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG 254 IgG2-IgGl.lf ASTKGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<VSVSQYNSCKTYRVVLNG

- 185 040561

PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG 255 IgG2CS-IgGl.lf ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VS WNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQT YTCNVDHKP SNTKVDKTVERKSCVECPPCPAPE IXGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG 256 IgGlf ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 257 IgGl.lf ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 258 IgG2.3 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VS WNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPP

- 186 040561

VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPMLDSDGSFFVMLYSKLTVDKSRWQALSPGNHFSK 259 IgG2 5 ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 260 IgG2.3Gl-KH ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 261 IgG2.5Gl-KH ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

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262 IgG2.3Gl-AY ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 263 IgG2.5Gl-AY ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 264 IgG2.3Gl.lf-KH ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 265 IgG2.5Gl.lf-KH ASTKGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VI<FNWYVDGVEVHNAI<TI<VSVSQYNSCKTYRVVLNG

- 188 040561

PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 266 IgG2.5Gl-V27 ASTKGPSVFPLAPSSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPE LLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVCSQVKS HELSPGKMT 267 IgG2.3-V13 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPP VAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 268 IgG2.3-V14 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPP VAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDG EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLT WHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 269 IgG2.3-V15 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF

- 189 040561

GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPP VAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDG EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLT WHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 270 IgG2.3-V16 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPP VAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDG EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLT WHQDWLNGKEYKCKVSNKGLPRPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 271 IgG2.3-V17 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPP VAGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDG EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLT WHQDWLNGKEYKCKVSNKGLPRPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 272 IgG2.3-V18 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE

- 190 040561

SNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 273 IgG2.3-V19 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 274 IgG2.3Gl-AY-V20 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPE LLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 275 IgG2.3Gl-AY-V21 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPE LLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDG EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 276 IgG2.3Gl-AY-V22 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPE LLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDG

- 191 040561

EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 277 IgG2.3Gl-AY-V23 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPE LLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDG EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 278 IgG2.3Gl-AY-V24 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPE LLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDG EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 279 IgG2.3Gl-AY-V25 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPE LLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDG EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK

- 192 040561

280 IgG2.3Gl-AY-V26 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPD LLGDDSVFLFPPKPKDTLMISRTPEVTCVVVDVSDEDG EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPRPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 281 IgG2.3Gl-AY-V28 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 282 OX40.6-Vh-hHC-IgG2.3 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYW VRQAPGKGLEWVSAIGIGGDTFYTDSVKGRFTISRDN AKNSLSLQMNSLRAEDTAVYYCARMGTGYFFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 283 OX40.8-Vh-hHC-IgG2.3 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGI<GLEWVAL1SYDGSRI<HYADSVI<GRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQG

- 193 040561

TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVEC PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR WSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 284 0X40.16-Vh-hHC-IgG2.3 EVQLVQ SGGGLVQPGGSLRLSC AGSGFTFS S YAMYW VRQAPGKGLEWVSAIDTDGGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 285 OX40.6-Vh-hHC- IgG2.3Gl EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYW VRQAPGKGLEWVSAIGIGGDTFYTDSVKGRFTISRDN AKNSLSLQMNSLRAEDTAVYYCARMGTGYFFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCV ECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

- 194 040561

286 OX40.8-Vh-hHC- IgG2.3Gl QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGKGLEWVALISYDGSRKHYADSVKGRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVEC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 287 OX40.16-Vh-hHC- IgG2.3Gl EVQLVQ SGGGLVQPGGSLRLSC AGSGFTFS S YAMYW VRQAPGKGLEWVSAIDTDGGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 288 OX40.6-Vh-hHC- IgG2.3Gl-V27 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYW VRQAPGKGLEWVSAIGIGGDTFYTDSVKGRFTISRDN AKNSLSLQMNSLRAEDTAVYYCARMGTGYFFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCV ECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVEHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI

