Anti-OX40 antibodies and methods of use

Abstract

The present disclosure provides antibodies and antigen-binding fragments thereof that bind to human OX40 (ACT35, CD134 or TNFRSF4), pharmaceutical compositions comprising the antibodies, and uses of the antibodies or the compositions for treating diseases such as cancer. In particular, the anti-OX40 antibodies of the present invention do not interfere with the binding of OX40 ligands to their receptors.

Description

Technical Field

[0001] Disclosed herein are antibodies or antigen-binding fragments thereof that bind to human OX40, compositions comprising the antibodies, and methods of use for treating cancer. Background Art

[0002] OX40 (also known as ACT35, CD134 or TNFRSF4) is a type I transmembrane glycoprotein of approximately 50 KD and a member of the tumor necrosis factor receptor superfamily (TNFRSF) (Croft, 2010; Gough and Weinberg, 2009). Mature human OX40 consists of 249 amino acid (AA) residues, with a cytoplasmic tail of 37 AA and an extracellular region of 185 AA. The extracellular domain of OX40 contains three complete and one incomplete cysteine-rich domain (CRD). The intracellular domain of OX40 contains a conserved signaling-related QEE motif that mediates binding to several TNFR-associated factors (TRAFs), including TRAF2, TRAF3 and TRAF5, thereby connecting OX40 to intracellular kinases (Arch and Thompson, 1998; Willoughby et al., 2017).

[0003] Initially in activated rat CD4 + OX40 was discovered on T cells, and mouse and human homologues were subsequently cloned from T cells (al-Shamkhani et al., 1996; Calderhead et al., 1993). + In addition to being expressed on T cells (including T helper (Th) 1 cells, Th2 cells, Th17 cells, and regulatory T (Treg) cells), it is also expressed on activated CD8 + OX40 expression is found on the surface of T cells, natural killer (NK) T cells, neutrophils, and NK cells (Croft, 2010). + and CD8 + Low OX40 expression is found on T cells and on most resting memory T cells (Croft, 2010; Soroosh et al., 2007). Surface expression of OX40 on naive T cells is transient. After TCR activation, OX40 expression on T cells increases greatly within 24 hours, and peaks within 2-3 days and lasts for 5-6 days (Gramaglia et al., 1998).

[0004] The ligand of OX40 (OX40L, also known as gp34, CD252 or TNFSF4) is the only ligand of OX40. Similar to other TNFSF (tumor necrosis factor superfamily) members, OX40L is a type II glycoprotein containing 183 AAs, with an intracellular domain of 23 AAs and an extracellular domain of 133 AAs (Croft, 2010; Gough and Weinberg, 2009). OX40L naturally forms a homopolymer trimer complex on the cell surface. The ligand trimer interacts with the three copies of OX40 mainly through the CRD1, CRD2 and part of the CRD3 region of the receptor without involving CRD4 at the ligand monomer-monomer interface (Compaan and Hymowitz, 2006). OX40L is mainly expressed on activated antigen presenting cells (APCs), including activated B cells (Stuber et al., 1995), mature conventional dendritic cells (DCs) (Ohshima et al., 1997), plasmacytoid DCs (pDCs) (Ito et al., 2004), macrophages (Weinberg et al., 1999) and Langerhans cells (Sato et al., 2002). In addition, OX40L has been found to be expressed on other cell types, such as NK cells, mast cells, activated T cell subsets, and vascular endothelial cells and smooth muscle cells (Croft, 2010; Croft et al., 2009).

[0005] OX40 trimerization via the OX40L trimerization or dimerization via agonistic antibodies facilitates recruitment and docking of aptamer molecules TRAF2, TRAF3 and / or TRAF5 to their intracellular QEE motifs (Arch and Thompson, 1998; Willoughby et al., 2017). The recruitment and docking of TRAF2 and TRAF3 can further lead to the activation of both the classical NF-κB1 and non-classical NF-κB2 pathways, which play a key role in regulating the survival, differentiation, expansion, cytokine production and effector functions of T cells (Croft, 2010; Gramaglia et al., 1998; Huddleston et al., 2006; Rogers et al., 2001; Ruby and Weinberg, 2009; Song et al., 2005a; Song et al., 2005b; Song et al., 2008).

