Fully human antibodies against OX40 and preparation methods and uses thereof

Abstract

The present application provides a fully human monoclonal antibody against member 4 of the tumor necrosis factor receptor superfamily (TNFRSF4) (also known as OX40 and CD134). The present application also provides a method for producing hybridomas using humanized transgenic rats, nucleic acid molecules encoding anti-OX40 antibodies, expression vectors and host cells for expressing anti-OX40 antibodies. The present invention also provides methods for verifying antibody function in vitro and antibody efficacy in vivo. The antibodies of the present invention provide very effective agents for treating a variety of cancers by regulating human immune function.

Description

[0001] This application is a divisional application. The Chinese application number of the parent application is 201910374787.0. The application date of the parent application is May 7, 2019. The name of the invention is “Fully human antibodies against OX40 and their preparation methods and uses”.

[0002] Priority information

[0003] This application claims priority to PCT application No. PCT / CN2018 / 086574 filed on May 11, 2018 and Chinese application No. 201810529840.5 filed on May 29, 2018.

[0004] Sequence Listing

[0005] This application contains a Sequence Listing, the entire contents of which are incorporated herein by reference. Technical Field

[0006] The present application generally relates to antibodies. More specifically, the present application relates to fully human monoclonal antibodies against OX40, methods for preparing the same, and uses thereof. Background Art

[0007] Increasing evidence from preclinical and clinical results indicates that targeting immune checkpoints is emerging as the most promising approach for treating cancer patients. Tumor necrosis factor receptor superfamily member 4 (TNFRSF4, also known as OX40, CD134, and ACT35) is one of the immune checkpoint proteins that plays a major role in T cell function by enhancing T cell receptor signaling and leading to its activation.

[0008] OX40 is mainly produced by activated CD4 + and CD8 +T cells, memory T cells, regulatory T (Treg) cells and natural killer (NK) cells express. The interaction of OX40 expressed on activated T cells with its ligand (OX40L) expressed on antigen presenting cells significantly promotes T cell activation, proliferation and migration, increases the survival of effector T cells, and enhances germinal center formation and dendritic cell maturation. In addition, OX40 signaling can inhibit the differentiation and expansion of Tregs, antagonize the generation of induced Tregs and block Treg inhibitory function. OX40 agonists have been demonstrated in a variety of preclinical mouse tumor models and clinical trials to be promising strategies for the treatment of cancer and infectious diseases. Pharmaceutical companies such as MedImmune, GlaxoSmithKline (GSK), Pfizer and Incyte have developed a variety of agonist agents targeting OX40. An agonist mouse antibody targeting OX40 (9B12, AgonOX) developed by MedImmune was used in a Phase I clinical trial in patients with advanced cancer. Patients treated with a course of the antibody "9B12" showed an acceptable toxicity profile, and at least one metastatic lesion regressed in 12 of 30 patients. Mechanistically, this treatment increased T and B cell responses to reporter antigen immunization (e.g., KLH), leading to preferential upregulation of OX40 on CD4+FoxP3+Treg cells in tumor-infiltrating lymphocytes and increased anti-tumor reactivity of T and B cells in melanoma patients. GSK is also developing GSK-3174998 for the potential treatment of cancers including solid tumors and hematological malignancies, which is a humanized IgG1 monoclonal antibody identified through a collaboration with the MD Anderson Cancer Center that activates OX-40 on the surface of T cells. Other drugs targeting OX40 in clinical development include Pfizer's fully human IgG2 agonist antibody PF-04518600, which is currently in clinical development for a broad spectrum of malignancies; and Incyte's INCAGN-1949, an anti-OX40 human IgG1 antibody with optimal agonist properties and the ability to selectively deplete regulatory T cells within tumors for the potential treatment of cancer.

[0009] There is some room for improvement for antibodies against OX40 as therapeutic agents. As agonists against co-stimulatory receptors, toxicity may be the most concerning issue, such as cytokine storm, which limits clinical application. In addition, the anti-OX40 antibodies currently tested in clinical trials are human-mouse chimeric or humanized antibodies, and the high immunogenicity caused by the mouse-derived protein sequence reduces the efficacy. Fully human antibodies overcome these shortcomings and show higher efficiency and lower toxicity in vivo.

[0010] In the present invention, by utilizing our proprietary hybridoma technology, we have generated fully human antibodies against OX40. The antibodies of the present invention have high binding affinity; specifically bind to human and monkey OX40 proteins; and effectively modulate immune responses, including enhancing T cell proliferation and increasing the production of cytokines IFN-γ and interleukin-2 and weakening the suppressive function of Treg cells. Summary of the invention

[0011] The present invention provides these and other objects, broadly speaking, compounds, methods, compositions and articles of manufacture relating to providing antibodies with improved efficacy. The benefits provided by the present invention are widely applicable to the fields of antibody therapy and diagnosis, and can be used in combination with antibodies capable of reacting with various targets. The present invention provides antibodies that bind to human OX40, preferably fully human monoclonal antibodies. Also provided are methods for producing hybridomas using humanized rats, nucleic acid molecules encoding anti-OX40 antibodies, vectors and host cells for expressing anti-OX40 antibodies. The present invention further provides methods for verifying antibody function in vitro and in vivo. The antibodies of the present invention provide effective agents for treating a variety of diseases (including cancer) by modulating human immune function.

[0012] In some aspects, the invention includes isolated antibodies or antigen-binding portions thereof.

[0013] In some embodiments, the isolated antibody, or antigen-binding portion thereof, has one or more of the following properties:

[0014] (a) 1×10 -8 M or lower K D Binds to human OX40;

[0015] (b) In CD4 + Inducing cytokine (e.g., IL-2 or IFN-γ) production in T cells;

[0016] (c) Enhancement of primary human CD4 + Proliferation of T cells;

[0017] (d) Enhancement of primary human CD4 + Proliferation of T effector cells;

[0018] (e) binds to human or cynomolgus OX40, respectively; or

[0019] (f) No cross-reactivity to human CD40, CD137 and CD271.

[0020] In some embodiments, the isolated antibody, or antigen binding portion thereof, binds to the CRD2 and / or CRD3 domains of OX40.

[0021] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0022] A) one or more heavy chain CDRs (CDRHs) selected from at least one of the following:

[0023] (i) a CDRH1 having at least 90% sequence identity to a CDRH1 set forth in one of the sequences selected from SEQ ID NOs: 1, 7, 13, 15, 21 and 27;

[0024] (ii) a CDRH2 having at least 90% sequence identity to a CDRH2 set forth in one of the sequences selected from SEQ ID NOs: 3, 9, 17, 23 and 29; and

[0025] (iii) a CDRH3 having at least 90% sequence identity to a CDRH3 set forth in one of the sequences selected from SEQ ID NOs: 5, 11, 19, 25, and 31;

[0026] B) one or more light chain CDRs (CDRLs) selected from at least one of the following:

[0027] (i) a CDRL1 having at least 90% sequence identity to a CDRL1 as set forth in one of the sequences selected from SEQ ID NOs: 2, 8, 14, 16, 22 and 28;

[0028] (ii) a CDRL2 having at least 90% sequence identity to the CDRL2 set forth in one of the sequences selected from SEQ ID NOs: 4, 10, 18, 24 and 30; and

[0029] (iii) a CDRL3 having at least 90% sequence identity to a CDRL3 set forth in one of the sequences selected from SEQ ID NOs: 6, 12, 20, 26 and 32; or

[0030] C) one or more CDRHs of A) and one or more CDRLs of B).

[0031] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0032] A) one or more heavy chain CDRs (CDRHs) selected from at least one of the following:

[0033] (i) a CDRH1 selected from SEQ ID NOs: 1, 7, 13, 15, 21 and 27, or a CDRH1 that differs from said CDRH1 in amino acid sequence by an amino acid addition, deletion or substitution of not more than 2 amino acids;

[0034] (ii) a CDRH2 selected from the group consisting of SEQ ID NOs: 3, 9, 17, 23 and 29, or a CDRH2 that differs in amino acid sequence from said CDRH2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and

[0035] (iii) a CDRH3 selected from the group consisting of SEQ ID NOs: 5, 11, 19, 25 and 31, or a CDRH3 that differs in amino acid sequence from said CDRH3 by an amino acid addition, deletion or substitution of not more than 2 amino acids;

[0036] B) one or more light chain CDRs (CDRLs) selected from at least one of the following:

[0037] (i) a CDLH selected from SEQ ID NOs: 2, 8, 14, 16, 22 and 28, or a CDRL1 that differs from said CDRL1 in amino acid sequence by an addition, deletion or substitution of no more than 2 amino acids;

[0038] (ii) a CDRL2 selected from the group consisting of SEQ ID NOs: 4, 10, 18, 24 and 30, or a CDRL2 that differs in amino acid sequence from said CDRL2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and

[0039] (iii) a CDRL3 selected from SEQ ID NOs: 6, 12, 20, 26 and 32, or a CDRL3 that differs from said CDRL3 in amino acid sequence by an amino acid addition, deletion or substitution of not more than 2 amino acids; or

[0040] C) one or more CDRHs of A) and one or more CDRLs of B).

[0041] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0042] A) a CDRH3 comprising SEQ ID NO: 5, 11, 19, 25 or 31; or

[0043] B) a CDRH3 having at least 90% sequence identity to a CDRH3 as set forth in one of the sequences selected from SEQ ID NO: 5, 11, 19, 25 and 31; or

[0044] C) a CDRH3 that differs in amino acid sequence from the CDRH3 of (A) by the addition, deletion or substitution of no more than 2 amino acids,

[0045] and wherein the isolated antibody or antigen-binding portion thereof is present at 1×10 -8 M or lower K D Binds human OX40.

[0046] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0047] (a) CDRH1 comprising or consisting of SEQ ID NO: 1;

[0048] (b) a CDRH2 comprising or consisting of SEQ ID NO: 3;

[0049] (c) a CDRH3 comprising or consisting of SEQ ID NO: 5;

[0050] (d) CDRL1 comprising or consisting of SEQ ID NO: 2;

[0051] (e) CDRL2 comprising or consisting of SEQ ID NO: 4; and

[0052] (f) CDRL3 comprising or consisting of SEQ ID NO: 6.

[0053] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0054] (a) CDRH1 comprising or consisting of SEQ ID NO: 7;

[0055] (b) a CDRH2 comprising or consisting of SEQ ID NO: 9;

[0056] (c) a CDRH3 comprising or consisting of SEQ ID NO: 11;

[0057] (d) CDRL1 comprising or consisting of SEQ ID NO: 8;

[0058] (e) CDRL2 comprising or consisting of SEQ ID NO: 10; and

[0059] (f) CDRL3 comprising or consisting of SEQ ID NO: 12.

[0060] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0061] (a) CDRH1 comprising or consisting of SEQ ID NO: 13;

[0062] (b) a CDRH2 comprising or consisting of SEQ ID NO: 9;

[0063] (c) a CDRH3 comprising or consisting of SEQ ID NO: 11;

[0064] (d) CDRL1 comprising or consisting of SEQ ID NO: 14;

[0065] (e) CDRL2 comprising or consisting of SEQ ID NO: 10; and

[0066] (f) CDRL3 comprising or consisting of SEQ ID NO: 12.

[0067] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0068] (a) CDRH1 comprising or consisting of SEQ ID NO: 15;

[0069] (b) a CDRH2 comprising or consisting of SEQ ID NO: 17;

[0070] (c) a CDRH3 comprising or consisting of SEQ ID NO: 19;

[0071] (d) CDRL1 comprising or consisting of SEQ ID NO: 16;

[0072] (e) CDRL2 comprising or consisting of SEQ ID NO: 18; and

[0073] (f) CDRL3 comprising or consisting of SEQ ID NO: 20.

[0074] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0075] (a) CDRH1 comprising or consisting of SEQ ID NO: 21;

[0076] (b) a CDRH2 comprising or consisting of SEQ ID NO: 23;

[0077] (c) a CDRH3 comprising or consisting of SEQ ID NO: 25;

[0078] (d) CDRL1 comprising or consisting of SEQ ID NO: 22;

[0079] (e) CDRL2 comprising or consisting of SEQ ID NO: 24; and

[0080] (f) CDRL3 comprising or consisting of SEQ ID NO: 26.

[0081] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0082] (a) CDRH1 comprising or consisting of SEQ ID NO: 27;

[0083] (b) a CDRH2 comprising or consisting of SEQ ID NO: 29;

[0084] (c) a CDRH3 comprising or consisting of SEQ ID NO: 31;

[0085] (d) CDRL1 comprising or consisting of SEQ ID NO: 28;

[0086] (e) CDRL2 comprising or consisting of SEQ ID NO: 30; and

[0087] (f) CDRL3 comprising or consisting of SEQ ID NO: 32.

[0088] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0089] (A) a heavy chain variable region (VH), wherein:

[0090] (i) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, 41 and 43;

[0091] (ii) comprises an amino acid sequence that is at least 85%, 90% or 95% identical to an amino acid sequence selected from SEQ ID NOs: 33, 35, 37, 39, 41 and 43; or

[0092] (iii) an amino acid sequence comprising one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4 or 5) amino acid additions, deletions and / or substitutions compared to an amino acid sequence selected from SEQ ID NOs: 33, 35, 37, 39, 41 and 43; and / or

[0093] (B) a light chain variable region (VL), wherein:

[0094] (i) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, 42 and 44;

[0095] (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, 42, and 44; or

[0096] (iii) an amino acid sequence comprising one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4 or 5) amino acid additions, deletions and / or substitutions compared to the amino acid sequence selected from SEQ ID NOs: 34, 36, 38, 40, 42 and 44.

[0097] In some embodiments, the invention comprises an isolated antibody, or antigen binding portion thereof, that competes for binding to the same epitope as the isolated antibody, or antigen binding portion thereof, defined above.

[0098] In some aspects, the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and / or a light chain variable region of an isolated antibody as disclosed herein.

[0099] In some aspects, the invention relates to vectors comprising a nucleic acid molecule encoding an antibody or antigen-binding portion thereof as disclosed herein.

[0100] In some aspects, the invention relates to a host cell comprising an expression vector as disclosed herein.

[0101] In some aspects, the invention relates to a pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as disclosed herein and a pharmaceutically acceptable carrier.

[0102] In some aspects, the invention relates to methods for preparing an anti-OX40 antibody or an antigen binding portion thereof, comprising expressing the antibody or the antigen binding portion thereof in a host cell and isolating the antibody or the antigen binding portion from the host cell.

[0103] In some aspects, the invention relates to methods of modulating an immune response in a subject, comprising administering to the subject an antibody, or antigen-binding portion thereof, as disclosed herein, such that the immune response in the subject is modulated.

[0104] In some aspects, the invention relates to a method for treating abnormal cell growth in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding portion thereof or a pharmaceutical composition as disclosed herein.

[0105] In some aspects, the invention relates to a method for inhibiting tumor cell growth in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding portion thereof or a pharmaceutical composition as disclosed herein.

[0106] In some aspects, the invention relates to a method for reducing tumor cell metastasis in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding portion thereof or a pharmaceutical composition as disclosed herein.

[0107] In some aspects, the invention relates to a method for weakening the suppressive function of Treg cells in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding portion thereof or a pharmaceutical composition as disclosed herein.

[0108] In some aspects, the invention relates to a method for treating or preventing a disease in a subject, including a proliferative disorder (such as cancer), an autoimmune disease, an inflammatory disease, or an infectious disease, comprising administering to the subject an effective amount of an antibody or antigen-binding portion thereof, or a pharmaceutical composition as disclosed herein.

[0109] In some aspects, the invention relates to the use of an antibody or antigen-binding portion thereof as disclosed herein in the preparation of a medicament for treating or preventing a disease, including a proliferative disorder (eg, cancer), an autoimmune disease, an inflammatory disease, or an infectious disease.

[0110] In some aspects, the invention relates to the use of an antibody or antigen binding portion thereof as disclosed herein in the preparation of a diagnostic agent for diagnosing a proliferative disease, including a proliferative disorder (eg, cancer), an autoimmune disease, an inflammatory disease, or an infectious disease.

[0111] In some aspects, the invention relates to the use of an antibody or antigen-binding portion thereof as disclosed herein for treating or preventing a disease, including a proliferative disorder (eg, cancer), an autoimmune disease, an inflammatory disease, or an infectious disease.

[0112] In some aspects, the present invention relates to kits or devices and related methods using antibodies or antigen-binding portions thereof as disclosed herein, and pharmaceutical compositions as disclosed herein, which can be used to treat diseases (including proliferative disorders (e.g., cancer), autoimmune diseases, inflammatory diseases, or infectious diseases). To this end, the present invention preferably provides an article that can be used to treat such disorders, comprising a container containing an antibody or antigen-binding portion thereof as disclosed herein and instructional materials for using an antibody or antigen-binding portion thereof as disclosed herein to treat, improve or prevent a proliferative disorder or its progression or recurrence.

[0113] The above is an overview and therefore contains simplifications, generalizations, and omissions of details as necessary; therefore, those skilled in the art will recognize that the overview is illustrative only and is not intended to be limiting in any way. Other aspects, features, and advantages of the methods, compositions, and / or devices and / or other subjects described herein will become apparent in the teachings shown herein. An overview is provided to simplify the introduction of a collection of some concepts, which will be further described in the detailed description below. This overview is not intended to determine the key features or essential features of the claimed subject matter, nor is it intended to be used as an auxiliary means for determining the scope of the claimed subject matter. In addition, the contents of all references, patents, and published patent applications cited throughout this application are incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 Graph showing comparison between variants of anti-OX40 antibody 1.62.3-ul-IgG1K following PTM mutation.

[0115] Figure 2 A graph showing antibodies binding to human OX40 transfected CHO-K1 cells.

[0116] Figure 3 Shown to bind activated human CD4 + Diagram of T cell antibodies.

[0117] Figure 4 A graph showing antibodies that compete with OX40L for binding to OX40.

[0118] Figure 5 A graph showing antibodies binding to cynomolgus OX40-transfected 293F cells.

[0119] Figure 6 Graph showing the results of family cross-binding testing of anti-OX40 antibodies to other TNFR family members including human CD40, CD137 and CD271 by ELISA.

[0120] Fig. 7A , 7B and 7C are shown for the benchmark antibody BMK1 ( Fig. 7A )、BMK7( Figure 7B ) and BMK10( Figure 7C ) is a diagram of epitope competition binding test (epitope binning) of antibodies.

[0121] Fig. 8A , 8B8C are graphs showing the effect of antibodies on OX40-stimulated NFkB luciferase activity in Jurkat cells using free antibodies or FcγR cross-linking via CD32b-expressing CHO-K1 cells or anti-human IgG Fc reagent. Fig. 8A ) free antibody or ( Figure 8B )F(ab')2 goat anti-human IgG or ( Figure 8C ) Reporter activity of cross-linked CHO-K1 cells expressing CD32b.

[0122] Fig. 9 Is a primary human CD4 that shows antibodies against CD3 induction + Diagram of the effect of IL-2 secretion by T cells.

[0123] Fig.10 Is a primary human CD4 that shows antibodies against CD3 induction + Diagram of the effect of IFN-γ secretion by T cells.

[0124] Fig.11 Is a primary human CD4 that shows antibodies against CD3 induction + Figure 2. Effect of T cell proliferation.