- 195 040561

SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 289 OX40.8-Vh-hHC- IgG2.3Gl-V27 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGKGLEWVALISYDGSRKHYADSVKGRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQG TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVEC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 290 OX40.16-Vh-hHC- IgG2.3Gl-V27 EVQLVQ SGGGLVQPGGSLRLSC AGSGFTFS S YAMYW VRQAPGKGLEWVSAIDTDGGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVE CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 291 OX40.6-Vh-hHC-IgG2.5 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSNYAMYW VRQAPGKGLEWVSAIGIGGDTFYTDSVKGRFTISRDN AKNSLSLQMNSLRAEDTAVYYCARMGTGYFFDYWG QGTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSNFGTQTCKNTKVERD

- 196 040561

VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVMKLSDGSFFLYGNTSKLTVDKKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVMKLSDGSFFLYGNTSKLTVDKKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY 292 OX40.8-Vh-hHC-IgG2.5 QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYALHW VRQAPGKGLEWVALISYDGSRKHYADSVKGRFSISRD NSKNTLYLQMNSLRAEDTAVYYCASLTMVREWGQG TLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR WSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DTAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 293 0X40.16-Vh-hHC-IgG2.5 EVQLVQ SGGGLVQPGGSLRLSC AGSGFTFS S YAMYW VRQAPGKGLEWVSAIDTDGGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ GTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 294 0X40.2 l-Vh-hHC-IgG2.5 EVQLVQ SGGGLVQPGGSLRLSC AGSGFTFS S YAMYW VRQAPGKGLEWVSAIDTDAGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ

- 197 040561

GTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 295 0X40.21-Vh-hHC- IgG2.5Gl EVQLVQ SGGGLVQPGGSLRLSC AGSGFTFS S YAMYW VRQAPGKGLEWVSAIDTDAGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ GTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 296 0X40.21-Vh-hHC- IgG2.5Gl-V27 EVQLVQ SGGGLVQPGGSLRLSC AGSGFTFS S YAMYW VRQAPGKGLEWVSAIDTDAGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ GTLVTVSSASTKGPSVFPLAPSSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVEHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

- 198 040561

297 IgG2.3Gl-V27 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFP AVLQS SGLYSLS S VVTVPS SNF GTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 298 CH2 c A330S/P331S human IgGl PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPSSIEKTISKAK 299 heavy chain - nivolumab QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHW VRQAPGKGL EWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLO MNSLRAEDT AVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPC SRSTSESTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGV EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKG LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGK 300 Light chain - nivolumab EIVLTQSPATLSLSPGERATLSCRASOSVSSYLAWYOO KPGQAPRLLI

- 199 040561

YD ASNRATGIPARF SGSGSGTDFTLTIS SLEPEDF AVYY CQQSSNWPR TFGQGTKVEIKRTVAAPSVFIFPPSDEOLKSGTASVV CLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC 301 Heavy chain variable region - nivolumab QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHW VRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISR DNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGT LVTVSS 302 Light chain variable region - nivolumab EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS LEPEDFAVYYCQQSSNWPRTFGQGTKVEIK 303 HCDR1 - nivolumab NSGMH 304 HCDR2 - nivolumab VIWYDGSKRYYADSVKG 305 HCDR3 - nivolumab NDDY 306 LCDR1 - nivolumab RASQSVSSYLA 307 LCDR2 - nivolumab DASNRAT 308 LCDR3 - nivolumab QQSSNWPRT 309 Heavy chain variable region - ipilimumab (from WO01/014424) QVQLVESGGGVVQPGRSLRLSC AASGFTF S S YTMHW VRQAPGI<GLEWVTFISYDGNNI<YYADSVI<GRFTISRD NSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWG QGTLVTVSS 310 Light chain variable region - ipilimumab (from WO01/014424) EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQ QKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTIS RLEPEDF AVYYCQQ YGS SPWTFGQGTKVEIK 311 HCDR1 - ipilimumab (from WO01/014424) SYTMH 312 HCDR2 - ipilimumab (from WO01/014424) FISYDGNNKYYADSVKG

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313 HCDR3 - ipilimumab (from WO01/014424) TGWLGPFDY 314 LCDR1 - ipilimumab (from WO01/014424) RASQSVGSSYLA 315 LCDR2 - ipilimumab (from WO01/014424) GAFSRAT 316 LCDR3 - ipilimumab (from WO01/014424) QQYGSSPWT 317 HCDR2 at 0X40.21 AIDTDAGTFYADSVRG 318 VH at 0X40.21 EVQLVQSGGGLVQPGGSLRLSCAGSGFTFSSYAMYW VRQAPGKGLEWVSAIDTDAGTFYADSVRGRFTISRDN AKNSLYLQMNSLRAEDTAVYFCARLGEGYFFDYWGQ GTLVTVSS

In table. 23 shows the sequences of the mature heavy and light chain variable regions and the signal peptide sequences.