[0006] In normal tissues, OX40 expression is low and mainly on lymphocytes in lymphoid organs (Durkop et al., 1995). However, upregulation of OX40 expression on immune cells is frequently observed in animal models and human patients with pathological conditions (Redmond and Weinberg, 2007), such as autoimmune diseases (Carboni et al., 2003; Jacquemin et al., 2015; Szypowska et al., 2014) and cancer (Kjaergaard et al., 2000; Vetto et al., 1997; Weinberg et al., 2000). Notably, increased expression of OX40 is associated with prolonged survival in patients with colorectal cancer and cutaneous melanoma, and is negatively correlated with the occurrence of distant metastases and more advanced tumor characteristics (Ladanyi et al., 2004; Petty et al., 2002; Sarff et al., 2008). It has also been shown that anti-OX40 antibody treatment can elicit anti-tumor efficacy in multiple mouse models (Aspeslagh et al., 2016), indicating the potential of OX40 as an immunotherapy target. In the first clinical trial in cancer patients conducted by Curti et al., evidence of anti-tumor efficacy and activation of tumor-specific T cells was observed with an agonistic anti-OX40 monoclonal antibody, indicating that OX40 antibodies can be used to promote anti-tumor T cell responses (Curti et al., 2013).

[0007] The mechanism of action of agonistic anti-OX40 antibodies in mediating anti-tumor efficacy has been initially studied in mouse tumor models (Weinberg et al., 2000). Until recently, the mechanism of action of agonistic anti-OX40 antibodies in tumors was attributed to their ability to trigger co-stimulatory signaling pathways in effector T cells and to their inhibitory effects on the differentiation and function of Treg cells (Aspeslagh et al., 2016; Ito et al., 2006; St Rose et al., 2013; Voo et al., 2013). Recent studies have shown that tumor-infiltrating Tregs express OX40 at higher levels than effector T cells (CD4 + and CD8 + Both) and peripheral Tregs (Lai et al., 2016; Marabelle et al., 2013b; Montler et al., 2016; Soroosh et al., 2007; Timperi et al., 2016). Therefore, the secondary effects by which anti-OX40 antibodies trigger anti-tumor responses rely on their ability to deplete intratumoral OX40 via antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). +Fc-mediated effector functions in Treg cells (Aspeslagh et al., 2016; Bulliard et al., 2014; Marabelle et al., 2013a; Marabelle et al., 2013b; Smyth et al., 2014). This work demonstrates that agonistic anti-OX40 antibodies with Fc-mediated effector functions can preferentially deplete intratumoral Tregs and enhance CD8 + The ratio of effector T cells to Tregs was increased, resulting in improved anti-tumor immune responses, increased tumor regression, and prolonged survival (Bulliard et al., 2014; Carboni et al., 2003; Jacquemin et al., 2015; Marabelle et al., 2013b). Based on these findings, there is an unmet medical need for the development of agonistic anti-OX40 antibodies that have both agonistic activity and Fc-mediated effector functions.

[0008] To date, agonist anti-OX40 antibodies in clinical practice are mainly ligand-competitive antibodies that block the OX40-OX40L interaction (e.g., WO 2016196228A1). Since the OX40-OX40L interaction is essential for enhancing effective anti-tumor immunity, the blockade of OX40-OX40L limits the efficacy of these ligand-competitive antibodies. Therefore, OX40 agonist antibodies that specifically bind to OX40 without interfering with the interaction between OX40 and OX40L can be used to treat cancer and autoimmune disorders. Summary of the invention

[0009] The present disclosure is directed to agonistic anti-OX40 antibodies and antigen-binding fragments thereof that activate OX40 and induce signaling in immune cells, thereby promoting anti-tumor immunity.

[0010] In one embodiment, the disclosure provides a monoclonal antibody or antigen binding fragment thereof that binds to human OX40. In one aspect, the antibodies of the disclosure do not compete with OX40L, or do not interfere with the binding of OX40 to its ligand OX40L.

[0011] The present disclosure includes the following embodiments.