[0125] Fig.12 The antibody showed the effect of CD3 / CD28 Dynabeads on primary human CD4 + Figure 3. Effect of T effector cell proliferation on the proliferation of T effector cells.

[0126] Fig.13A Is a display of activated human CD4 + Figure 1. OX40 expression on T cells. Fig. 13B is a graph showing OX40 expression on Jurkat cells overexpressing OX40.

[0127] Fig.14A This is a diagram showing the ADCC effect of OX40 antibody on Jurkat cells overexpressing OX40. Fig. 14B The OX40 antibody is shown to be effective against activated human CD4 + Diagram of ADCC effect of T cells.

[0128] Fig.15A This is a diagram showing the CDC effect of OX40 antibody on Jurkat cells overexpressing OX40. Fig. 15B The OX40 antibody is shown to be effective against activated human CD4 + Diagram of the CDC effect of T cells.

[0129] Fig.16A and Fig. 16Bis a graph showing tumor growth in MC38 tumor-bearing mice following administration of antibody 1.134.9-ul-IgG1L.

[0130] Fig.17 is a graph showing changes in body weight of MC38 tumor-bearing mice after administration of antibody 1.134.9-u1-IgG1L. DETAILED DESCRIPTION

[0131] Although the present invention can be implemented in many different forms, what is disclosed here is its specific illustrative embodiment that verifies the principle of the present invention.It should be emphasized that the present invention is not limited to the specific embodiment illustrated.In addition, any section title used herein is only for organizational purposes and is not to be interpreted as limiting the subject matter described.

[0132] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, terms in the singular shall include plural forms, and terms in the plural shall include singular forms. More specifically, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural indicators unless the context clearly indicates otherwise. Thus, for example, reference to "a protein" includes multiple proteins; reference to "a cell" includes a mixture of cells, etc. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include all values ​​between the endpoints and the endpoints.

[0133] Generally, terms relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization and techniques described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6 thed., WB Saunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The terms and laboratory procedures and techniques related to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those well known and commonly used in the art. In addition, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0134] definition

[0135] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0136] As used herein, the term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) chains and two light (L) chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of antibodies can be divided into kappa and lambda light chains. The heavy chains can be divided into μ, δ, γ, α and ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. In the light and heavy chains, the variable region is connected to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and the heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1, C H 2 and C H3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ) H and V L The V region can be further divided into regions of hypervariability, called complementarity determining regions (CDR), separated by regions that are relatively conserved, called framework regions (FR). H and V L It consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus. The variable region (V H and V L ) form antigen binding sites respectively. The distribution of amino acids in various regions or domains follows the definition in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al., (1989) Nature 342: 878-883. Antibodies can have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0137] The terms "antigen-binding portion" or "antigen-binding fragment" of an antibody, which may be used interchangeably in the context of this application, refer to polypeptides comprising fragments of a full-length antibody that retain the ability to specifically bind to an antigen that is specifically bound by the full-length antibody and / or which compete with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Under certain conditions, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and such polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability on the polypeptide. Antigen-binding fragments of antibodies can be obtained from a given antibody (e.g., a monoclonal anti-human OX40 antibody provided in the present application) by conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and can be screened for specificity in the same manner as intact antibodies.

[0138] As used herein, the term "monoclonal antibody" or "mAb" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0139] As used herein, the term "human antibody" or "fully human antibody" is intended to include antibodies with variable regions in which both the framework and 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. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0140] As used herein, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.

[0141] The term "humanized antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0142] The term "chimeric antibody" as used herein refers to antibodies in which the variable region sequences are from one species and the constant region sequences are from another species, for example, antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0143] As used herein, the term "recombinant antibody" refers to an antibody prepared, expressed, created or isolated by recombinant means, such as an antibody isolated from an animal that is transgenic for the immunoglobulin genes of another species, an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant combinatorial antibody library, or an antibody prepared, expressed, created or isolated by any other means involving splicing of immunoglobulin gene sequences to other DNA sequences.

[0144] As used herein, the term "anti-OX40 antibody" or "OX40 antibody" refers to an antibody as defined herein that is capable of binding to an OX40 receptor, eg, a human OX40 receptor.

[0145] The terms "OX40", "OX40 receptor", "OX40 protein", "tumor necrosis factor receptor superfamily member 4 (TNFRSF4)" or "CD134", used interchangeably herein, are members of the tumor necrosis factor (TNF) receptor superfamily. The term "OX40" may include human OX40 receptor, as well as variants, isoforms and species homologs thereof. Thus, the antibodies or antigen-binding portions thereof as defined and disclosed herein may also bind to OX40 from species other than human, such as macaque OX40.

[0146] As used herein, the term "human OX40" refers to human sequence OX40, such as the complete amino acid sequence of human OX40 with Genbank Accession No. CAE11757.1. The human OX40 sequence may differ from the human OX40 with Genbank Accession No. CAE11757.1, such as having conservative mutations in non-conserved regions, and the OX40 has substantially the same biological function as the human OX40 with Genbank Accession No. CAE11757.1.

[0147] As used herein, the term "mouse OX40" refers to the complete amino acid sequence of mouse sequence OX40, such as mouse OX40 having Genbank Accession No. CAA59476.1.

[0148] As used herein, the term "cynomolgus monkey OX40" refers to the complete amino acid sequence of cynomolgus monkey OX40, such as cynomolgus monkey OX40 having Genbank Accession No. XP_001090870.1.

[0149] As used herein, the term "Ka" is intended to refer to the association rate of a particular antibody-antigen interaction, while the term "Kd" as used herein is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using methods well established in the art. As used herein, the term "Kd" is intended to refer to the association rate of a particular antibody-antigen interaction. D " is intended to represent the dissociation constant for a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). A preferred method for determining the Kd of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as system.

[0150] As used herein, the term "high affinity" of an IgG antibody refers to an affinity of 1×10 -7 M or less, more preferably 5×10 -8 M or less, even more preferably 1×10 -8 M or less, even more preferably 5×10 -9 M or less, even more preferably 1×10 -9 M or lower K D of antibodies.

[0151] As used herein, the term "EC 50 EC, also known as the "half effective concentration", is the concentration of a drug, antibody or toxicant that induces a response that is 50% between baseline and maximum after a specified exposure time. In the context of this application, EC 50 The unit is "nM".

[0152] As used herein, the term "competitive binding" refers to the interaction of two antibodies in their binding to a binding target. If the binding of a first antibody to its cognate epitope in the presence of a second antibody is detectably reduced compared to the binding of the first antibody in the absence of the second antibody, then the first antibody competes with the second antibody for binding. The opposite situation can be established but not necessarily so, that is, the binding of the second antibody to its epitope in the presence of the first antibody can also be detectably reduced, but not necessarily so. In other words, the first antibody can inhibit the binding of the second antibody to its epitope, while the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, in the case where each antibody can detectably inhibit the binding of another antibody to its cognate epitope, whether to the same, greater or lesser extent, the antibodies are considered to "cross-compete" with each other for the binding of their respective epitopes.

[0153] As used herein, the ability to "inhibit binding" refers to the ability of an antibody or antigen-binding fragment thereof to inhibit the binding of two molecules (e.g., human OX40 and human anti-OX40 antibody) to any detectable level. In certain embodiments, the binding of two molecules may be inhibited by the antibody or antigen-binding fragment thereof by at least 50%. In certain embodiments, such inhibition may be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0154] As used herein, the term "epitope" refers to the antigenic portion to which an immunoglobulin or antibody specifically binds. "Epitope" is also referred to as an "antigenic determinant". An epitope or antigenic determinant is generally composed of chemically active surface groups of molecules such as amino acids, carbohydrates or sugar side chains, and generally has a specific three-dimensional structure and a specific charge characteristic. For example, an epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or discontinuous amino acids in a unique three-dimensional conformation, which can be a "linear" or "conformational" epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all interaction sites between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction site spans amino acid residues separated from each other in a protein. Depending on the competitiveness of the same epitope detected by conventional techniques known to those skilled in the art, antibodies can be screened. For example, competition or cross-competition studies can be performed to obtain antibodies that compete or cross-compete with each other for binding antigens (e.g., RSV fusion proteins). A high-throughput method for obtaining antibodies that bind to the same epitope is described in International Patent Application WO 03 / 48731, which is based on their cross-competition.

[0155] As used herein, the term "isolated" refers to a state obtained from a natural state by artificial means. If a certain "isolated" substance or component exists in nature, it may be because its natural environment has changed, or the substance is separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide exists naturally in a living organism, and the same high-purity polynucleotide or polypeptide separated from the natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances, nor does it exclude other impure substances that do not affect the activity of the isolated substance.

[0156] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to an OX40 protein is substantially free of antibodies that specifically bind to antigens other than the OX40 protein). However, an isolated antibody that specifically binds to a human OX40 protein may have cross-reactivity to other antigens, such as OX40 proteins from other species. In addition, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0157] As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector can be transformed, transduced or transfected into a host cell to express the genetic material elements carried in the host cell. Vectors are well known to those skilled in the art, including but not limited to plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). The vector may contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may contain an origin of replication.

[0158] As used herein, the term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment or peptide. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rat, mouse, guinea pig or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells and insect cells, as well as cells contained in transgenic animals or cultured tissues. The term covers not only specific test cells, but also the offspring of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such offspring may be different from parental cells, but are still included in the scope of the term "host cell".

[0159] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules determined by alignment and comparison of the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecules being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAM J. Applied Math. 48:1073.

[0160] As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It refers not only to the property of antigens to stimulate the activation, proliferation and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response of antibodies or sensitized T lymphocytes that can be formed in the immune system of an organism after stimulating the organism with an antigen. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host and the means of immunity.

[0161] As used herein, the term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. In a specific embodiment of the invention, the human XO40 gene is transfected into 293F cells.

[0162] As used herein, the term "hybridoma" and the term "hybridoma cell line" are used interchangeably. When referring to the term "hybridoma" and the term "hybridoma cell line", they also include subclones and progeny cells of the hybridoma.

[0163] As used herein, the term "SPR" or "surface plasmon resonance" refers to and includes an optical phenomenon that allows analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For a detailed description, see Examples 5 and U., et al. (1993) Ann. Biol. Clin. 51: 19-26; U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0164] As used herein, the term "fluorescence activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers one cell at a time according to the specific light scattering and fluorescence characteristics of each cell (Flow Metric. "Sorting Out Fluorescence Activated Cell Sorting". 2017-11-09). Instruments for performing FACS are known to those skilled in the art and can be commercially available to the public. Examples of such instruments include FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (Foster City, Calif.), Epics C from Coulter Epics Division (Hialeah, FLa), and MoFlo from Cytomation (Colorado Springs, Colo.).

[0165] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are absolutely required for this killing. NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). In order to assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, can be performed. Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0166] The term "complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), can be performed.

[0167] The term "subject" includes any human or non-human animal, preferably a human.

[0168] As used herein, the term "cancer" refers to any neoplastic or malignant cell growth, proliferation, or metastasis-mediated medical condition, both solid tumors and non-solid tumors such as leukemia.

[0169] The terms "treatment" and "treatment" as used herein in the context of treating a condition generally refer to treatment and therapy of a human or animal in which some desired therapeutic effect is achieved, for example, inhibition of disease progression, including a decrease in the rate of progression, stagnation of the rate of progression, regression of the disease, improvement of the disease, and cure of the disease. Treatment as a preventive measure (i.e., prevention) is also included. For cancer, "treatment" may refer to inhibiting or slowing tumor or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, "treatment" includes removing all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of a tumor, or some combination thereof.

[0170] As used herein, the term "therapeutically effective amount" relates to an amount of an active compound, or a material, composition or dosage form comprising an active compound, which, when administered according to the desired treatment regimen, is effective for producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. For example, when used in conjunction with treating an OX40-related disease or condition, "effective amount" refers to an amount or concentration of an antibody or antigen-binding portion thereof that is effective for treating the disease or condition.

[0171] As used herein, the terms "prevent," "control," or "prevention" with respect to a disease condition in a mammal means preventing or delaying the onset of the disease or preventing the manifestation of clinical or subclinical symptoms thereof.

[0172] As used herein, the term "pharmaceutically acceptable" means that the carrier, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0173] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0174] As used herein, the term "adjuvant" refers to a non-specific immunopotentiator that can enhance the immune response to the antigen in the organism or change the type of immune response when it is delivered to the organism together with the antigen or delivered to the organism in advance. There are a variety of adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0175] Anti-OX40 Antibodies

[0176] In some aspects, the invention includes isolated antibodies or antigen-binding portions thereof.

[0177] In the context of the present application, "antibodies" may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including mutant proteins and variants thereof; and derivatives thereof (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecules, as long as they show preferential association or binding with OX40 protein. In addition, unless the context otherwise dictates, the term also includes antibodies of all classes (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a human monoclonal antibody.

[0178] Human antibodies can be produced using various techniques known in the art. One technique is phage display, in which a (preferably human) antibody library is synthesized on phage, the library is screened with the antigen of interest or its antibody binding portion, and the phage that binds the antigen is isolated, from which immunoreactive fragments can be obtained. Methods for preparing and screening such libraries are well known in the art, and kits for producing phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, catalog number 27-9400-01; and Stratagene SurfZAP TM Phage Display Kit, Catalog No. 240612). There are other methods and reagents available for generating and screening antibody display libraries (see, e.g., Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).

[0179] Human antibodies can also be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated and human immunoglobulin genes have been introduced). Upon challenge, human antibody production is observed that is very similar to that observed in humans in all aspects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and about 6,075,181 and 6,150,584 of the US Patents on the technology; and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995). Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes (such B lymphocytes can be obtained from individuals suffering from neoplastic diseases or who may have been immunized in vitro) that produce antibodies against the target antigen. See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol, 147 (l): 86-95 (1991); and USPN 5,750,373.

[0180] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals (e.g. ) or some combination thereof. For example, monoclonal antibodies can be produced using hybridomas and art-recognized biochemical and genetic engineering techniques, as described in detail in An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1 st ed.2009; Shire et.al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science+Business Media LLC,1 st ed.2010;Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.1988;Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. It should be understood that the selected binding sequence can be further altered, for example, to increase affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its in vivo immunogenicity, generate multispecific antibodies, etc., and antibodies comprising altered target binding sequences are also antibodies of the present invention. In a preferred embodiment, anti-human OX40 monoclonal antibodies are prepared by using hybridomas.

[0181] Generation of hybridomas producing human monoclonal antibodies of the invention

[0182] In order to obtain hybridomas that produce antibodies of the invention, such as human monoclonal antibodies of the invention, spleen cells and / or lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas are screened for the production of antigen-specific antibodies. The production of hybridomas is well known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0183] Generation of transfectomas producing monoclonal antibodies of the invention

[0184] The antibodies of the present invention can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors so that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is linked to the vector so that the transcriptional and translational control sequences within the vector play their intended functions of regulating transcription and translation of the antibody gene.

[0185] The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. These regulatory sequences are described in, for example, Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Exemplary regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP), and polyoma virus. Alternatively, nonviral regulatory sequences can be used, such as the ubiquitin promoter or the β-globin promoter; also, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al. (1988) MoI. Cell. Biol. 8:466-472). The expression vector and expression control sequences are selected to be compatible with the expression host cell used.

[0186] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors. In some embodiments, the variable region is used to produce the full-length antibody gene of any antibody isotype by inserting it into the expression vector of the heavy chain constant region and the light chain constant region that have encoded the desired isotype, so that the VH segment is operably connected to the CH segment and the VL segment in the carrier are operably connected to the CL segment in the carrier. In addition or alternatively, the recombinant expression vector can encode a signal peptide that promotes the secretion of antibody chains from host cells. The antibody chain gene can be cloned into a vector so that the signal peptide is connected to the amino terminus of the antibody chain gene in frame. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0187] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector of the present invention can also carry additional sequences, such as sequences (such as replication origins) and selectable marker genes that regulate the replication of the vector in the host cell. The selectable marker gene helps to select the host cell into which the vector has been introduced (see, for example, U.S. Patent No. 4,399,216; 4,634,665 and 5,179,017). For example, the selectable marker gene usually confers resistance to drugs (such as G418, hygromycin or methotrexate) to the host cell into which the vector has been introduced. The selectable marker gene can include a dihydrofolate reductase (DHFR) gene (for dhfr-host cells with methotrexate selection / amplification) and a neo gene (for G418 selection).

[0188] To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. The antibodies of the present invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells, which can assemble and secrete appropriately folded and immunologically active antibodies.

[0189] Mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary (CHO cells) (including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220) used with a DHFR selection marker (e.g., as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159: 601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036 and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell or secreted into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

[0190] Anti-OX40 antibodies with certain properties

[0191] The antibodies of the present invention are characterized by specific functional characteristics or properties of the antibodies. In some embodiments, the isolated antibodies or antigen-binding portions thereof have one or more of the following properties:

[0192] (a) 1×10-8 M or lower K D Binds to human OX40;

[0193] (b) Induction of CD4 + production of cytokines (e.g., IL-2 or IFN-γ) in T cells;

[0194] (c) Enhancement of primary human CD4 + Proliferation of T cells;

[0195] (d) Enhancement of primary human CD4 + Proliferation of T effector cells;

[0196] (e) binds to human or cynomolgus OX40, respectively; or

[0197] (f) No cross-reactivity to human CD40, CD137 and CD271.

[0198] The antibodies of the invention bind to human OX40 with high affinity. The binding of the antibodies of the invention to OX40 can be assessed using one or more techniques well established in the art, such as ELISA. The binding specificity of the antibodies of the invention can also be determined by, for example, monitoring the binding of the antibodies to cells expressing the OX40 protein by flow cytometry. For example, the antibodies can be tested by flow cytometry assays, in which the antibodies react with cell lines expressing human OX40, such as CHO cells that have been transfected to express OX40 on their cell surfaces. Other suitable cells for flow cytometry assays include anti-CD3-stimulated CD4 expressing native OX40. + Additionally or alternatively, the binding of the antibodies can be tested in a BIAcore binding assay, including binding kinetics (e.g., K d Other suitable binding assays include ELISA assays, for example using recombinant OX40 protein. For example, the antibodies of the invention are expressed at 1×10 -8 M or lower K D Bind to human OX40 protein, at 1×10 -9 M or lower K D Bind to human OX40 protein, 5×10 -10 M or lower K D Bind to human OX40 protein, at 2×10 -10 M or lower K D Bind to human OX40 protein, at 1×10 -10 M or lower K D Bind to human OX40 protein, 5×10 -11 M or lower K D Bind to human OX40 protein, at 3×10 -11M or lower K D Bind to human OX40 protein, or at 2×10 -11 M or lower K D Binds to human OX40 protein.