Equivalents.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments disclosed herein. Such equivalents are intended to be covered by the following claims.

Claims (12)

CLAIM 1. A method for enhancing an antigen-specific response of T cells, comprising contacting a T cell with an isolated antibody that binds to human OX40 (anti-OX40 antibody) such that the antigen-specific response of T cells is enhanced, and wherein the anti-OX40 antibody comprises a variable region CDR1 heavy chain containing the sequence of SEQ ID NO: 87, a heavy chain variable region CDR2 containing the sequence of SEQ ID NO: 317, and a heavy chain variable region CDR3 containing the sequence 89, and a light chain variable region CDR1 containing the sequence of SEQ ID NO: 90 , a light chain variable region CDR2 containing the sequence of SEQ ID NO: 91 and a light chain variable region CDR3 containing the sequence of SEQ ID NO: 92. 2. A method of reducing or altering the suppressive effects of regulatory T cells in a tumor in a subject in need thereof, comprising administering a therapeutically effective amount of an isolated antibody that binds to human OX40 (anti-OX40 antibody) such that the suppressive effects of regulatory T cells are reduced or are changed, and wherein the anti-OX40 antibody comprises a heavy chain variable region CDR1 containing the sequence of SEQ ID NO: 87, a heavy chain variable region CDR2 containing the sequence of SEQ ID NO: 317, and a heavy chain variable region CDR3 containing the sequence 89, and a CDR1 light chain variable region containing the sequence of SEQ ID NO: 90, a light chain variable region CDR2 containing the sequence of SEQ ID NO: 91 and a light chain variable region CDR3 containing the sequence of SEQ ID NO: 92. 3. A method for inhibiting tumor cell growth in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated antibody that binds to human OX40 (anti-OX40 antibody) such that tumor cell growth is inhibited, and wherein the anti-OX40 antibody comprises CDR1 heavy chain variable region containing the sequence of SEQ ID NO: 87, a heavy chain variable region CDR2 containing the sequence of SEQ ID NO: 317, and a heavy chain variable region CDR3 containing the sequence 89, and a light chain variable region CDR1 containing the sequence of SEQ ID NO: : 90, a light chain variable region CDR2 containing the sequence of SEQ ID NO: 91 and a light chain variable region CDR3 containing the sequence of SEQ ID NO: 92. 4. The method according to any one of claims 1 to 3, wherein the anti-OX40 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 318 and the light chain variable region comprises the sequence of SEQ ID NO: 94. 5. The method according to any one of claims 1-4, wherein the anti-OX40 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the sequence of SEQ ID NO: 124 and the light chain comprises the sequence - 201 040561 SEQ ID NO: 116. 6. The method according to any one of claims 1, 4 and 5, wherein the contact is carried out in vitro or in the subject in vivo. 7. The method according to any one of claims 1 and 4-6, wherein the T cells are effector T cells (Teff cells), helper T cells (Th cells), cytotoxic T cells (Tc cells), or combinations thereof . 8. The method according to any one of claims 1 to 7, further comprising administering one or more additional therapeutic agents to the subject. 9. The method of claim 8, wherein the one or more additional therapeutic agents are an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-TGFe antibody, or combinations thereof. 10. The method of claim 8 or 9, wherein one or more additional therapeutic agents are administered to the subject simultaneously with the anti-OX40 antibody. 11. The method according to any one of claims 1 and 4-10, wherein the subject has a tumor. 12. The method of any one of claims 2-11, wherein the tumor is associated with a cancer selected from cervical cancer, bladder cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, or a combination thereof.