[0199] Anti-OX40 antibodies comprising CDRs having sequence identity to a specific sequence

[0200] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0201] A) one or more heavy chain CDRs (CDRHs) selected from at least one of the following:

[0202] (i) a CDRH1 having at least 90% sequence identity to a CDRH1 set forth in one of the sequences selected from SEQ ID NOs: 1, 7, 13, 15, 21 and 27;

[0203] (ii) a CDRH2 having at least 90% sequence identity to a CDRH2 set forth in one of the sequences selected from SEQ ID NOs: 3, 9, 17, 23 and 29; and

[0204] (iii) a CDRH3 having at least 90% sequence identity to a CDRH3 set forth in one of the sequences selected from SEQ ID NOs: 5, 11, 19, 25, and 31;

[0205] B) one or more light chain CDRs (CDRLs) selected from at least one of the following:

[0206] (i) a CDRL1 having at least 90% sequence identity to a CDRL1 as set forth in one of the sequences selected from SEQ ID NOs: 2, 8, 14, 16, 22 and 28;

[0207] (ii) a CDRL2 having at least 90% sequence identity to the CDRL2 set forth in one of the sequences selected from SEQ ID NOs: 4, 10, 18, 24 and 30; and

[0208] (iii) a CDRL3 having at least 90% sequence identity to a CDRL3 set forth in one of the sequences selected from SEQ ID NOs: 6, 12, 20, 26 and 32; or

[0209] C) one or more CDRHs of A) and one or more CDRLs of B).

[0210] Unless otherwise indicated, the assignment of amino acids to each CDR may be based on one of the numbering schemes provided by Kabat et al. (1991) Sequences of Proteins of Immunological Interest (5 th Ed.), USDept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242; Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID: 2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, Ed. (2007) Handbook of Therapeutic Antibodies, 3 rd Ed., Wily-VCHVerlag GmbH and Co.

[0211] The variable region and CDR in the antibody sequence can be identified according to the general rules (as described above, such as the Kabat numbering system) developed in the art or by comparing the sequence with the database of known variable regions. The method of identifying these regions is described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. The exemplary database of antibody sequence is described in and available from the "Abysis" website (maintained by AC Martin of Department of Biochemistry & Molecular Biology University College London, London, England) and the VBASE2 website www.vbase2.org on www.bioinf.org.uk / abs, such as Retter et al., Nucl. Acids Res., 33 (Database issue): described in D671-D674 (2005). Preferably, the Abysis database is used to analyze the sequence, which integrates sequence data from Kabat, IMGT and protein database (PDB) with structural data from PDB, see Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg.uk / abs). The Abysis database website also includes general rules that have been developed to identify CDRs that can be used according to the teachings of this article. Unless otherwise stated, all CDRs described herein are obtained according to the Abysis database website of Kabat.

[0212] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available from http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6.

[0213] Additionally or alternatively, the protein sequences of the invention can be further used as a "query sequence" to perform a search against a public database to, for example, identify related sequences. Such a search can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215: 403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be used, as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0214] In other embodiments, the CDR amino acid sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above corresponding sequences. As an illustrative example, the antibody may comprise a CDRH1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a CDRH1 as set forth in one of the sequences selected from SEQ ID NOs: 1, 7, 13, 15, 21 and 27.

[0215] Anti-OX40 antibodies comprising CDRs with amino acid additions, deletions and / or substitutions

[0216] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0217] A) one or more heavy chain CDRs (CDRHs) selected from at least one of the following:

[0218] (i) a CDRH1 selected from SEQ ID NOs: 1, 7, 13, 15, 21 and 27, or a CDRH1 that differs from said CDRH1 in amino acid sequence by an amino acid addition, deletion or substitution of not more than 2 amino acids;

[0219] (ii) a CDRH2 selected from the group consisting of SEQ ID NOs: 3, 9, 17, 23 and 29, or a CDRH2 that differs in amino acid sequence from said CDRH2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and

[0220] (iii) a CDRH3 selected from the group consisting of SEQ ID NOs: 5, 11, 19, 25 and 31, or a CDRH3 that differs in amino acid sequence from said CDRH3 by an amino acid addition, deletion or substitution of not more than 2 amino acids;

[0221] B) one or more light chain CDRs (CDRLs) selected from at least one of the following:

[0222] (i) a CDLH selected from SEQ ID NOs: 2, 8, 14, 16, 22 and 28, or a CDRL1 that differs from said CDRL1 in amino acid sequence by an addition, deletion or substitution of no more than 2 amino acids;

[0223] (ii) a CDRL2 selected from the group consisting of SEQ ID NOs: 4, 10, 18, 24 and 30, or a CDRL2 that differs in amino acid sequence from said CDRL2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and

[0224] (iii) a CDRL3 selected from SEQ ID NOs: 6, 12, 20, 26 and 32, or a CDRL3 that differs from said CDRL3 in amino acid sequence by an amino acid addition, deletion or substitution of not more than 2 amino acids; or

[0225] C) one or more CDRHs of A) and one or more CDRLs of B).

[0226] In some embodiments, the CDR of the isolated antibody or its antigen-binding portion thereof comprises no more than 1 conservative substitution of amino acid. The term "conservative substitution" as used herein refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is substituted by another amino acid residue with a similar side chain, such as a physical or functionally similar residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent bonds or hydrogen bonds, etc.) to a corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., 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), which are incorporated herein by reference).

[0227] Anti-OX40 Antibodies Comprising CDRs

[0228] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0229] (a) CDRH1 comprising SEQ ID NO: 1;

[0230] (b) CDRH2 comprising SEQ ID NO: 3;

[0231] (c) a CDRH3 comprising SEQ ID NO: 5;

[0232] (d) CDRL1 comprising SEQ ID NO: 2;

[0233] (e) CDRL2 comprising SEQ ID NO: 4; and

[0234] (f) CDRL3 comprising SEQ ID NO:6.

[0235] In a specific embodiment, the isolated antibody, or antigen-binding portion thereof, comprises:

[0236] (a) CDRH1 consisting of SEQ ID NO: 1;

[0237] (b) CDRH2 consisting of SEQ ID NO: 3;

[0238] (c) CDRH3 consisting of SEQ ID NO: 5;

[0239] (d) CDRL1 consisting of SEQ ID NO: 2;

[0240] (e) CDRL2 consisting of SEQ ID NO: 4; and

[0241] (f) CDRL3 consisting of SEQ ID NO:6.

[0242] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0243] (a) CDRH1 comprising SEQ ID NO: 7;

[0244] (b) a CDRH2 comprising SEQ ID NO: 9;

[0245] (c) a CDRH3 comprising SEQ ID NO: 11;

[0246] (d) CDRL1 comprising SEQ ID NO: 8;

[0247] (e) CDRL2 comprising SEQ ID NO: 10; and

[0248] (f) CDRL3 comprising SEQ ID NO:12.

[0249] In a specific embodiment, the isolated antibody, or antigen-binding portion thereof, comprises:

[0250] (a) CDRH1 consisting of SEQ ID NO: 7;

[0251] (b) CDRH2 consisting of SEQ ID NO: 9;

[0252] (c) CDRH3 consisting of SEQ ID NO: 11;

[0253] (d) CDRL1 consisting of SEQ ID NO: 8;

[0254] (e) CDRL2 consisting of SEQ ID NO: 10; and

[0255] (f) CDRL3 consisting of SEQ ID NO:12.

[0256] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0257] (a) CDRH1 comprising SEQ ID NO: 13;

[0258] (b) a CDRH2 comprising SEQ ID NO: 9;

[0259] (c) a CDRH3 comprising SEQ ID NO: 11;

[0260] (d) CDRL1 comprising SEQ ID NO: 14;

[0261] (e) CDRL2 comprising SEQ ID NO: 10; and

[0262] (f) CDRL3 comprising SEQ ID NO:12.

[0263] In a specific embodiment, the isolated antibody, or antigen-binding portion thereof, comprises:

[0264] (a) CDRH1 consisting of SEQ ID NO: 13;

[0265] (b) CDRH2 consisting of SEQ ID NO: 9;

[0266] (c) CDRH3 consisting of SEQ ID NO: 11;

[0267] (d) CDRL1 consisting of SEQ ID NO: 14;

[0268] (e) CDRL2 consisting of SEQ ID NO: 10; and

[0269] (f) CDRL3 consisting of SEQ ID NO:12.

[0270] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0271] (a) CDRH1 comprising SEQ ID NO: 15;

[0272] (b) a CDRH2 comprising SEQ ID NO: 17;

[0273] (c) a CDRH3 comprising SEQ ID NO: 19;

[0274] (d) CDRL1 comprising SEQ ID NO: 16;

[0275] (e) CDRL2 comprising SEQ ID NO: 18; and

[0276] (f) CDRL3 comprising SEQ ID NO:20.

[0277] In a specific embodiment, the isolated antibody, or antigen-binding portion thereof, comprises:

[0278] (a) CDRH1 consisting of SEQ ID NO: 15;

[0279] (b) CDRH2 consisting of SEQ ID NO: 17;

[0280] (c) CDRH3 consisting of SEQ ID NO: 19;

[0281] (d) CDRL1 consisting of SEQ ID NO: 16;

[0282] (e) CDRL2 consisting of SEQ ID NO: 18; and

[0283] (f) CDRL3 consisting of SEQ ID NO:20.

[0284] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0285] (a) CDRH1 comprising SEQ ID NO: 21;

[0286] (b) a CDRH2 comprising SEQ ID NO: 23;

[0287] (c) a CDRH3 comprising SEQ ID NO: 25;

[0288] (d) CDRL1 comprising SEQ ID NO: 22;

[0289] (e) CDRL2 comprising SEQ ID NO: 24; and

[0290] (f) CDRL3 comprising SEQ ID NO:26.

[0291] In a specific embodiment, the isolated antibody, or antigen-binding portion thereof, comprises:

[0292] (a) CDRH1 consisting of SEQ ID NO: 21;

[0293] (b) CDRH2 consisting of SEQ ID NO: 23;

[0294] (c) CDRH3 consisting of SEQ ID NO: 25;

[0295] (d) CDRL1 consisting of SEQ ID NO: 22;

[0296] (e) CDRL2 consisting of SEQ ID NO: 24; and

[0297] (f) CDRL3 consisting of SEQ ID NO:26.

[0298] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0299] (a) CDRH1 comprising SEQ ID NO: 27;

[0300] (b) a CDRH2 comprising SEQ ID NO: 29;

[0301] (c) a CDRH3 comprising SEQ ID NO: 31;

[0302] (d) CDRL1 comprising SEQ ID NO: 28;

[0303] (e) CDRL2 comprising SEQ ID NO: 30; and

[0304] (f) CDRL3 comprising SEQ ID NO:32.

[0305] In a specific embodiment, the isolated antibody, or antigen-binding portion thereof, comprises:

[0306] (a) CDRH1 consisting of SEQ ID NO: 27;

[0307] (b) CDRH2 consisting of SEQ ID NO: 29;

[0308] (c) CDRH3 consisting of SEQ ID NO: 31;

[0309] (d) CDRL1 consisting of SEQ ID NO: 28;

[0310] (e) CDRL2 consisting of SEQ ID NO: 30; and

[0311] (f) CDRL3 consisting of SEQ ID NO:32.

[0312] Anti-OX40 antibody comprising a heavy chain variable region and a light chain variable region

[0313] In some embodiments, the isolated antibody, or antigen-binding portion thereof, comprises:

[0314] (A) a heavy chain variable region (VH), wherein:

[0315] (i) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, 41 and 43;

[0316] (ii) comprises an amino acid sequence that is at least 85%, 90% or 95% identical to an amino acid sequence selected from SEQ ID NOs: 33, 35, 37, 39, 41 and 43; or

[0317] (iii) an amino acid sequence comprising one or more amino acid additions, deletions and / or substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, 41 and 43; and / or

[0318] (B) a light chain variable region, wherein:

[0319] (i) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, 42 and 44;

[0320] (ii) comprises an amino acid sequence that is at least 85%, 90% or 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, 42 and 44; or

[0321] (iii) an amino acid sequence comprising an addition, deletion and / or substitution of one or more amino acids compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, 42 and 44.

[0322] In a specific embodiment, the isolated antibody or antigen-binding portion thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:33 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:34.

[0323] In a specific embodiment, the isolated antibody or antigen-binding portion thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:35 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:36.

[0324] In a specific embodiment, the isolated antibody or antigen-binding portion thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:37 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:38.

[0325] In a specific embodiment, the isolated antibody or antigen-binding portion thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:39 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:40.

[0326] In a specific embodiment, the isolated antibody or antigen-binding portion thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:41 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:42.

[0327] In a specific embodiment, the isolated antibody, or antigen-binding portion thereof, comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:43 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:44.

[0328] In other embodiments, the amino acid sequences of the heavy chain variable region and / or the light chain variable region can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned respective sequences. As an illustrative example, the antibody can comprise a heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 33.

[0329] In some further embodiments, the isolated antibody or antigen binding portion thereof may comprise conservative substitutions or modifications of amino acids in the variable region of the heavy chain and / or light chain. It is understood in the art that certain conservative sequence modifications may be made that do not eliminate antigen binding. See, e.g., Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0330] As described above, the term "conservative substitution" as used herein refers to an amino acid substitution that does not adversely affect or change the basic properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is substituted with another amino acid residue having a similar side chain, such as a physical or functionally similar residue (e.g., having a similar size, shape, charge, chemical properties including the ability to form covalent bonds or hydrogen bonds, etc.) to a corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., 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), which are incorporated herein by reference).

[0331] Epitope competition binding and epitope mapping

[0332] It will be further understood that the disclosed antibodies will associate or bind to discrete epitopes or immunogenic determinants presented by the selected target or fragment thereof. In some embodiments, epitopes or immunogenic determinants include chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups. In some embodiments, epitopes can have specific three-dimensional structural characteristics and / or specific charge characteristics. Therefore, as used herein, the term "epitope" includes any protein determinant that can specifically bind to an immunoglobulin or T cell receptor or otherwise interact with a molecule. In some embodiments, when an antibody preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules, the antibody is considered to specifically bind (or immunospecifically bind or react) to the antigen. In some embodiments, when the equilibrium dissociation constant (K D ) is less than or equal to 10-6 M or less than or equal to 10 -7 M, more preferably when K D Less than or equal to 10 -8 M, and even more preferably when K D Less than or equal to 10 -9 When M, the antibody is said to bind specifically to the antigen.

[0333] Epitopes formed by consecutive amino acids (sometimes referred to as "linear" or "continuous" epitopes) are usually retained when the protein is denatured, while epitopes formed by tertiary folding are usually lost after protein denaturation. In any case, an antibody epitope usually contains at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation.

[0334] In this regard, it should be understood that in some embodiments, an epitope may be associated with or located in one or more regions, domains or motifs of, for example, a PD-1 protein. Similarly, the art-recognized term "motif" will be used according to its common meaning and should generally refer to a short conserved region of a protein of typically ten to twenty consecutive amino acid residues.

[0335] In any case, once the desired epitope on the antigen is determined, it is possible to produce antibodies against the epitope, for example by immunizing with a peptide comprising the epitope using the technology described in the present invention. Alternatively, in the discovery process, the generation and characterization of antibodies can clarify information about the desired epitope located in a specific domain or motif. From this information, it is possible to competitively screen antibodies that bind to the same epitope. The method for achieving this is to conduct competitive studies to find antibodies that competitively bind to each other, i.e., antibodies compete to bind to the antigen. A high-throughput method for competitive binding of antibodies based on their cross-competition is described in WO 03 / 48731. Competitive binding or domain level or epitope mapping include antibody competition or other methods of antigen fragment expression on yeast are well known in the art.

[0336] As used herein, the term "competitive binding" refers to a method for grouping or classifying antibodies based on antigen binding characteristics and competition. Although these techniques are useful for defining and classifying the antibodies of the present invention, these bins do not always bind directly to epitopes, and this initial determination of epitope binding can be further improved and confirmed by other recognized methods in the art and as described herein. However, it is understood that the empirical allocation of antibodies to various bins provides information that can indicate the therapeutic potential of the disclosed antibodies.

[0337] More specifically, whether a selected reference antibody (or fragment thereof) binds to the same epitope or cross-competes for binding (i.e., is in the same bin) as a second test antibody can be determined by using methods known in the art and shown in the Examples herein.

[0338] Other compatible epitope mapping techniques include alanine scanning mutants, peptide blotting (Reineke (2004) Methods Mol Biol 248: 443-63) (specifically incorporated herein by reference in its entirety) or peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens (Tomer (2000) Protein Science 9: 487-496) (specifically incorporated herein by reference in its entirety) can be used.

[0339] Nucleic acid molecules encoding the antibodies of the present invention

[0340] In some aspects, the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region and / or a light chain variable region of an isolated antibody as disclosed herein.

[0341] Nucleic acids of the present invention can be obtained using standard molecular biology techniques. For hybridoma-expressed antibodies (e.g., hybridomas prepared by transgenic mice carrying human immunoglobulin genes as further described below), cDNA encoding the light and heavy chains of antibodies prepared by hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be recovered from gene libraries.

[0342] The isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene by operably linking the nucleic acid encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat et al. (1991), supra), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but more preferably an IgG1 or IgG4 constant region.

[0343] By operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, the isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequence of human light chain constant region genes is known in the art (see, e.g., Kabat et al., supra), and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a kappa or lambda constant region.

[0344] Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes or scFv genes. In these operations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein is intended to mean that the two DNA fragments are connected so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0345] In some embodiments, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region of an isolated antibody as disclosed herein.

[0346] In some specific embodiments, the isolated nucleic acid molecule encodes the heavy chain variable region of the isolated antibody and comprises a nucleic acid sequence selected from the group consisting of:

[0347] (A) nucleic acid sequences encoding the heavy chain variable regions shown in SEQ ID NOs: 33, 35, 37, 39, 41 and 43;

[0348] (B) a nucleic acid sequence as shown in SEQ ID NO: 45, 47, 49, 51, 53 or 55; or

[0349] (C) A nucleic acid sequence that hybridizes to the complementary strand of the nucleic acid sequence of (A) or (B) under highly stringent conditions.

[0350] For example, the nucleic acid molecule consists of SEQ ID NO: 45, 47, 49, 51, 53, or 55. Alternatively, the nucleic acid molecule shares at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 45, 47, 49, 51, 53, or 55. In some embodiments, the percent identity results from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0351] In some embodiments, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the light chain variable region of an isolated antibody as disclosed herein.

[0352] In some specific embodiments, the isolated nucleic acid molecule encodes the light chain variable region of an isolated antibody, which comprises a nucleic acid sequence selected from the group consisting of:

[0353] (A) a nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO: 34, 36, 38, 40, 42 or 44;

[0354] (B) a nucleic acid sequence as shown in SEQ ID NO: 46, 48, 50, 52, 54 or 56; or

[0355] (C) A nucleic acid sequence that hybridizes to the complementary strand of the nucleic acid sequence of (A) or (B) under highly stringent conditions.

[0356] For example, the nucleic acid molecule consists of SEQ ID NO: 46, 48, 50, 52, 54, or 56. Alternatively, the nucleic acid molecule shares at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 46, 48, 50, 52, 54, or 56. In some embodiments, the percent identity results from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0357] Exemplary high stringency conditions include hybridization at 45°C in 5X SSPE and 45% formamide, and final washing at 65°C in 0.1X SSC. It is understood in the art that conditions of equal stringency can be achieved by changing the temperature and buffer or salt concentration, as described in Ausubel et al. (eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pages 6.0.3 to 6.4.10. The changes in hybridization conditions can be determined empirically or accurately calculated based on the length and percentage of the guanosine / cytosine (GC) base pairing of the probe. Hybridization conditions can be calculated as described in Sambrook et al. (eds.), Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pages 9.47 to 9.51.

[0358] Pharmaceutical composition

[0359] In some aspects, the invention relates to a pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as disclosed herein and a pharmaceutically acceptable carrier.

[0360] Components of the composition

[0361] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent or vaccine, such that the anti-OX40 antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, combinations of various components known in the art or more.

[0362] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene and / or propyl arsenate. As disclosed in the present invention, in a solvent containing an antibody or antigen-binding fragment of the composition disclosed in the present invention containing one or more antioxidants such as methionine that reduce the antibody or its antigen-binding fragment, it may be oxidized. Redox can prevent or reduce the reduction of binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the present invention provides a composition comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants such as methionine. The present invention further provides a variety of methods, wherein the antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine. Thus, the antibody or antigen-binding fragment thereof can be protected from oxidation to extend its shelf life and / or increase its activity.

[0363] To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN-80), partitioning agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multiple dose containers comprising phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.

[0364] Administration, formulation and dosage

[0365] The pharmaceutical compositions of the present invention can be administered to a subject in need thereof in vivo by various routes, including but not limited to oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The compositions of the present invention can be formulated into solid, semisolid, liquid or gaseous preparations; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. Suitable preparations and routes of administration can be selected according to the intended application and treatment regimen.

[0366] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalants and controlled release forms thereof.

[0367] Preparations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickeners, and solutes that make the preparation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohol, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such preparations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosage regimens (including dosage, time, and repetition) will depend on specific individuals and individual medical history as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0368] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferating or tumorigenic cells, maintaining the reduction of such tumor cells, reducing the proliferation of tumor cells or delaying the development of metastasis. In some embodiments, the dose administered can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a sustained continuous release formulation of the therapeutic composition of the present invention may be suitable.

[0369] Those skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dosage generally involves a balance between the level of therapeutic benefit and any risk or harmful side effects. The selected dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound, administration, administration time, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, sex, age, weight, condition, general health and previous medical history of the patient. The amount of the compound and the route of administration are ultimately determined by a physician, veterinarian or clinician, but the dosage is generally selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects.

[0370] Generally, the antibodies of the present invention or their antigen-binding portions can be administered in various ranges. These include about 5 μg / kg body weight to about 100 mg / kg body weight per dose; about 50 μg / kg body weight to about 5 mg / kg body weight per dose; about 100 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose and about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In certain embodiments, the dosage is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, at least about 10 mg / kg body weight.

[0371] In any case, the antibodies or antigen-binding portions thereof of the present invention are preferably administered to subjects in need as needed. The frequency of administration can be determined by those skilled in the art, such as the attending physician based on the considerations of the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, etc.

[0372] In certain preferred embodiments, the course of treatment involving the antibodies or antigen-binding portions thereof of the invention will include multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibodies or antigen-binding portions thereof of the invention may be administered every day, every two days, every four days, every week, every ten days, every two weeks, every three weeks, every month, every six weeks, every two months, every ten weeks or every three months. In this regard, it will be appreciated that the dosage may be changed or the interval adjusted based on patient response and clinical practice.

[0373] The dosage and regimen of the disclosed therapeutic composition can also be determined empirically in individuals given one or more administrations. For example, an individual may be given incremental doses of a therapeutic composition produced as described herein. In selected embodiments, the dosage may be gradually increased or decreased or mitigated, respectively, based on empirically determined or observed side effects or toxicity. In order to assess the efficacy of the selected composition, markers of a specific disease, disorder or condition may be tracked as described above. For cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; improvements assessed by direct tumor biopsy and microscopic examination of tumor specimens; measurement of indirect tumor markers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein, relief of pain or paralysis; improvements in speech, vision, breathing or other disabilities associated with the tumor; increased appetite; or improvements in quality of life or prolonged survival as measured by accepted tests. Those skilled in the art will appreciate that the dosage will vary depending on the individual, the type of tumor condition, the stage of the tumor condition, whether the tumor condition has begun to metastasize to other locations in the individual, and the treatments used in the past and the treatments used concurrently.

[0374] Compatible formulations for parenteral administration (e.g., intravenous injection) will include an antibody or antigen-binding portion thereof as disclosed herein at a concentration of about 10 μg / ml to about 100 mg / ml. In certain selected embodiments, the concentration of the antibody or antigen-binding portion thereof will include 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml or 1 mg / ml. In other preferred embodiments, the ADC concentration will include 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml or 100 mg / ml.

[0375] Application of the present invention

[0376] The antibodies, antibody compositions and methods of the invention have many in vitro and in vivo uses, including, for example, detection of OX40 or enhancement of immune responses. For example, these molecules can be administered to cultured cells in vitro or ex vivo, or, for example, administered to human subjects in vivo, to enhance immunity in various situations. The immune response can be modulated, for example, enhanced, stimulated or upregulated.

[0377] Preferred subjects include human patients in need of an enhanced immune response. The method is particularly suitable for treating human patients with conditions that can be treated by enhancing an immune response (e.g., a T cell-mediated immune response). In a specific embodiment, the method is particularly suitable for treating cancer in vivo. In order to achieve antigen-specific enhancement of immunity, the anti-OX40 antibody can be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a subject carrying a tumor or a virus). When the anti-OX40 antibody is administered together with another agent, the two can be administered in any order or simultaneously.

[0378] The present invention further provides a method for detecting the presence of human OX40 antigen in a sample or measuring the amount of human OX40 antigen, comprising contacting a sample and a control sample with a human monoclonal antibody or an antigen-binding portion thereof that specifically binds to human OX40 under conditions that allow formation of a complex between the antibody or a portion thereof and human OX40. The formation of the complex is then detected, wherein differential complex formation between the samples compared to the control sample indicates the presence of human OX40 antigen in the sample. In addition, the anti-OX40 antibodies of the present invention can be used to purify human OX40 by immunoaffinity purification.

[0379] Treating medical conditions including cancer

[0380] In some aspects, the invention provides a method of treating a disorder in a mammal, comprising administering a therapeutically effective amount of an antibody or antigen-binding portion thereof disclosed herein to a subject (eg, a human) in need of treatment. For example, the disorder is cancer.

[0381] Various cancers involving OX40, whether malignant or benign, and whether primary or secondary, can be treated or prevented using the methods provided by the present disclosure. The cancer can be a solid cancer or a hematological malignancy. Examples of such cancers include lung cancers such as bronchial cancers (e.g., squamous cell carcinomas, small cell carcinomas, large cell carcinomas, and adenocarcinomas), alveolar cell carcinomas, bronchial adenomas, cartilaginous hamartomas (non-cancerous) and sarcomas (cancerous); cardiac cancers such as myxomas, fibromas, and rhabdomyomas; bone cancers such as osteochondromas, condromas, chondroblastomas, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, chondrosarcomas, multiple myeloma, osteosarcomas, fibrosarcomas, malignant fibrous histiocytomas, Ewing's tumors (Ewing's sarcomas), and reticulum cell sarcomas; brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytomas, and gliomas. , astrocytoma, oligodendroglioma, medulloblastoma, chordoma, neurilemoma, ependymoma, meningioma, pituitary adenoma, pinealoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germ cell tumor, teratoma, dermoid cyst and hemangioma; cancers of the digestive system such as colon cancer, leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colorectal polyp and colorectal cancer; liver cancer such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrocarcinoma, bile duct cancer, hepatoblastoma and angiosarcoma; kidney cancer such as renal adenocarcinoma, renal cell carcinoma, adrenal tumor and transitional cell carcinoma of the renal pelvis; bladder cancer; blood liquid cancers such as acute lymphocytic (lymphoblastic) leukemia, acute myeloid (myelocytic, myeloid, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), chronic myeloid (myeloid, myeloid, granulocytic) lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia, and chronic myelogenous leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; Cancers of the cervix, ovaries, ovaries, ovaries, uterus, vagina, fallopian tubes, and hydatidiform moles are also included in the invention. Cancers of the cervix, ovaries ...

[0382] In some embodiments, examples of cancer include, but are not limited to, B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-lytic cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL) leukemia; hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with nevus hamartomatosis, edema (such as associated with brain tumors), B-cell proliferative disorders, and Meig's syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, Frontline low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B-cell lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, Nodal marginal zone-MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasma cell myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular NHL, mantle cell lymphoma, follicle center lymphoma (follicular), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive frontline NHL and aggressive relapsed NHL), relapsed or refractory NHL after autologous stem cell transplantation (NHL relapsing after or refractory to autologous stem cell transplantation), primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-lytic cell NHL, bulky disease NHL, Burkitt's lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sezary syndrome, cutaneous (cutaneous) lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

[0383] In some embodiments, examples of cancer also include, but are not limited to, B-cell proliferative disorders, which also include, but are not limited to, lymphomas (e.g., B-cell non-Hodgkin lymphoma (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small non-lytic cell lymphoma / Burkitt's lymphoma (including endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma, and non-Burkitt's lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B-cell lymphoma, and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B-cell diffuse large cell lymphoma (DLCL) ), diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B-cell lymphoma, angiocentric lymphoma - pulmonary B-cell lymphoma), f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasmacytoma, plasma cell myeloma, multiple myeloma, plasmacytoma and / or j) Hodgkin's disease.

[0384] In some other embodiments, the condition is an autoimmune disease. Examples of autoimmune diseases treatable with antibodies or antigen-binding portions thereof include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. Antibodies or antigen-binding portions thereof may also be used to treat or prevent infectious diseases, inflammatory diseases (e.g., allergic asthma), and chronic graft-versus-host disease.

[0385] Stimulation of immune response

[0386] In some aspects, the present invention also provides a method for enhancing (e.g., stimulating) an immune response in a subject, comprising administering an antibody or antigen-binding portion thereof of the present invention to a subject so that the immune response in the subject is enhanced. For example, the subject is a mammal. In a specific embodiment, the subject is a human.

[0387] The term "enhancing an immune response" or grammatical variations thereof means any response that stimulates, induces, increases, improves or enhances the immune system of a mammal. The immune response can be a cellular response (i.e., cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (i.e., an antibody-mediated response), and can be a primary or secondary immune response. Examples of enhancing an immune response include increased CD4 +The generation of helper T cell activity and cytolytic T cells. The enhancement of immune response can be assessed using many in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production or IFN-γ production), tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, expression of cell surface markers and cytotoxicity. Generally, the disclosed method enhances the immune response of mammals when compared with the immune response of untreated mammals or mammals not treated using the methods disclosed herein. In one embodiment, antibodies or their antigen-binding portions are used to enhance the immune response of people to microbial pathogens (e.g., viruses). In another embodiment, antibodies or their antigen-binding portions are used to enhance the immune response of people to vaccines. In one embodiment, the method enhances cellular immune response, particularly cytotoxic T cell response. In another embodiment, the cellular immune response is a T helper cell response. In yet another embodiment, the immune response is cytokine production, particularly IFN-γ production or IL-2 production. Antibodies or their antigen-binding portions can be used to enhance the immune response of people to microbial pathogens (e.g., viruses) or vaccines.

[0388] The antibodies, or antigen-binding portions thereof, can be used alone as a monotherapy or can be used in combination with chemotherapy or radiation therapy.

[0389] Used in combination with chemotherapy

[0390] The antibodies, or antigen-binding portions thereof, can be used in combination with anti-cancer agents, cytotoxic agents, or chemotherapeutic agents.

[0391] The term "anticancer agent" or "antiproliferative agent" means any agent that can be used to treat a cell proliferative disorder such as cancer, and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiotherapy and radiotherapeutic agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents and immunotherapeutic agents. It should be understood that in selected embodiments as described above, such anticancer agents may comprise a conjugate and may be combined with the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, the selected anticancer agent is connected to an unpaired cysteine ​​of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such an engineered conjugate is explicitly contemplated within the scope of the present invention. In other embodiments, the disclosed anticancer agent will be administered in combination with a site-specific conjugate comprising a different therapeutic agent as described above.

[0392] As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell destruction. In certain embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxins and exotoxins, Staphylococcus enterotoxin A), fungi (e.g., alpha-sarcin, restrictocin), plants (abrin, ricin, modeccin, viscumin, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, hordeotoxin, Aleurites fordii proteins, dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotonin, phytoncidin inhibitor, gelonin, mitegellin, restrictocin, phenomycin, neomycin and trichothecenes), or animals (e.g., cytotoxic RNases, such as extracellular pancreatic RNase; DNase I, including fragments and / or variants thereof).

[0393] For purposes of the present invention, "chemotherapeutic agents" include chemical compounds (e.g., cytotoxic agents or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. These chemicals are generally directed to the intracellular processes required for cell growth or division, and are therefore particularly effective for cancer cells that typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. Typically, chemotherapeutic agents may include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become resistant or produce tumorigenic offspring (e.g., TICs). These agents are typically used in combination, and are typically the most effective, for example, in regimens such as CHOP or FOLFIRI.

[0394] Examples of anticancer agents that can be used in combination with the site-specific constructs of the invention (either as a component of the site-specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethyleneimine and methylmelamine, acetogenins, camptothecins, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, sarcodict yin), spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicins, chromoprotein enediyne antibiotic chromophores, aclacinomycins, actinomycins, anthramycins, azoserine, bleomycins, actinomycin C, carabicin, carminomycin, carmomycin, chromomycins, dactinomycin, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, Doxorubicin, epirubicin, esorubicin, idarubicin, mexilomycin, mitomycin, mycophenolic acid, nogamycin, olivetocin, peplomycin, potfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, tuberculocin, ubenimex, netastatin, zorubicin; anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, anti-adrenergics, folic acid supplements such as furinic acid acid), aceglucuronide, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatrexate, defofamine, colcemid, diazocone, elfornithine, elliptonium acetate, apocillin, etogluconate, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids, mitoxantrone, mitoxantrone, mopidanmol, nitraerine, pentostatin, methamine, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazine, procarbazine, polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizoran; spirogermanium; tenuzocic acid; triazoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, baculosporin A, and serpentin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine, Vinorelbine; Novoline; Teniposide; Edatrexate; Daunorubicin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11); Topoisomerase inhibitor RFS 2000; Difluoromethylornithine; Retinoids; Capecitabine; Combretastatin; Leucovorin; Oxaliplatin; Inhibitors of PKC-α, Raf, H-Ras, EGFR and VEGF-A (which reduce cell proliferation), and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in this definition are anti-hormonal agents used to modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; as well as Troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, rIL-2; Topoisomerase 1 inhibitors; rmRH; vinorelbine and esperamicin, and pharmaceutically acceptable salts, acids or derivatives of any one of the foregoing.

[0395] Used in combination with radiation therapy

[0396] The present invention also provides a combination of an antibody or an antigen-binding portion thereof with radiotherapy (i.e., any mechanism for inducing DNA damage locally in tumor cells, such as γ-irradiation, X-rays, UV-irradiation, microwaves, electron emission, etc.). Combination therapy using directed delivery of radioisotopes to tumor cells is also contemplated, and the disclosed conjugates can be used in combination with targeted anticancer agents or other targeted means. Typically, radiotherapy is administered in a pulsed manner over a period of about 1 week to about 2 weeks. Radiotherapy can be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple sequential doses.

[0397] diagnosis

[0398] The present invention provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods for screening cells from patients to identify tumor cells including tumorigenic cells. Such methods include identifying individuals with cancer for treatment or monitoring the progression of cancer, including contacting the patient or a sample obtained from the patient (in vivo or in vitro) with an antibody as described herein, and detecting the presence or absence or binding level of the antibody bound to a bound or free target molecule in the sample. In some embodiments, the antibody will include a detectable label or reporter molecule as described herein.

[0399] In some embodiments, binding of an antibody to specific cells in a sample can indicate that the sample is likely to contain tumorigenic cells, thereby indicating that an individual with cancer can be effectively treated with an antibody described herein.

[0400] Samples can be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblotting assays, Western blot analysis, and flow cytometry assays. Compatible in vivo diagnostics or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scans), positron emission tomography (e.g., PET scans), radiography, ultrasound, etc., known to those skilled in the art.

[0401] Pharmaceutical packaging and kits

[0402] Also provided are pharmaceutical packaging and kits comprising one or more containers containing one or more doses of an antibody or an antigen-binding portion thereof. In certain embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or an antigen-binding portion thereof, with or without one or more other agents. For other embodiments, such a unit dose is supplied in a disposable prefilled syringe for injection. In other embodiments, the composition contained in the unit dose may contain saline, sucrose or the like; a buffer such as phosphate, etc.; and / or be formulated in a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition may be provided as a lyophilized powder, which may be reconstituted after adding a suitable liquid (e.g., sterile water or saline). In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the encapsulated conjugate composition is used to treat a selected tumor disease condition.

[0403] The present invention also provides a kit for producing a single-dose or multi-dose administration unit of a site-specific conjugate and optionally one or more anticancer agents. The kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of a variety of materials, such as glass or plastic, and contains a pharmaceutically effective amount of the disclosed conjugate or non-conjugated conjugate. In other preferred embodiments, the container includes a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). Such a kit generally contains a pharmaceutically acceptable preparation of an engineered conjugate in a suitable container, and optionally contains one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable preparations for diagnosis or combined therapy. For example, in addition to the antibody or antigen-binding portion thereof of the present invention, such a kit may contain any one or more anticancer agents, such as chemotherapeutic agents or radiotherapeutic agents; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents.

[0404] More specifically, the kit can have a single container containing the disclosed antibody or antigen-binding portion thereof, with or without additional components, or they can have different containers for each required reagent. In the case of providing a combination therapeutic agent for conjugation, a single solution can be premixed in a molar equivalent combination or one component more than another. Alternatively, the conjugate of the kit and any optional anticancer agent can be stored separately in different containers before being administered to the patient. The kit can also include a second / third container device for containing a sterile pharmaceutically acceptable buffer or other diluent such as antibacterial water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution and glucose solution.

[0405] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit may be provided as dry powders. When the reagents or components are provided in dry powder form, the powder may be reconstituted by adding a suitable solvent. It is contemplated that the solvent may also be provided in another container.

[0406] As briefly described above, the kit may also contain a means for administering the antibody or antigen-binding portion thereof and any optional components to a patient, such as one or more needles, IV bags or syringes, or even eye drops, pipettes or other similar devices, by which the formulation can be injected or introduced into an animal or applied to a diseased area of ​​the body. The kit of the invention will typically also include a device for containing vials or the like and other tightly closed components for commercial sale, such as an injection or blow molded plastic container, in which the desired vials and other devices are placed and retained.

[0407] Sequence Listing Overview

[0408] This application is accompanied by a sequence listing containing a number of nucleic acid and amino acid sequences. Tables A, B and C below provide an overview of the sequences contained.

[0409] The six illustrative antibodies as disclosed herein are fully human anti-OX40 monoclonal antibodies, respectively designated as "1.62.3-u1-IgG1K", "1.62.3-u1-3-IgG1K", "1.7.10-u1-IgG1K", "1.134.9-u1-IgG1L", "1.186.19-u1-IgG1K" and "1.214.23-u1-IgG1K".

[0410] Table A

[0411] CDR amino acid sequences

[0412]

[0413] Table B

[0414] Variable region amino acid sequence

[0415]

[0416]

[0417] Table C

[0418] Variable region nucleotide sequence

[0419]

[0420]

[0421] Example

[0422] The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention. These examples are not intended to represent that the experiments below are all or the only experiments performed.

[0423] Example 1

[0424] Preparation of materials

[0425] 1.1 Generation of immunogens

[0426] The cDNA encoding the extracellular domain (ECD) of the OX40 protein was synthesized by Sangon Biotech (GenBank refCAB96543.1) and inserted into the modified expression vector pcDNA3.3 (ThermoFisher). Plasmid DNA was prepared on a large scale and the inserted DNA sequence was verified by sequencing. The fusion protein OX40 ECD coupled to a human Fc or His tag was obtained by transfecting the human OX40 ECD gene into Freestyle 293F (ThermoFisher) or Expi-293F cells (ThermoFisher). After 5 days, the supernatant was harvested from the transiently transfected cell culture. The fusion protein was purified and quantified for immunization and screening.

[0427] 1.2 Production of benchmark antibodies

[0428] Four reference antibodies, namely BMK1, BMK5, BMK7 and BMK10, are used as positive controls in the examples. BMK1 is synthesized according to the clone of 11D4 of U.S. Patent No. US8236930B2 (Pfizer). BMK5 is synthesized according to the clone of 106-222 of U.S. Patent Application No. US20140308276 (University of Texas System). BMK7 is synthesized according to the clone of OX40mAb24 of PCT Publication No. WO2016057667 (MedImmune). BMK10 is synthesized according to the clone of 1A7.gr1 of PCT Publication No. WO2015153513 (Genentech).

[0429] 1.3 Establishment of stable cell lines

[0430] In order to obtain tools for antibody screening and validation, we have generated OX40 transfected cell lines. In brief, CHO-K1 or 293F cells were transfected with a modified expression vector pcDNA3.3 containing the full-length OX40 gene using Lipofectamine 2000 or PlasFect transfection kit according to the manufacturer's protocol. After 48-72 hours of transfection, the transfected cells were cultured in a culture medium containing blasticidin for screening. Over time, this will screen cells with an expression plasmid stably integrated into their genomic DNA. Check the OX40 expression of the cells at the same time. Once expression is confirmed, a single target clone is selected and expanded to a large amount by limiting dilution. The monoclonal cell line established is then maintained in a culture medium containing blasticidin and cultured.

[0431] Example 2

[0432] Antibody hybridoma production

[0433] 2.1 Immunization and cell fusion

[0434] OMT rats were used to prepare fully human monoclonal antibodies against OX40, which contained chimeric polynucleotides that can be used for optimal production of functional immunoglobulins with human idiotypes. As described in PCT Publication WO2014 / 093908, the rat strain carries human heavy and light chain transgenes. To produce fully human monoclonal antibodies against OX40, 6-8 week old OMT rats were immunized in the footpad with 20 μg of human OX40 ECD protein mixed with aluminum phosphate (Alum-Phos) as an adjuvant, and TiterMax mixed with 20 μg of human OX40 ECD protein was injected subcutaneously for the first boost, and immunization was repeated every two weeks with Alum-Phos and TiterMax mixed with human OX40ECD protein. Serum antibody titers were measured by enzyme-linked immunosorbent assay (ELISA) every 1 or 2 weeks. When the serum antibody titer was high enough, the rats were given a final booster immunization with 40 μg of human OX40 ECD protein mixed with DPBS without adjuvant. Cell fusion was performed as follows: myeloma cells SP2 / 0 were prepared, thawed and revived one week before fusion, and passaged at 1:2 every day until the day before fusion to keep the cells in the logarithmic growth phase. B lymphocytes isolated from the lymph nodes of immunized OMT rats were mixed with myeloma cells (at a ratio of 1:1.1). The cell mixture was washed and incubated at 2x10 6 Resuspend 10 cells / mL in ECF solution and prepare for ECF. After cell electrofusion, transfer the cell suspension from the fusion chamber immediately to a sterile tube containing more culture medium and incubate in a 37°C incubator for at least 24 hours. Mix the cell suspension and transfer to a 96-well plate (1x10 4 The 96-well plates were incubated at 37°C, 5% CO2 and monitored regularly. When the clones were large enough, 100 uL of supernatant was transferred from the tissue culture plates to 96-well assay plates for antibody screening.

[0435] 2.2 High-throughput screening of hybridoma supernatants

[0436] ELISA was used as the first screening method to test the binding of hybridoma supernatants to human and monkey OX40 proteins. In short, plates (Nunc) were coated overnight at 4°C with soluble proteins of human or macaque OX40 ECD at 1ug / mL. After blocking and washing, hybridoma supernatants were transferred to the coated plates and incubated at room temperature for 2 hours. The plates were then washed and subsequently incubated for 1 hour with secondary antibodies (goat anti-rat IgG HRP (Bethyl)). After washing, TMB substrate was added and the interaction was terminated with 2M HCl. The absorbance at 450nm was read using a microplate reader (Molecular Device).

[0437] To confirm the natural binding of anti-OX40 antibodies to conformational OX40 molecules expressed on the cell membrane, flow cytometry (FACS) analysis was performed on OX40-transfected cell lines. 293F cells expressing human OX40 were cultured at 1x10 5 The density of cells / well was transferred to a 96-well U-bottom plate (Corning). The hybridoma supernatant was then mixed with the cells and incubated at 4°C for 1 hour. After washing with 1x PBS / 1% BSA, the secondary antibody goat-anti-rat Alexa647 (Jackson ImmunoResearchLab) was applied and incubated with the cells at 4°C in the dark for half an hour. The cells were then washed and resuspended in 1x PBS / 1% BSA or fixed with 4% paraformaldehyde and analyzed by flow cytometry (BD). Antibodies that bind to the 293F cell line that is not transfected with OX40 were used as negative controls. The biological activity of the antibodies was tested using Jurkat NFkB-luciferase reporter T cells as a second screening method. In brief, Jurkat NFkB-luciferase reporter cells overexpressing human OX40 / CD40 fusion protein were constructed as described above. The cells were cultured in complete RPMI1640 medium containing 10% FBS and 0.5mg / mL hygromycin B (as screening). OX40 Jurkat reporter cells were collected and cultured at 4 × 10 4 Cells / well were added to a 96-well plate. Cross-linked antibody F(ab')2 goat anti-rat IgG (Jackson ImmunoResearch Lab) and hybridoma supernatant diluted in RPMI 1640 complete medium were added to the cells, and then the cells were incubated overnight at 37°C, 5% CO2. The next day, reconstructed luciferase substrate (Promega) was added to each well (50 μL / well) and mixed thoroughly. Luciferase intensity was read using a microplate reader (Molecular Device).

[0438] 2.3 Hybridoma subcloning:

[0439] Once specific binding and biological activity have been verified by the first and confirmation screening, the positive hybridoma cell lines are used for subcloning. In brief, for each hybridoma cell line, cells are counted and diluted to a density of 1 cell per 200 μL cloning medium. The cell suspension is inoculated into two 96-well plates at 200 μL / well. The plates are cultured at 37°C, 5% CO2 until they are ready to be checked by ELISA assay. The exhausted supernatant (ESN) of the selected monoclonal clones is collected, and the antibodies are purified for further characterization.

[0440] Example 3

[0441] Construction and purification of fully human antibody molecules

[0442] 3.1 Hybridoma Sequencing

[0443] RNA was extracted from monoclonal hybridoma cells using RNeasy Plus Mini Kit (Qiagen) and Trizol reagent. The heavy chain variable region (VH) and light chain variable region (VL) of the OX40 chimeric antibody were amplified as follows. Briefly, RNA was first reverse transcribed into cDNA using reverse transcriptase as described below.

[0444] Table 1. cDNA amplification reaction (20 μL)

[0445]

[0446] Table 2. cDNA amplification reaction conditions

[0447] Step 1 Step 2 Step 3 Step 4 temperature 25 50 85 4 time 10min 50min 5min ∞

[0448] The resulting cDNA was used as a template for subsequent PCR amplification using primers specific for the target gene. The PCR reaction was performed as follows.

[0449] Table 3. PCR reaction system (50 μL)

[0450] Components Dosage cDNA 2.0μL Premix Ex Taq 25μL 5'-Degenerate Primer Set (10pM) 2.5μL 3'-Constant region degenerate primer (10pM) 1.0μL <![CDATA[ddH2O]]> 19.5μL

[0451] Table 4. PCR reaction conditions

[0452]

[0453] The PCR product (10 μL) was inserted into the pMD18-T vector; and 10 μL of the ligation product was transformed into Top 10 competent cells. The transformed cells were plated on 2-YT+Cab plates and cultured overnight. Positive clones were checked by PCR using M13-48 and M13-47 primers and then sequenced.

[0454] Hybridoma clones 1.7.10, 1.62.3, 1.134.9, 1.186.19, and 1.214.23 were selected for sequence optimization and further evaluation.

[0455] 3.2 Antibody sequence optimization

[0456] Antibody sequence optimization is performed by introducing appropriate modifications at specific sites into the nucleotide sequence encoding the antibody. Mutations that remove PTM (post-translational modification) sites are introduced by site-directed mutagenesis using the QuickChange mutagenesis kit according to the manufacturer's protocol.

[0457] The amino acid NGG in the CDR1 of the heavy chain of hybridoma clone 1.62.3 was identified as a deamidation site, so antisense mutagenic nucleotides were designed to introduce the following mutations into the "1.62.3-u1-IgG1K" heavy chain: N to Q (NGG-QGG), N to S (NGG-SGG), or G to A (NGG-NAG). The amino acid C in the CDR1 of the light chain of clone 1.62.3 was identified as a cysteine ​​residue, so cysteine ​​(C to S) was mutated to serine. All mutations were verified by sequencing.

[0458] Figure 1 Comparison between variants after PTM mutation is shown. The variants are named antibodies "1.62.3-u1-1-IgG1K", "1.62.3-u1-2-IgG1K" and "1.62.3-u1-2-IgG1K". Antibody "1.62.3-u1-1-IgG1K" contains a mutation from N to Q, "1.62.3-u1-2-IgG1K" contains a mutation from N to S, and "1.62.3-u1-3-IgG1K" contains a mutation from G to A.

[0459] 3.3 Construction and purification of fully human antibody molecules

[0460] The VH and VL of the OX40 hybridoma antibody were amplified as described above. The synthetic genes were re-cloned into the modified human IgG1 expression vector pcDNA3.4 (ThermoFisher) to express the fully human antibody. Expi-293F cells were transiently transfected with the vector for antibody expression. The culture supernatant containing the antibody was collected and purified using protein A chromatography.

[0461] Fully human monoclonal antibodies 1.7.10-u1-IgG1K, 1.62.3-u1-IgG1K (sequence optimized clones were named "1.62.3-u1-3-IgG1K"), 1.134.9-u1-IgG1L, 1.186.19-u1-IgG1K and 1.214.23-u1-IgG1K were obtained from hybridoma clones 1.7.10, 1.62.3, 1.134.9, 1.118.19 and 1.214.23 hybridomas, respectively. Their sequences are summarized in Tables A, B and C.

[0462] Example 4

[0463] Antibody characterization

[0464] 4.1 Testing the complete binding kinetics affinity by surface plasmon resonance (SPR)

[0465] The affinity and binding kinetics of antibodies against OX40 were characterized by SPR assays using Biacore T200 (GE). Anti-human IgG antibodies were pre-immobilized on a sensor chip (CM5) and anti-OX40 antibodies diluted in buffer (1×HBS-EP+, GE) were captured when injected into the chip. Different concentrations of human or monkey OX40 protein and buffer were then flowed through the sensor chip at a flow rate of 30 μL / min for an association phase of 180 s, followed by dissociation. The binding and dissociation curves were fitted by a 1:1 Langmuir binding model using BIAevaluation T200 software.

[0466] The experimental results are shown in Table 5 below.

[0467] Table 5. Full kinetic binding affinity for human OX40 determined by SPR

[0468] Abs ka(1 / Ms) kd(1 / s) <![CDATA[K D (M)]]> 1.7.10-u1-IgG1K <![CDATA[8.60×10 5 ]]> <![CDATA[1.28×10 -3 ]]> <![CDATA[1.49×10 -9 ]]> 1.62.3-u1-IgG1K <![CDATA[5.26×10 5 ]]> <![CDATA[2.43×10 -4 ]]> <![CDATA[4.61×10 -10 ]]> 1.62.3-u1-3-IgG1K <![CDATA[6.97×10 5 ]]> <![CDATA[1.45×10 -3 ]]> <![CDATA[2.08×10 -9 ]]> 1.134.9-u1-IgG1L <![CDATA[2.07×10 5 ]]> <![CDATA[3.70×10 -5 ]]> <![CDATA[1.79×10 -10 ]]> 1.186.19-u1-IgG1K <![CDATA[3.90×10 5 ]]> <![CDATA[4.51×10 -4 ]]> <![CDATA[1.16×10 -9 ]]> 1.214.23-u1-IgG1K <![CDATA[4.26×10 5 ]]> <![CDATA[2.14×10 -4 ]]> <![CDATA[5.02×10 -10 ]]> BMK7 <![CDATA[2.44×10 5 ]]> <![CDATA[1.54×10 -3 ]]> <![CDATA[6.30×10 -9 ]]> BMK10 <![CDATA[4.42×10 5 ]]> <![CDATA[2.25×10 -5 ]]> <![CDATA[5.10×10 -10 ]]>

[0469] As shown in Table 5, the illustrative antibodies of the present invention (including 1.7.10-u1-IgG1K, 1.62.3-u1-IgG1K, 1.62.3-u1-3-IgG1K, 1.134.9-u1-IgG1L, 1.186.19-u1-IgG1K and 1.214.23-u1-IgG1K) bind to human OX40 with high specificity. D 1.79×10 -10 M to 2.08×10 -9 M.

[0470] 4.2 Binding affinity analysis by flow cytometry

[0471] CHO-K1 cells expressing human OX40 were cultured at 5×10 4 The cells were transferred to a 96-well U-bottom plate (Corning) at a density of 1 cells / mL. The test antibody was diluted in a 1:2 gradient in wash buffer (1x PBS / 1% BSA) and incubated with the cells at 4°C for 1 hour. The secondary antibody goat anti-human IgG Fc FITC (3.2 moles FITC / mole IgG, Jackson Immunoresearch Lab) was added and incubated with the cells at 4°C in the dark for half an hour. The cells were then washed once, resuspended in 1x PBS / 1% BSA and analyzed by flow cytometry (BD). Based on the quantitative beads Quantum TM The fluorescence intensity was converted into the number of molecules bound per cell using the MESF kit (Bangs Laboratories, Inc.). The K of each antibody was calculated using Graphpad Prism5. D value.

[0472] The data of the binding of anti-human OX40 antibodies to CHO-K1 cells expressing human OX40 determined by flow cytometry are shown in Tables 6 and Figure 2 The data demonstrate that the illustrative antibodies 1.7.10-u1-IgG1K, 1.62.3-u1-3-IgG1K, 1.134.9-u1-IgG1L, 1.186.19-u1-IgG1K, and 1.214.23-u1-IgG1K have good binding efficiency to CHO-K1 cells expressing human OX40.

[0473] Table 6. Binding affinity of anti-OX40 antibodies to cell surface human OX40 tested by flow cytometry

[0474]

[0475]

[0476] As shown in Table 6 and Figure 2 As shown in , antibodies 1.134.9-u1-IgG1L and 1.214.23-u1-IgG1K bind to cell surface human OX40 with high affinity comparable to or even higher than that of BMK7 and BMK10.

[0477] 4.3 Binding of anti-OX40 antibodies to OX40

[0478] Cell-based FACS was used to test the binding activity of anti-OX40 antibodies to OX40. Briefly, CHO-K1 cells expressing human OX40 or activated human CD4 + T cells were expressed at 1x10 5 The density of cells / well was transferred to a 96-well U-bottom plate (Corning). The test antibody was serially diluted in a wash buffer (1x PBS / 1% BSA) and incubated with the cells at 4°C for 1 hour. After washing with 1x PBS / 1% BSA, a secondary goat anti-human IgG Fc-PE (Jackson ImmunoResearch Lab) was applied and incubated with the cells at 4°C in the dark for 1 hour. The cells were then washed and resuspended in 1xPBS / 1% BSA or fixed with 4% paraformaldehyde and then analyzed by flow cytometry (BD).

[0479] Figure 3 The results show that anti-OX40 antibodies can inhibit activated human CD4 +Data for binding of T cells. The data show that illustrative antibodies 1.7.10-u1-IgG1K, 1.62.3-u1-IgG1K, 1.134.9-u1-IgG1L, 1.186.19-u1-IgG1K and 1.214.23-u1-IgG1K bind to cell surface human OX40 in a dose-dependent manner.

[0480] 4.4 Competition for ligand binding to OX40

[0481] An ELISA-based competition assay was used to test whether anti-OX40 antibodies could competitively block the binding of OX40 to OX40 ligand (OX40L). Briefly, ELISA plates (Nunc) were coated with 1 μg / mL of human OX40 ECD overnight at 4°C. The antibodies were gradient diluted in blocking buffer and mixed with a constant concentration of OX40L. After blocking and washing, the antibody / OX40L mixture was added to the ELISA plate and then incubated for 1 hour at room temperature. The plate was then washed and subsequently incubated with an HRP-conjugated secondary antibody for 1 hour to detect the binding of OX40L to OX40 ECD. After washing, TMB substrate was added and the interaction was terminated with 2M HCl. The absorbance at 450nm and 540nm was read using a microplate reader (Molecular Device).

[0482] like Figure 4 As shown, illustrative antibodies 1.7.10-u1-IgG1K, 1.62.3-u1-IgG1K, 1.134.9-u1-IgG1L, 1.186.19-u1-IgG1K, and 1.214.23-u1-IgG1K compete with OX40L for binding to human OX40.

[0483] 4.5 Ortholog (cross-species) binding test

[0484] Cross-reactivity of anti-OX40 antibodies with macaque OX40 was measured by cell-based FACS. Briefly, 293F cells expressing macaque OX40 were cultured at 2×10 5 The density of cells / well was transferred to a 96-well U-bottom plate (Corning). The test antibody was diluted in a wash buffer (1x PBS / 1% BSA) and incubated with the cells at 4°C for 1 hour. After washing with 1x PBS / 1% BSA, the secondary antibody goat anti-human IgG Fc-PE (Jackson ImmunoResearch Lab) was applied and incubated with the cells at 4°C in the dark for 1 hour. The cells were then washed and resuspended in 1x PBS / 1% BSA or fixed with 4% paraformaldehyde and analyzed by flow cytometry (BD).

[0485] like Figure 5 As shown, illustrative antibodies 1.7.10-u1-IgG1K, 1.62.3-u1-3-IgG1K, 1.134.9-u1-IgG1L, 1.186.19-u1-IgG1K and 1.214.23-u1-IgG1K cross-reactively bind to 293F cells transfected with macaque OX40.

[0486] 4.6 Homologous (family crossover) binding

[0487] Human OX40, CD40, 4-1BB (CD137) and CD271 ECD were coated on ELISA plates (Nunc) overnight at 4°C. After blocking and washing, the test antibodies were diluted in blocking buffer and added to the ELISA plates and incubated at room temperature for 1 hour. The ELISA plates were then washed and subsequently incubated with secondary goat anti-human IgG Fc-HRP (Bethyl) for 1 hour. After washing, TMB substrate was added and the interaction was terminated with 2M HCl. The absorbance at 450nm and 540nm was read using a microplate reader (Molecular Device).

[0488] The results of family cross-binding testing of anti-OX40 antibodies to human CD40, 4-1BB (CD137), and CD271 ECD by ELISA are shown in Figure 6 The results demonstrated that OX40 antibodies 1.7.10-u1-IgG1K, 1.62.3-u1-IgG1K, 1.134.9-u1-IgG1L, 1.186.19-u1-IgG1K, and 1.214.23-u1-IgG1K specifically bound to OX40 (ie, CD134), and did not bind to human CD40, CD137, and CD271.

[0489] 4.7 Epitope Binning for BMK Antibodies

[0490] The binding epitope of the anti-OX40 antibody was competitively bound with the reference antibody by ELISA. The test antibody was coated on an ELISA plate (Nunc) overnight at 4°C. After blocking and washing, a constant concentration of human OX40 protein diluted in blocking buffer was added to the ELISA plate and incubated for 1 hour at room temperature. The biotinylated reference antibody was then diluted in gradients and added to each well and incubated for another 1 hour. The ELISA plate was then washed and then the secondary antibody HRP-conjugated streptavidin (LifeTechnology) was added and incubated for 1 hour. After washing, TMB substrate was added and the interaction was terminated with 2M HCl. The absorbance at 450nm and 540nm was read using a microplate reader (Molecular Device).

[0491] Fig. 7A , 7B and 7C show the antibodies 1.7.10-u1-IgG1K, 1.62.3-u1-IgG1K, 1.134.9-u1-IgG1L, 1.186.19-u1-IgG1K and 1.214.23-u1-IgG1K against the benchmark antibody BMK1 ( Fig. 7A )、BMK7( Figure 7B ) and BMK10( Figure 7C ) epitope binding competition. Fig. 7A As shown, the epitopes of antibodies 1.7.10-u1-IgG1K, 1.62.3-u1-IgG1K, 1.134.9-u1-IgG1L, 1.186.19-u1-IgG1K, and 1.214.23-u1-IgG1K are different from those of BMK1. Figure 7B and 7C As shown, the epitopes of antibodies 1.62.3-u1-IgG1K and 1.134.9-u1-IgG1L are different from those of BMK7 and BMK10, while the epitopes of antibodies 1.7.10-u1-IgG1K, 1.186.19-u1-IgG1K, and 1.214.23-u1-IgG1K are similar or close to those of BMK7 and BMK10.

[0492] 4.8 Bioactivity Assay Using Jurkat NFkB-Luciferase Reporter T Cells

[0493] The ability of anti-OX40 antibodies to activate the human OX40 signaling pathway was evaluated using an engineered Jurkat cell line expressing an OX40 / CD40 fusion protein and a NFkB-luciferase reporter gene. This experiment examined the biological activity of anti-OX40 antibodies cross-linked using an anti-human IgG Fc reagent or cells expressing human Fcγ receptors. Jurkat NFkB-luciferase reporter cells were cultured in complete RPMI 1640 medium containing 10% FBS and 0.5 mg / mL hygromycin B (as selection).

[0494] To determine the bioactivity of anti-OX40 antibodies under cross-linking conditions, antibody cross-linking was mediated using CHO-K1 cells expressing CD32b or F(ab')2 goat anti-human IgG (Jackson ImmunoResearch Lab), which aggregates and activates the OX40 receptor on Jurkat reporter cells. OX40 Jurkat reporter cells were collected and added to 96-well plates. OX40 antibodies diluted in gradients in complete medium were added to the cells in the presence of CHO-K1 cells expressing CD32b, parental CHO-K1 cells, or cross-linker antibodies, and the plates were incubated at 37°C, 5% CO2 for 6 hours or overnight. Reconstituted luciferase substrate (Promega) was added to each well and mixed thoroughly. Luciferase intensity was read using a microplate reader (Molecular Device). The bioactivity of anti-OX40 antibodies under free conditions was also tested.

[0495] Fig. 8A , 8B 8C show the effect of the antibody on OX40-stimulated NFkB luciferase activity in Jurkat cells using free antibody or FcγR cross-linking test antibody via CHO-K1 cells expressing CD32b or anti-human IgG Fc reagent. Fig. 8A ) free antibody or ( Figure 8B ) antibody cross-linked with F(ab')2 goat anti-human IgG or ( Figure 8C ) Reporter gene activity of antibodies cross-linked to CHO-K1 cells expressing CD32b.

[0496] like Fig. 8A , 8B As shown in Figure 8C, the cross-linking antibody can effectively activate OX40 signaling.

[0497] 4.9 Testing the in vitro functionality of anti-OX40 antibodies by cell-based assays

[0498] Human CD4 + The human CD4 T Cell Enrichment Kit (StemCell) was used to isolate the human CD4 T cells used in this example from human PBMCs. + T cells. Resuspend the cells in RPMI 1640 complete medium.

[0499] 4.9.1 Effect of anti-OX40 antibodies on interleukin-2 (IL-2) production

[0500] In this assay, flat-bottom 96-well plates (Corning) that were not tissue culture treated were pre-coated with anti-CD3 antibody overnight at 4°C. On the day of the experiment, the plates were washed with complete RPMI 1640 medium to remove unbound antibody. Freshly isolated human CD4 + T cells were expressed at 1x10 5 The density of cells / well was added to each well in a volume of 100 μL. Then a constant concentration of cross-linking antibody F(ab')2 goat anti-human IgG and gradient dilution of OX40 antibody were mixed in 100 μL and added to each well of the plate. The plate was cultured at 37°C, 5% CO2 for 3 days, and then the supernatant was harvested to measure the content of IL-2 by ELISA.

[0501] Fig. 9 Display of antibodies against CD3-induced primary human CD4 + The effects of IL-2 secretion by T cells were demonstrated to enhance the secretion of IL-2 by primary human CD4 + IL-2 secretion capacity of T cells.

[0502] 4.9.2 Effects of anti-OX40 antibodies on cytokine IFNγ secretion and CD4 + Effects on T cell proliferation

[0503] To directly evaluate the effects of anti-OX40 antibodies on enhancing IFNγ production and CD4 + We investigated the role of OX40 signaling in the proliferation of human CD4 T cells by combining it with the CD3 / T cell receptor (TCR) complex. + T cell assay. Briefly, a non-tissue culture treated flat-bottom 96-well plate (Corning) was pre-coated with 100 μL of a mixture of a constant concentration of anti-CD3 and different concentrations of anti-OX40 antibodies. The plate was incubated overnight at 4°C and then washed with RPMI 1640 complete medium to remove unbound antibodies. Freshly isolated human CD4 + T cells were expressed at 1x10 5 Cells were added to each well at a density of 10 cells / well in a volume of 200 μL. The plates were incubated at 37°C, 5% CO2 for 3 days and the supernatant was harvested for IFNγ measurement by ELISA. The cell pellet was harvested as follows 3 H-thymidine measurement of CD4 + T cell proliferation: 0.5 μCi / well 3H-thymidine (PerkinElmer) was added to the cell culture plates. The plates were incubated at 37°C in 5% CO2 for 16 to 18 hours, and then the number of proliferating cells was measured using a Topcount NXT scintillation counter (Perkin Elmer). 3 H-thymidine incorporation.

[0504] Fig.10 Antibodies against CD3 induced primary human CD4 + The effects of IFN-γ secretion on T cells were confirmed by the exemplary antibodies 1.7.10-u1-IgG1K, 1.62.3-u1-3-IgG1K, 1.134.9-u1-IgG1L, 1.186.19-u1-IgG1K and 1.214.23-u1-IgG1 to enhance the secretion of IFN-γ from primary human CD4 + IFN-γ secretion capacity of T cells.

[0505] Fig.11 Antibodies against CD3 induced primary human CD4 + The illustrative antibodies 1.7.10-u1-IgG1K, 1.62.3-u1-3-IgG1K, 1.134.9-u1-IgG1L, 1.186.19-u1-IgG1K and 1.214.23-u1-IgG1 were shown to enhance the proliferation of primary human CD4 + The proliferation capacity of T cells.

[0506] 4.9.3 Effect of human anti-OX40 antibody on the suppressive function of Tregs

[0507] Tregs are a subset of T cells that are key immune regulators and play an important role in maintaining self-tolerance. + CD25 + Regulatory T cells are associated with tumor growth, as increased numbers of Tregs are found in patients with a variety of cancers and are associated with poor prognosis. To directly evaluate the effect of anti-OX40 antibodies on the immunosuppressive response, we compared the function of Tregs in the presence and absence of anti-OX40 antibodies. CD4 + CD25 + Treg and CD4 + CD25 - Effector T (Teff) cells. Teff cells were divided into 1x10 5 cells / 50 μL / well and seeded at 1x10 5Treg / 50 μL / well were co-cultured in 96-well round-bottom plates (BD). The cells were then stimulated with human allogeneic dendritic cells (DC) induced from monocytes at 1 DC / 10 Teff cells in the presence of cross-linking antibody and anti-OX40 antibody. No antibody or isotype antibody was used as negative control. The co-cultures were incubated at 37°C, 5% CO2 for 5 days. Cell pellets were collected on day 5 to determine the 3 Teff proliferation measured by H-thymidine incorporation.

[0508] Fig.12 Shows antibody-induced activation of primary human CD4 dendritic cells in the presence of Treg cells + Antibodies 1.134.9-u1-IgG1L and 1.214.23-u1-IgG1K can restore CD4 + CD25 - T cell proliferation.

[0509] 4.10ADCC and CDC testing:

[0510] OX40 is expressed on a variety of cell types. To assess their ability to trigger Fc effector functions, we evaluated whether anti-OX40 antibodies could induce ADCC and CDC effects on cells expressing OX40.

[0511] Fig.13A Display of activated human CD4 + Expression of OX40 on T cells, Fig. 13B Shown is the expression of OX40 on Jurkat cells overexpressing OX40.

[0512] 4.10.1 ADCC test:

[0513] Jurkat cells expressing OX40 or activated human CD4 + T cells were used as targets, and various concentrations of anti-OX40 antibodies were pre-incubated for 30 minutes in 96-well round-bottom plates (BD); allogeneic PBMCs were then added as effectors at an effector / target ratio of 50:1. The plates were kept at 37°C, 5% CO2 for 4 hours. Target cell lysis was determined by an LDH-based cytotoxicity detection kit (Roche). The absorbance at 492 nm was read using a microplate reader (Molecular Device).

[0514] Fig.14A and 14B The results show that OX40 antibody can inhibit the expression of OX40 in Jurkat cells ( Fig.14A ) or activated human CD4 +T cells ( Fig. 14B ) has an ADCC effect. Fig.14A and 14B As shown, the illustrative antibodies 1.134.9-u1-IgG1L and 1.214.23-u1-IgG1K of the present invention are effective against Jurkat cells overexpressing OX40 and activated human CD4 + The ADCC effect of T cells is low.

[0515] 4.10.2CDC test:

[0516] Jurkat cells expressing OX40 or activated human CD4 + T cells were used as targets, and various concentrations of anti-OX40 antibodies were mixed in 96-well round-bottom plates (BD). Human complement was added at a final dilution of 1:50. The 96-well plates were incubated at 37°C, 5% CO2 for 2 hours. Target cell lysis was determined by CellTiter-Glo (Promega). Luminescence was read using a microplate reader (Molecular Device).

[0517] Fig.15A and 15B The results show that OX40 antibody can inhibit the expression of OX40 in Jurkat cells ( Fig.15A ) or activated human CD4 + T cells ( Fig. 15B )’s CDC effect. Fig.15A and 15B As shown, the illustrative antibodies 1.134.9-u1-IgG1L and 1.214.23-u1-IgG1K of the present invention are effective against Jurkat cells overexpressing OX40 and activated human CD4 + The CDC effect of T cells is low.

[0518] 4.11 Domain Mapping

[0519] To examine the binding domain of the OX40 antibody, a series of human / mouse OX40 chimeric variants were used. The OX40 antibody specifically binds to human OX40, while having no cross-reactivity with mouse OX40 and human CD40, despite sharing 60% and 23% identity in their amino acid (or, referred to herein as "aa") sequences, respectively. Briefly, twenty-two variants (named variants "x1", "x2" ... "x22") were constructed by replacing the following residues of the extracellular domain of human OX40 (hPro1) with the corresponding mouse OX40 (mPro1) amino acids or human CD40 amino acids (hPro40).

[0520] Variant x1: xPro1.FL-x1: CRDmox40_1 (human OX40 aa 29 to 65 replaced with mouse counterparts)

[0521] Variant x2: xPro1.FL.x2: CRDmox40_2 (human OX40 aa 66 to 107 replaced with mouse counterparts)

[0522] Variant x3: xPro1.FL-x3:CRDmox40_3 (human OX40 aa 108 to 146 replaced with mouse counterparts)

[0523] Variant x4: xPro1.FL-x4:CRDmox40_4 (human OX40 aa 147 to 214 replaced with mouse counterparts)

[0524] Variant x5: xPro1.FL-x5:CRDmox40_1-2 (human OX40 aa 29 to 107 replaced with mouse counterparts)

[0525] Variant x6: xPro1.FL-x6:CRDmox40_2-3 (human OX40 aa 66 to 146 replaced with mouse counterparts)

[0526] Variant x7: xPro1.FL-x7:CRDmox40_3-4 (human OX40 aa 108 to 214 replaced with mouse counterparts)

[0527] Variant x8: xPro1.FL-x8:CRDmox40_1-3 (human OX40 aa 29 to 146 replaced with mouse counterparts)

[0528] Variant x9: xPro1.FL-x9:CRDmox40_2-4 (human OX40 aa 66 to 214 replaced with mouse counterparts)

[0529] Variant x10: xPro1.FL-x10:CRDmox40_1,2,4 (human OX40 amino acids 29-107 and 147-214 replaced with mouse counterparts)

[0530] Variant x11: xPro1.FL-x11:CRDmox40_1,3,4 (human OX40 amino acids 29 to 65 and 108 to 214 replaced with mouse counterparts)

[0531] Variant x12: xPro1.FL-x12:CRDhcd40_1 (human OX40 aa 29 to 65 replaced with human CD40 aa counterparts)

[0532] Variant x13: xPro1.FL-x13:CRDhcd40_2 (human OX40 aa 66 to 107 replaced with human CD40 aa counterparts)

[0533] Variant x14: xPro1.FL-x14:CRDhcd40_3 (human OX40 aa 108 to 146 replaced with human CD40 aa counterparts)

[0534] Variant x15: xPro1.FL-x15: CRD hcd40_4 (human OX40 aa 147 to 214 replaced with human CD40 aa counterparts)

[0535] Variant x16: xPro1.FL-x16:CRDhcd40_1-2 (human OX40 aa 29 to 107 replaced with human CD40 aa counterparts)

[0536] Variant x17: xPro1.FL-x17:CRDhcd40_2-3 (human OX40 aa 66 to 146 replaced with human CD40 aa counterparts)

[0537] Variant x18: xPro1.FL-x18:CRDhcd40_3-4 (human OX40 aa 108 to 214 replaced with human CD40 aa counterparts)

[0538] Variant x19: xPro1.FL-x19:CRDhcd40_1-3 (human OX40 aa 29 to 146 replaced with human CD40 aa counterparts)

[0539] Variant x20: xPro1.FL-x20:CRDhcd40_2-4 (human OX40 aa 66 to 214 replaced with human CD40 aa counterparts)

[0540] Variant x21: xPro1.FL-x21:CRDhcd40_1,2,4 (human OX40 aa 29 to 107 and 147 to 214 were replaced with human CD40 aa counterparts)

[0541] Variant x22: xPro1.FL-x22:CRDhcd40_1,3,4 (human OX40 aa 29-65 and 108-214 were replaced with human CD40 aa counterparts)

[0542] Twenty-two variants from "x1" to "x22" were cloned into pcDNA3.0 vector and used for 293F cell transfection. Briefly, 293F cells were diluted to 1x10 6 The density of individual cells / mL, and the aliquot of 3mL per well is added in 24-well plates.293fectin reagent (Life Technologies) is used for transfection.For each transfection, 3 μg DNA is diluted in 150 μL Opti-MEMI reduced serum culture medium (Life Technologies), then combined with 6 μL 293fectin reagents diluted in advance in 150 μL Opti-MEMI reduced serum culture medium.DNA / Lipofectamine mixture is placed 20 minutes at 25 ℃, then added in the culture.Transfection is analyzed by flow cytometry transfected cells after 48 hours.

[0543] The binding of antibodies to chimeric OX40 variants was analyzed by flow cytometry. Briefly, 1 μg / mL antibody (but 2 μg / mL for BMK10) was incubated with transfected 293F cells expressing chimeric OX40 at 4°C for 1 hour, followed by incubation with 3 μg / mL goat anti-human IgG Fc R-PE (Jackson ImmunoResearch Lab) at 4°C for 40 minutes. Cells were analyzed using a flow cytometer.

[0544] The results are shown in Tables 7-9 below.

[0545] Table 7. Binding of variants to OX40 antibodies

[0546]

[0547] Table 8. Binding of variants to benchmark antibodies BMK1 or BMK5

[0548]

[0549]

[0550] Table 9. Binding of variants to benchmark antibodies BMK7 or BMK10

[0551]

[0552]

[0553] Table 10 shows the domains of OX40 involved in antigen binding (colored in grey).

[0554] Table 10. Domains of OX40 involved in antigen binding (colored in gray)

[0555]

[0556] Note: CRD stands for cysteine-rich domain, where "CRD1" refers to amino acids 29-65 of human OX40, "CRD2" refers to amino acids 66-107 of human OX40, "CRD3" refers to amino acids 108-146 of human OX40, and "CRD4" refers to amino acids 147-214 of human OX40.

[0557] OX40 antibody inhibits the growth of MC38 colon cancer in the human OX40 transgenic model

[0558] This study evaluated the in vivo antitumor efficacy of antibody 1.134.9-u1-IgG1L in the MC38 colon cancer model in OX40 humanized B-hTNFRSF4 mice.

[0559] OX40 humanized B-hTNFRSF4 mice were purchased from Biocytogen Co., Ltd. The mice were housed in individual ventilated cages at a constant temperature and humidity, with 5 animals per cage.

[0560] MC38 cells were cultured at 37°C, 5% CO2 in DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Cells in the exponential growth phase were harvested and counted for tumor inoculation.

[0561] Each mouse was inoculated with MC38 tumor cells (3x10 5 ) were inoculated subcutaneously to form tumors. When the average tumor volume reached 65 mm 3 At 1 , animals were randomized and treatment was started for efficacy studies. All test antibodies and control antibodies were injected intraperitoneally (IP) twice a week (BIW) for three weeks ("BIW x 3"). Detailed information is provided in Table 11.

[0562] Table 11

[0563]

[0564]

[0565] All procedures related to the handling, care and treatment of animals in the study were performed in accordance with the guidelines approved by the WuXi Apptec Institutional Animal Care Committee (IACUC) and in compliance with the International Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). During routine monitoring, animals were examined daily to assess any effects of tumor growth and treatment on normal behaviors such as activity, food and water consumption (by observation only), weight gain / loss (weight measured once a day), loss of eye / hair gloss, and any other abnormal effects, as described in the protocol. Deaths and observed clinical signs were recorded according to the number of animals within each subgroup.

[0566] Tumor size was measured three times weekly in two dimensions using calipers and expressed in mm using the following formula: 3 Measuring volume: V = 0.5a × b 2 , where a and b are the long and short diameters of the tumor, respectively. The tumor size is then used to calculate the T / C (%) value. The relative tumor proliferation rate T / C (%) is calculated using the following formula: T / C% = T RTV / C RTV x 100%(T RTV Represents the relative tumor volume of the treatment group; C RTV Relative tumor volume is calculated based on the tumor measurement value. The calculation formula is: RTV = V t / V0, V0 is the average tumor volume on the day of treatment initiation, V t is the average tumor volume measured at one time, T RTV Use with C RTV Data for the same day.

[0567] Use the formula: TGI (%) = [1-(T i -T0) / (V i -V0)]×100 to calculate the TGI of each group; T i is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on the first day of treatment, V i is with T i The mean tumor volume of the vehicle control group on the same day, V0 is the mean tumor volume of the vehicle group on the first day of treatment.

[0568] Summary statistics of tumor volume for each group at each time point are provided, including the mean and standard error of the mean (SEM). Statistical analysis of tumor volume differences between groups and drug interaction analysis were performed on the data obtained at the optimal treatment time point. One-way ANOVA was performed to compare tumor volumes between three or more groups. When a significant F statistic (ratio of treatment variance to error variance) was obtained, the Games-Howell test was used for intergroup comparisons; if not, Dunnett-t (two-sided) was used. All data were analyzed using SPSS 17.0. A p value less than 0.05 (p<0.05) was considered statistically significant.

[0569] The experimental data are shown in Tables 12 and 13 and Figures 16 and 17.

[0570] Table 12. Changes in mean tumor volume over time in mice bearing MC38 tumors after administration of 1.134.9-ul-IgG1L

[0571]

[0572] Table 13. Tumor growth inhibition rate on day 12 after administration of 1.134.9-u1-IgG1L

[0573]

[0574] It can be seen that the OX40 antibody 1.134.9-u1-IgG1L produced significant antitumor activity against B-hTNFRSF4 mice carrying MC38 colon cancer, and the tumor-bearing animals had good tolerance to the antibody.

[0575] 4.13 Epitope Mapping

[0576] In order to check the binding epitope of OX40 antibody, alanine scanning experiment was performed on human OX40, and their influence on antibody binding was evaluated. The alanine residues on human OX40 were mutated to glycine codons, and all other residues (except cysteine ​​residues, and based on OX40-OX40R complex (PDB:2HEV), solvent accessible surface area (solventaccessible surface area, SASA) <10 (OX40 amino acid) (SASA>10 is set as surface amino acid)) were mutated to alanine codons. For each residue of human OX40 extracellular domain (ECD), two continuous PCR steps were used to carry out point amino acid replacement. The pcDNA3.3-OX40-ECD.His plasmid encoding the ECD of human OX40 and the C-terminal His tag was used as a template, and a set of mutagenic primers was used for the first step PCR using QuikChange Lightning Multi-site Directed Mutagenesis Kit (Agilenttechnologies, Palo Alto, CA). After the mutant chain synthesis reaction, the parent template was digested using Dpn I endonuclease. In a second PCR step, a linear DNA expression cassette consisting of the CMV promoter, the extracellular domain (ECD) of OX40, a His-tag, and herpes simplex virus thymidine kinase (TK) polyadenylation was amplified and transiently expressed in 293F cells (Life Technologies, Gaithersburg, MD) at 37°C and quantified by His-tag quantitative ELISA.

[0577] The monoclonal antibody 1.134.9-u1-IgG1L (2 μg / mL) was coated in a plate for ELISA binding assay. After interaction with the supernatant containing quantitative OX40 mutants or human OX40-ECD.His protein, an HRP-conjugated anti-His antibody (1:5000, GenScript-A00612, CHN) was added as a detection antibody. The absorbance was standardized according to the average value of the control mutant. After setting an additional critical value for the binding fold change (<0.75), the final epitope residues were identified by considering domain mapping, epitope mapping, and crystal structures (which do not include amino acids that play a role in structural stability, such as those belonging to CRD3 and CRD4).

[0578] The normalized fold change of OX40 point mutations on antibody binding is shown in Table 14. Hotspot residues were identified by considering domain mapping, alanine scanning (cutoff: binding fold change <0.75, SASA>10) and crystal structures (PDB:2HEV) (which do not include amino acids that play a role in structural stability, such as those belonging to CRD3 and CRD4). As shown in Table 15, 1.134.9-u1-IgG1L has 8 hotspot positions.

[0579] Table 14. Normalized fold change of OX40 point mutations on antibody binding

[0580]

[0581]

[0582]

[0583]

[0584] a The fold changes in binding are relative to the binding of some silent alanine substitution.

[0585] Table 15. 8 hotspot positions of 1.134.9-u1-IgG1L

[0586]

[0587] The critical value of change fold is <0.75, SASA>10.

[0588] Those skilled in the art will further recognize that the present invention may be implemented in other specific forms without departing from its spirit or central features. Since the foregoing description of the present invention discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. On the contrary, the scope and content of the present invention should be indicated with reference to the appended claims.

[0589] In summary, the present invention provides the following embodiments:

[0590] 1. An isolated antibody or antigen-binding portion thereof, wherein the isolated antibody or antigen-binding portion thereof comprises:

[0591] A) one or more heavy chain CDRs (CDRHs) selected from at least one of the following:

[0592] (i) a CDRH1 having at least 90% sequence identity to a CDRH1 set forth in one of the sequences selected from SEQ ID NOs: 1, 7, 13, 15, 21 and 27;

[0593] (ii) a CDRH2 having at least 90% sequence identity to a CDRH2 set forth in one of the sequences selected from SEQ ID NOs: 3, 9, 17, 23 and 29; and

[0594] (iii) a CDRH3 having at least 90% sequence identity to a CDRH3 set forth in one of the sequences selected from SEQ ID NOs: 5, 11, 19, 25, and 31;

[0595] B) one or more light chain CDRs (CDRLs) selected from at least one of the following:

[0596] (i) a CDRL1 having at least 90% sequence identity to a CDRL1 as set forth in one of the sequences selected from SEQ ID NOs: 2, 8, 14, 16, 22 and 28;

[0597] (ii) a CDRL2 having at least 90% sequence identity to the CDRL2 set forth in one of the sequences selected from SEQ ID NOs: 4, 10, 18, 24 and 30; and

[0598] (iii) a CDRL3 having at least 90% sequence identity to a CDRL3 set forth in one of the sequences selected from SEQ ID NOs: 6, 12, 20, 26 and 32; or

[0599] C) one or more CDRHs of A) and one or more CDRLs of B).

[0600] 2. The isolated antibody or antigen-binding portion thereof according to embodiment 1, wherein the isolated antibody or antigen-binding portion thereof comprises:

[0601] A) one or more heavy chain CDRs (CDRHs) selected from at least one of the following:

[0602] (i) a CDRH1 selected from SEQ ID NOs: 1, 7, 13, 15, 21 and 27, or a CDRH1 that differs from said CDRH1 in amino acid sequence by an amino acid addition, deletion or substitution of not more than 2 amino acids;

[0603] (ii) a CDRH2 selected from the group consisting of SEQ ID NOs: 3, 9, 17, 23 and 29, or a CDRH2 that differs in amino acid sequence from said CDRH2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and

[0604] (iii) a CDRH3 selected from the group consisting of SEQ ID NOs: 5, 11, 19, 25 and 31, or a CDRH3 that differs in amino acid sequence from said CDRH3 by an amino acid addition, deletion or substitution of not more than 2 amino acids;

[0605] B) one or more light chain CDRs (CDRLs) selected from at least one of the following:

[0606] (i) a CDRL1 selected from SEQ ID NOs: 2, 8, 14, 16, 22 and 28, or a CDRL1 that differs from said CDRL1 in amino acid sequence by an amino acid addition, deletion or substitution of no more than 2 amino acids;

[0607] (ii) a CDRL2 selected from the group consisting of SEQ ID NOs: 4, 10, 18, 24 and 30, or a CDRL2 that differs in amino acid sequence from said CDRL2 by an amino acid addition, deletion or substitution of not more than 2 amino acids; and

[0608] (iii) a CDRL3 selected from SEQ ID NOs: 6, 12, 20, 26 and 32, or a CDRL3 that differs from said CDRL3 in amino acid sequence by an amino acid addition, deletion or substitution of not more than 2 amino acids; or

[0609] C) one or more CDRHs of A) and one or more CDRLs of B).

[0610] 3. The isolated antibody or antigen-binding portion thereof according to embodiment 1 or 2, wherein the isolated antibody or antigen-binding portion thereof comprises:

[0611] (a) CDRH1 comprising or consisting of SEQ ID NO: 1;

[0612] (b) a CDRH2 comprising or consisting of SEQ ID NO: 3;

[0613] (c) a CDRH3 comprising or consisting of SEQ ID NO: 5;

[0614] (d) CDRL1 comprising or consisting of SEQ ID NO: 2;

[0615] (e) CDRL2 comprising or consisting of SEQ ID NO: 4; and

[0616] (f) CDRL3 comprising or consisting of SEQ ID NO: 6.

[0617] 4. The isolated antibody or antigen-binding portion thereof according to embodiment 1 or 2, wherein the isolated antibody or antigen-binding portion thereof comprises:

[0618] (a) CDRH1 comprising or consisting of SEQ ID NO: 7;

[0619] (b) a CDRH2 comprising or consisting of SEQ ID NO: 9;

[0620] (c) a CDRH3 comprising or consisting of SEQ ID NO: 11;

[0621] (d) CDRL1 comprising or consisting of SEQ ID NO: 8;

[0622] (e) CDRL2 comprising or consisting of SEQ ID NO: 10; and

[0623] (f) CDRL3 comprising or consisting of SEQ ID NO: 12.

[0624] 5. The isolated antibody or antigen-binding portion thereof according to embodiment 1 or 2, wherein the isolated antibody or antigen-binding portion thereof comprises:

[0625] (a) CDRH1 comprising or consisting of SEQ ID NO: 13;

[0626] (b) a CDRH2 comprising or consisting of SEQ ID NO: 9;

[0627] (c) a CDRH3 comprising or consisting of SEQ ID NO: 11;

[0628] (d) CDRL1 comprising or consisting of SEQ ID NO: 14;

[0629] (e) CDRL2 comprising or consisting of SEQ ID NO: 10; and

[0630] (f) CDRL3 comprising or consisting of SEQ ID NO: 12.

[0631] 6. The isolated antibody or antigen-binding portion thereof according to embodiment 1 or 2, wherein the isolated antibody or antigen-binding portion thereof comprises:

[0632] (a) CDRH1 comprising or consisting of SEQ ID NO: 15;

[0633] (b) a CDRH2 comprising or consisting of SEQ ID NO: 17;

[0634] (c) a CDRH3 comprising or consisting of SEQ ID NO: 19;

[0635] (d) CDRL1 comprising or consisting of SEQ ID NO: 16;

[0636] (e) CDRL2 comprising or consisting of SEQ ID NO: 18; and

[0637] (f) CDRL3 comprising or consisting of SEQ ID NO: 20.

[0638] 7. The isolated antibody or antigen-binding portion thereof according to embodiment 1 or 2, wherein the isolated antibody or antigen-binding portion thereof comprises:

[0639] (a) CDRH1 comprising or consisting of SEQ ID NO: 21;

[0640] (b) a CDRH2 comprising or consisting of SEQ ID NO: 23;

[0641] (c) a CDRH3 comprising or consisting of SEQ ID NO: 25;

[0642] (d) CDRL1 comprising or consisting of SEQ ID NO: 22;

[0643] (e) CDRL2 comprising or consisting of SEQ ID NO: 24; and

[0644] (f) CDRL3 comprising or consisting of SEQ ID NO: 26.

[0645] 8. An isolated antibody or antigen binding portion thereof according to embodiment 1 or 2, wherein the isolated antibody or antigen binding portion thereof comprises:

[0646] (a) CDRH1 comprising or consisting of SEQ ID NO: 27;

[0647] (b) a CDRH2 comprising or consisting of SEQ ID NO: 29;

[0648] (c) a CDRH3 comprising or consisting of SEQ ID NO: 31;

[0649] (d) CDRL1 comprising or consisting of SEQ ID NO: 28;

[0650] (e) CDRL2 comprising or consisting of SEQ ID NO: 30; and

[0651] (f) CDRL3 comprising or consisting of SEQ ID NO: 32.

[0652] 9. The isolated antibody or antigen-binding portion thereof according to embodiment 1 or 2, wherein the isolated antibody or antigen-binding portion thereof comprises:

[0653] (A) a heavy chain variable region, which:

[0654] (i) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, 41 and 43;

[0655] (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, 41, and 43; or

[0656] (iii) an amino acid sequence comprising one or more amino acid additions, deletions and / or substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 35, 37, 39, 41 and 43; and / or

[0657] (B) a light chain variable region, which:

[0658] (i) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, 42 and 44;

[0659] (ii) comprises an amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, 42, and 44; or

[0660] (iii) an amino acid sequence comprising an addition, deletion and / or substitution of one or more amino acids compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, 38, 40, 42 and 44.

[0661] 10. The isolated antibody or antigen binding portion thereof of embodiment 9, wherein the isolated antibody or antigen binding portion thereof comprises:

[0662] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34; or

[0663] (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 36; or

[0664] (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 38; or

[0665] (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40; or

[0666] (e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42; or

[0667] (f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 43 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 44.

[0668] 11. An isolated antibody or antigen binding portion thereof according to any one of the preceding embodiments, which has one or more of the following properties:

[0669] (a) 1×10 -8 M or lower K D Binds to human OX40;

[0670] (b) In CD4 + Inducing cytokine (e.g., IL-2 or IFN-γ) production in T cells;

[0671] (c) Enhancement of primary human CD4 + Proliferation of T cells;

[0672] (d) Enhancement of primary human CD4 + Proliferation of T effector cells;

[0673] (e) binds to human or cynomolgus OX40, respectively; or

[0674] (f) No cross-reactivity to human CD40, CD137 and CD271.

[0675] 12. The isolated antibody or antigen binding portion thereof according to any one of the preceding embodiments, wherein the antibody is a monoclonal antibody, a chimeric antibody or a humanized antibody.

[0676] 13. The isolated antibody, or antigen binding portion thereof, according to any one of the preceding embodiments, wherein the antibody is a fully human monoclonal antibody.

[0677] 14. The isolated antibody, or antigen binding portion thereof, according to any one of the preceding embodiments, wherein the antibody is a fully human monoclonal antibody produced by a transgenic mammal.

[0678] 15. The isolated antibody or antigen binding portion thereof according to any one of the preceding embodiments, wherein the antibody is a fully human monoclonal antibody produced by a transgenic rat.

[0679] 16. The isolated antibody or antigen binding portion thereof according to any one of the preceding embodiments, wherein the antibody is a fully human monoclonal antibody produced by transgenic rats with recombinant immunoglobulin loci.

[0680] 17. The isolated antibody, or antigen binding portion thereof, according to any one of the preceding embodiments, wherein the antibody is fused to the constant region of IgG.

[0681] 18. An isolated antibody, or antigen binding portion thereof, according to any one of the preceding embodiments, wherein the antibody is fused to the constant region of human IgG.

[0682] 19. The isolated antibody, or antigen binding portion thereof, according to any one of the preceding embodiments, wherein the antibody is fused to the constant region of human IgG1 or human IgG4.

[0683] 20. The isolated antibody, or antigen binding portion thereof, according to any one of the preceding embodiments, wherein the antibody binds to the CRD2 and / or CRD3 domains of OX40.

[0684] 21. An isolated antibody, or antigen binding portion thereof, that competes for binding to the same epitope as the isolated antibody, or antigen binding portion thereof, of any preceding embodiment.

[0685] 22. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region and / or the light chain variable region of the isolated antibody as defined in any one of embodiments 1-21.

[0686] 23. An isolated nucleic acid molecule according to embodiment 22, encoding the heavy chain variable region of the isolated antibody as defined in any one of embodiments 1-21, and comprising a nucleic acid sequence selected from the group consisting of:

[0687] (A) a nucleic acid sequence encoding a heavy chain variable region as shown in SEQ ID NO: 33, 35, 37, 39, 41 or 43;

[0688] (B) a nucleic acid sequence as shown in SEQ ID NO: 45, 47, 49, 51, 53 or 55; or

[0689] (C) A nucleic acid sequence that hybridizes to the complementary strand of the nucleic acid sequence of (A) or (B) under highly stringent conditions.

[0690] 24. An isolated nucleic acid molecule according to embodiment 22, encoding the light chain variable region of the isolated antibody as defined in any one of embodiments 1-21, and comprising a nucleic acid sequence selected from the group consisting of:

[0691] (A) a nucleic acid sequence encoding a light chain variable region as shown in SEQ ID NO: 34, 36, 38, 40, 42 or 44;

[0692] (B) a nucleic acid sequence as shown in SEQ ID NO: 46, 48, 50, 52, 54 or 56; or

[0693] (C) A nucleic acid sequence that hybridizes to the complementary strand of the nucleic acid sequence of (A) or (B) under highly stringent conditions.

[0694] 25. A vector comprising the nucleic acid molecule of any one of embodiments 22-24.

[0695] 26. A host cell comprising the vector of embodiment 25.

[0696] 27. A pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as defined in any one of embodiments 1-21 and a pharmaceutically acceptable carrier.

[0697] 28. A method for preparing an antibody or antigen binding portion thereof as defined in any one of embodiments 1-21, comprising the following steps:

[0698] - expressing an antibody or antigen binding portion thereof as defined in any one of embodiments 1 to 21 in a host cell of embodiment 26; and

[0699] - isolating the antibody or antigen-binding portion thereof from said host cell.

[0700] 29. Use of an antibody, or antigen binding portion thereof, as defined in any one of embodiments 1 to 21 for the preparation of a medicament for modulating an immune response in a subject.

[0701] 30. The use according to embodiment 29, wherein T cell proliferation is enhanced in a subject administered with the medicament.

[0702] 31. The use according to embodiment 29, wherein the anti-CD3 induced primary human CD4 + T cell proliferation is enhanced in a subject administered with the medicament.

[0703] 32. The use according to embodiment 29, wherein primary human CD4 + The proliferation of T effector cells is enhanced in a subject administered with the medicament.

[0704] 33. The use according to embodiment 29, wherein the production of the cytokine IFN-γ is increased in a subject administered with the medicament.

[0705] 34. The use according to embodiment 29, wherein the production of cytokine IL-2 is increased in a subject administered with the drug.

[0706] 35. Use of an antibody, or antigen binding portion thereof, as defined in any one of embodiments 1 to 21 for the preparation of a medicament for treating abnormal cell growth in a subject.

[0707] 36. Use of an antibody or antigen binding portion thereof as defined in any one of embodiments 1-21 for the preparation of a medicament for attenuating the suppressive function of Treg cells in a subject.

[0708] 37. Use of an antibody or antigen binding portion thereof as defined in any one of embodiments 1 to 21 for the preparation of a medicament for inhibiting the growth of tumor cells in a subject.

[0709] 38. Use of an antibody or antigen binding portion thereof as defined in any one of embodiments 1-21 for the preparation of a medicament for reducing tumor cell metastasis in a subject.

[0710] 39. Use of an antibody or antigen binding portion thereof as defined in any one of embodiments 1-21 for the preparation of a medicament for treating or preventing a proliferative disorder (eg cancer), an autoimmune disease, an inflammatory disease or an infectious disease.

[0711] 40. The use according to embodiment 39, wherein the cancer is a solid cancer.

[0712] 41. The use according to embodiment 39 or 40, wherein the cancer is colon cancer.

[0713] 42. Use of an antibody, or an antigen binding portion thereof, as defined in any one of embodiments 1 to 21 for the preparation of a diagnostic agent for diagnosing a proliferative disorder (eg cancer), an autoimmune disease, an inflammatory disease or an infectious disease.

[0714] 43. An antibody, or antigen binding portion thereof, as defined in any one of embodiments 1 to 21 for use in treating or preventing a proliferative disorder (eg cancer), an autoimmune disease, an inflammatory disease or an infectious disease.

[0715] 44. An antibody, or antigen binding portion thereof, as defined in any one of embodiments 1 to 21 for use in diagnosing a proliferative disorder (eg cancer), an autoimmune disease, an inflammatory disease or an infectious disease.

[0716] 45. A kit for treating or diagnosing a proliferative disorder (eg, cancer), an autoimmune disease, an inflammatory disease, or an infectious disease, comprising a container comprising at least one antibody, or antigen-binding portion thereof, as defined in any one of embodiments 1-21. Sequence Listing <110> Tongrun Macau One Person Limited Company <120> Fully human antibody against human OX40 and preparation method and use thereof <130> IDC226023 <150> PCT / CN2018 / 086574 <151> 2018-05-11 <150> CN201810529840.5 <151> 2018-05-29 <160> 56 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 1.7.10-u1-HCDR1 of IgG1K <400> 1 Gly Phe Thr Phe Ser Asp Tyr Tyr Met Ser 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 1.7.10-u1-LCDR1 of IgG1K <400> 2 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 3 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 1.7.10-u1-HCDR2 of IgG1K <400> 3 Tyr Ile Ser Gly Ser Gly Asn Thr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 4 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 1.7.10-u1-LCDR2 of IgG1K <400> 4 Ala Ala Ser Ser Leu Gln Gly 1 5 <210> 5 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 1.7.10-u1-HCDR3 of IgG1K <400> 5 Glu Arg Gly Ala Ala Gly Thr Gly Trp Phe Asp Pro 1 5 10 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 1.7.10-u1-LCDR3 of IgG1K <400> 6 Gln Gln Val Asn Ser Phe Pro Trp Thr 1 5 <210> 7 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 1.62.3-u1-HCDR1 of IgG1K <400> 7 Gly Gly Ser Ile Ser Asn Gly Gly Tyr Tyr Trp Ser 1 5 10 <210> 8 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 1.62.3-u1-LCDR1 of IgG1K <400> 8 Lys Ser Ser Gln Ser Val Val Phe Ser Ser Asn Asn Lys Ile Cys Leu 1 5 10 15 Ala <210> 9 <211> 16 <212> PRT <213> Artificial sequence <220> <223> HCDR2 of 1.62.3-u1-IgG1K or 1.62.3-u1-3-IgG1K <400> 9 Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 10 <211> 7 <212> PRT <213> Artificial sequence <220> <223> LCDR2 of 1.62.3-u1-IgG1K or 1.62.3-u1-3-IgG1K <400> 10 Trp Ser Ser Thr Arg Glu Ser 1 5 <210> 11 <211> 10 <212> PRT <213> Artificial sequence <220> <223> HCDR3 of 1.62.3-u1-IgG1K or 1.62.3-u1-3-IgG1K <400> 11 Asp Glu Trp Glu Leu Arg Gly Phe Asp Tyr 1 5 10 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LCDR3 of 1.62.3-u1-IgG1K or 1.62.3-u1-3-IgG1K <400> 12 Gln Gln Tyr Tyr Ser Ser Pro Trp Thr 1 5 <210> 13 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 1.62.3-u1-3-HCDR1 of IgG1K <400> 13 Gly Gly Ser Ile Ser Asn Ala Gly Tyr Tyr Trp Ser 1 5 10 <210> 14 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 1.62.3-u1-3-LCDR1 of IgG1K <400> 14 Lys Ser Ser Gln Ser Val Val Phe Ser Ser Asn Asn Lys Ile Ser Leu 1 5 10 15 Ala <210> 15 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 1.134.9-u1-HCDR1 of IgG1L <400> 15 Gly Gly Ser Ile Ser Ser Tyr Asn Trp Trp Ser 1 5 10 <210> 16 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 1.134.9-u1-LCDR1 of IgG1L <400> 16 Gln Gly Asp Asn Leu Arg Thr Tyr Tyr Ala Ser 1 5 10 <210> 17 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 1.134.9-u1-HCDR2 of IgG1L <400> 17 Glu Ile Tyr His Gly Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 18 <211> 7 <212> PRT <213> Artificial sequence <220> <223> LCDR2 of 1.134.9-u1-IgG1L <400> 18 Gly Arg Asn Lys Arg Pro Ser 1 5 <210> 19 <211> 13 <212> PRT <213> Artificial sequence <220> <223> 1.134.9-u1-HCDR3 of IgG1L <400> 19 Ala Pro Gly Asp Trp Gly Gly Ser Pro Tyr Phe Asp Phe 1 5 10 <210> 20 <211> 11 <212> PRT <213> Artificial sequence <220> <223> LCDR3 1.134.9-u1-IgG1L <400> 20 Asn Ser Arg Asp Ser Ser Gly Asn Pro Val Val 1 5 10 <210> twenty one <211> 10 <212> PRT <213> Artificial sequence <220> <223> 1.186.19-u1-HCDR1 of IgG1K <400> twenty one Gly Phe Thr Phe Ser Asp Tyr Tyr Met Gly 1 5 10 <210> twenty two <211> 11 <212> PRT <213> Artificial sequence <220> <223> 1.186.19-u1-LCDR1 of IgG1K <400> twenty two Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> twenty three <211> 17 <212> PRT <213> Artificial sequence <220> <223> 1.186.19-u1-HCDR2 of IgG1K <400> twenty three Tyr Ile Ser Gly Ser Gly Asn Thr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> twenty four <211> 7 <212> PRT <213> Artificial sequence <220> <223> 1.186.19-u1-LCDR2 of IgG1K <400> twenty four Ala Ala Ser Ser Leu Gln Gly 1 5 <210> 25 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 1.186.19-u1-HCDR3 of IgG1K <400> 25 Glu Arg Gly Ala Ala Gly Ala Gly Trp Phe Asp Pro 1 5 10 <210> 26 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LCDR3 of 1.186.19-u1-IgG1K <400> 26 Gln Gln Val Asn Ser Phe Pro Trp Thr 1 5 <210> 27 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 1.214.23-u1-HCDR1 of IgG1K <400> 27 Gly Gly Ser Ile Ser Asn Arg Asn Trp Trp Ser 1 5 10 <210> 28 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 1.214.23-u1-LCDR1 of IgG1K <400> 28 Arg Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ala 1 5 10 <210> 29 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 1.214.23-u1-HCDR2 of IgG1K <400> 29 Glu Ile Phe His Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 30 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 1.214.23-u1-LCDR2 of IgG1K <400> 30 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 31 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR3 of 1.214.23-u1-IgG1K <400> 31 Ser Phe Ala Val Ala Leu Asp Ser 1 5 <210> 32 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LCDR3 of 1.214.23-u1-IgG1K <400> 32 Gln Gln Leu Phe Ser Tyr Pro Ile Thr 1 5 <210> 33 <211> 121 <212> PRT <213> Artificial sequence <220> <223> VH of 1.7.10-u1-IgG1K <400> 33 Gln Val His Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Gly Ser Gly Asn Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Arg Gly Ala Ala Gly Thr Gly Trp Phe Asp Pro Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 34 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL of 1.7.10 - u1 - IgG1K <400> 34 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Gly Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Asn Ser Phe Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 35 <211> 120 <212> PRT <213> Artificial sequence <220> <223> VH of 1.62.3-u1-IgG1K <400> 35 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Gly 20 25 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Glu Trp Glu Leu Arg Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 36 <211> 113 <212> PRT <213> Artificial sequence <220> <223> VL of 1.62.3-u1-IgG1K <400> 36 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Val Phe Ser 20 25 30 Ser Asn Asn Lys Ile Cys Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ser Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 37 <211> 120 <212> PRT <213> Artificial sequence <220> <223> VH of 1.62.3-u1-3-IgG1K <400> 37 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Ala 20 25 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Asp Glu Trp Glu Leu Arg Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> VL of 1.62.3-u1-3-IgG1K <400> 38 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Val Phe Ser 20 25 30 Ser Asn Asn Lys Ile Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ser Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 39 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> VH of 1.134.9-u1-IgG1L <400> 39 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Glu Ile Tyr His Gly Gly Asn Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Met Ser Val Asp Asn Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Gly Asp Trp Gly Gly Ser Pro Tyr Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 40 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL of 1.134.9-u1-IgG1L <400> 40 Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Asn Leu Arg Thr Tyr Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Ile Leu Leu Ile Tyr 35 40 45 Gly Arg Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Ala Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn Pro 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 41 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> VH of 1.186.19-u1-IgG1K <400> 41 Gln Val His Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Gly Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Gly Ser Gly Asn Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Glu Arg Gly Ala Ala Gly Ala Gly Trp Phe Asp Pro Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 42 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL of 1.186.19-u1-IgG1K <400> 42 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Gly Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr His Cys Gln Gln Val Asn Ser Phe Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 43 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> VH of 1.214.23-u1-IgG1K <400> 43 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly 1 5 10 15 Thr Leu Ser Leu Thr Cys Val Val Ser Gly Gly Ser Ile Ser Asn Arg 20 25 30 Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Glu Ile Phe His Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Val Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Phe Ala Val Ala Leu Asp Ser Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 44 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL of 1.214.23-u1-IgG1K <400> 44 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ala Trp Cys Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Thr Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Phe Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 45 <211> 363 <212> DNA <213> Artificial sequence <220> <223> VH of 1.7.10 - u1 - IgG1K <400> 45 caggtgcacc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtggta gtggtaacac catttactac 180 gcagactctg tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgacgac acggccgtat atttctgtgc gagagagaga 300 ggagcagctg gtacagggtg gttcgacccc tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 46 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL of 1.7.10 - u1 - IgG1K <400> 46 gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaggtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttacta ttgtcaacag gttaacagtt tcccgtggac gttcggccaa 300 gggaccaagg tggaaatcaa a 321 <210> 47 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> VH of 1.62.3-u1-IgG1K <400> 47 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt aatggtggtt actactggag ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaaac tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagat 300 gagtgggagc tacgggggtt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360 <210> 48 <211> 339 <212> DNA <213> Artificial sequence <220> <223> VL of 1.62.3 - u1 - IgG1K <400> 48 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgttgta ttcagctcca acaataagat ctgcttagct 120 tggtaccagc agaaaccagg acagcctcct aagctgctca tttactggtc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatagttct 300 ccgtggacgt tcggccaagg gaccaaggtg gaaatcaaa 339 <210> 49 <211> 360 <212> DNA <213> Artificial sequence <220> <223> VH of 1.62.3 - u1 - 3 - IgG1K <400> 49 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt aatgccggtt actactggag ctggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa gaaccagttc 240 tccctgaaac tgagctctgt gactgccgcg gacacggccg tgtattactg tgcgagagat 300 gagtgggagc tacgggggtt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360 <210> 50 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> VL of 1.62.3-u1-3-IgG1K <400> 50 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgttgta ttcagctcca acaataagat cagcttagct 120 tggtaccagc agaaaccagg acagcctcct aagctgctca tttactggtc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatagttct 300 ccgtggacgt tcggccaagg gaccaaggtg gaaatcaaa 339 <210> 51 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> VH of 1.134.9-u1-IgG1L <400> 51 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggggac cctgtccctc 60 acctgcgctg tctctggtgg ctccatcagt agttataact ggtggagttg ggtccgccag 120 cccccaggga agggactgga gtggattggg gaaatctatc atggtgggaa caccaactac 180 aacccgtccc tcaagagtcg agtcaccatg tcagtagaca actccaagaa ccagttctcc 240 ctgaagctga gctctgtgac cgccgcggac acggccgtat attactgtgc gagagccccc 300 ggggactggg gaggttcccc ctattttgac ttctggggcc agggaaccct ggtcaccgtc 360 tcctca 366 <210> 52 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> VL of 1.134.9-u1-IgG1L <400> 52 ggttctgtgg tttcttctga actgactcag gaccctgctg tgtctgtggc cttgggacag 60 acagtcagga tcacatgcca gggagacaac ctcagaacct attatgcaag ctggtaccag 120 cagaagccag gacaggcccc tatacttctc atctatggta gaaacaagcg gccctcaggg 180 atcccagacc gattctctgg ctccagctcg ggaaacacag cttccttgac catcactggg 240 gctcaggcgg aagatgaggc tgcgtactac tgtaactccc gggacagcag tggtaatcct 300 gtggtattcg gcggagggac caagctgacc gtccta 336 <210> 53 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> VH of 1.186.19-u1-IgG1K <400> 53 caggtgcacc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gactactaca tgggctggat ccgccaggct 120 ccagggcagg ggctggagtg ggtttcatac attagtggta gtggtaacac catttactac 180 gcagactctg tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgacgac acggccgttt atttctgtgc gaaagagaga 300 ggagcagctg gtgcagggtg gttcgacccc tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 54 <211> 321 <212> DNA <213> Artificial sequence <220> <223> VL of 1.186.19-u1-IgG1K <400> 54 gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtca gggtattagc agctggttag cctggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaggtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttacca ttgtcaacag gttaacagtt tcccgtggac gttcggccaa 300 gggaccaagg tggaaatcaa a 321 <210> 55 <211> 351 <212> DNA <213> Artificial sequence <220> <223> VH of 1.214.23-u1-IgG1K <400> 55 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggggac cctgtccctc 60 acctgtgttg tctccggtgg ctccatcagc aatagaaact ggtggagttg ggtccgccag 120 cccccaggga aggggctgga gtggattggg gaaatctttc atagtgggaa caccaactac 180 aacccgtccc tcaagagtcg cgtcaccata tcagtagaca agtccaagaa ccagttctcc 240 ctgaaggtga actctgtgac cgccgcggac acggccgtgt attactgtgc gaaatccttt 300 gcagtggccc ttgactcctg gggccaggga accctggtca ccgtctcctc a 351 <210> 56 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> VL of 1.214.23-u1-IgG1K <400> 56 gacatccagt tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca ggacattaac agttatttag cctggtgtca gcaaaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatcctctt tgcaaagtgg ggtcccatca 180 aggttcagcg gcactggatc tgggacagag ttcactctca caatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcaacag ctttttagtt acccgatcac cttcggccaa 300 gggacacgac tggagattaa a 321

Claims

1. An isolated anti-OX40 antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof comprises: (a) CDRH1 shown in SEQ ID NO: 1; (b) CDRH2 shown in SEQ ID NO: 3; (c) CDRH3 shown in SEQ ID NO: 5; (d) CDRL1 shown in SEQ ID NO: 2; (e) CDRL2 shown in SEQ ID NO: 4; and (f) CDRL3 shown in SEQ ID NO:

6.

2. The anti-OX40 antibody or antigen-binding portion thereof of claim 1, wherein the antibody or antigen-binding portion thereof comprises a heavy chain variable region as set forth in SEQ ID NO: 33 and a light chain variable region as set forth in SEQ ID NO:

34.

3. The anti-OX40 antibody or antigen binding portion thereof of claim 1, wherein the antibody is a monoclonal antibody, a chimeric antibody or a humanized antibody.

4. The anti-OX40 antibody, or antigen binding portion thereof, of claim 3, wherein the antibody is a fully human monoclonal antibody.

5. The anti-OX40 antibody or antigen binding portion thereof of claim 3, wherein the antibody is a fully human monoclonal antibody produced by a transgenic mammal.

6. The anti-OX40 antibody or antigen binding portion thereof of claim 5, wherein the antibody is a fully human monoclonal antibody produced by transgenic rats.

7. The anti-OX40 antibody or antigen binding portion thereof of claim 6, wherein the antibody is a fully human monoclonal antibody produced by transgenic rats with recombinant immunoglobulin loci.

8. The anti-OX40 antibody, or antigen binding portion thereof, of claim 1, wherein the antibody, or antigen binding portion thereof, is fused to the constant region of IgG.

9. The anti-OX40 antibody, or antigen binding portion thereof, of claim 8, wherein the antibody, or antigen binding portion thereof, is fused to the constant region of human IgG.

10. The anti-OX40 antibody, or antigen binding portion thereof, of claim 9, wherein the antibody, or antigen binding portion thereof, is fused to the constant region of human IgG1 or human IgG4.

11. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region and the light chain variable region of the anti-OX40 antibody or antigen binding portion thereof of any one of claims 1-10.

12. The nucleic acid molecule according to claim 11, comprising a nucleic acid sequence selected from the group consisting of: (A) a nucleic acid sequence encoding the heavy chain variable region as shown in SEQ ID NO: 33; or (B) The nucleic acid sequence shown in SEQ ID NO:

45.

13. The nucleic acid molecule according to claim 11, comprising a nucleic acid sequence selected from the group consisting of: (A) a nucleic acid sequence encoding the light chain variable region as shown in SEQ ID NO: 34; or (B) The nucleic acid sequence shown in SEQ ID NO:

46.

14. A vector comprising the nucleic acid molecule according to any one of claims 11 to 13. A host cell comprising the vector according to claim 14 .

16. A pharmaceutical composition comprising at least one anti-OX40 antibody or antigen-binding portion thereof according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

17. A method for preparing the anti-OX40 antibody or antigen binding portion thereof according to any one of claims 1 to 10, comprising the following steps: - expressing the anti-OX40 antibody or antigen binding portion thereof according to any one of claims 1 to 10 in the host cell according to claim 15; and - isolating said antibody or antigen-binding portion thereof from said host cell.

18. Use of the anti-OX40 antibody or antigen binding portion thereof of any one of claims 1-10 in the preparation of a medicament for treating or preventing an OX40-related disease or disorder, wherein the OX40-related disease or disorder is cancer.

19. The use according to claim 18, wherein the cancer is a solid cancer.

20. The use according to claim 19, wherein the cancer is colon cancer.

21. Use of the anti-OX40 antibody, or antigen binding portion thereof, of any one of claims 1-10 in the preparation of a diagnostic agent for diagnosing an OX40-related disease or disorder, wherein the OX40-related disease or disorder is cancer.

22. The use according to claim 21, wherein the cancer is a solid cancer.

23. The use according to claim 22, wherein the cancer is colon cancer.

24. A kit for diagnosing an OX40-related disease or disorder, comprising a container comprising at least one anti-OX40 antibody, or antigen-binding portion thereof, according to any one of claims 1-10, wherein the OX40-related disease or disorder is cancer.

25. The kit of claim 24, wherein the cancer is a solid cancer.

26. The kit of claim 25, wherein the cancer is colon cancer.

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