[0012] An antibody or antigen-binding fragment thereof, which specifically binds to human OX40 and comprises: (i) a heavy chain variable region comprising (a) HCDR (heavy chain complementary determining region) 1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 24, and (c) HCDR3 of SEQ ID NO: 5; and a light chain variable region comprising (d) LCDR (light chain complementary determining region) 1 of SEQ ID NO: 25, (e) LCDR2 of SEQ ID NO: 19, and (f) LCDR3 of SEQ ID NO: 8; (ii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 18, (c) HCDR3 of SEQ ID NO: 5; and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 6, (e) LCDR2 of SEQ ID NO: 19, and (f) LCDR3 of SEQ ID NO: 8; (iii) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 18, (c) HCDR3 of SEQ ID NO: 5; and a light chain variable region comprising (d) LCDR1 of SEQ ID NO: 6, (e) LCDR2 of SEQ ID NO: 19, and (f) LCDR3 of SEQ ID NO: 8 NO:3, (b) HCDR1 of SEQ ID NO:13, (c) HCDR3 of SEQ ID NO:5; and a light chain variable region comprising (d) LCDR1 of SEQ ID NO:6, (e) LCDR2 of SEQ ID NO:7, and (f) LCDR3 of SEQ ID NO:8; or (iv) a heavy chain variable region comprising (a) HCDR1 of SEQ ID NO:3, (b) HCDR2 of SEQ ID NO:4, (c) HCDR3 of SEQ ID NO:5; and a light chain variable region comprising (d) LCDR1 of SEQ ID NO:6, (e) LCDR2 of SEQ ID NO:7, and (f) LCDR3 of SEQ ID NO:8.

[0013] The antibody or antigen-binding fragment of claim 1, comprising: (i) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 26, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 28; (ii) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: NO:22; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:14, and a light chain variable region (VL) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:16; or (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:17. NO:9 has a heavy chain variable region (VH) that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence, and a light chain variable region (VL) that comprises an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:11.

[0014] The antibody or antigen-binding fragment, wherein one, two, three, four, five, six, seven, eight, nine or ten amino acids in SEQ ID NO: 26, 28, 20, 22, 14, 16, 9 and / or 11 have been inserted, deleted or substituted.

[0015] The antibody or antigen-binding fragment comprises: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 26 and a light chain variable region (VL) comprising SEQ ID NO: 28; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 20 and a light chain variable region (VL) comprising SEQ ID NO: 22; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 14 and a light chain variable region (VL) comprising SEQ ID NO: 16; or (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 9 and a light chain variable region (VL) comprising SEQ ID NO: 11.

[0016] The antibody or antigen-binding fragment is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single-chain antibody (scFv), a Fab fragment, a Fab' fragment or a F(ab')2 fragment.

[0017] The antibody or antigen-binding fragment has OX40 agonist activity.

[0018] The antibody or antigen-binding fragment binds to human OX40 at an epitope comprising one or more amino acid residues selected from H153 to D170 of human OX40.

[0019] The antibody or antigen-binding fragment binds to human OX40 at an epitope comprising one or more amino acid residues selected from H153, T154, I165, E167 and D170 of human OX40.

[0020] The antibody or antigen-binding fragment binds to human OX40 at an epitope comprising one or more amino acid residues selected from H153, I165 and E167 of human OX40. The antibody or antigen-binding fragment binds to human OX40 at an epitope comprising one amino acid residue selected from H153, I165 and E167 of human OX40.

[0021] The antibody or antigen-binding fragment binds to human OX40 at or within SEQ ID NO.30.

[0022] The antibody or antigen-binding fragment thereof has an equilibrium dissociation constant (K) equal to or higher than 7.28 nM, 9.47 nM, 13.5 nM or 17.1 nM as measured by surface plasmon resonance (SPR). D ) binds to human OX40.

[0023] The antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0024] The antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or no glycosylation or is hypofucosylated.

[0025] The antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof comprises an added bisecting GlcNac structure.

[0026] The antibody or antigen-binding fragment, wherein the Fc domain belongs to IgG1.

[0027] The antibody or antigen-binding fragment, wherein the Fc domain belongs to IgG4.

[0028] The antibody or antigen-binding fragment, wherein the IgG4 has a S228P substitution (according to the EU numbering system).

[0029] The antibody or antigen-binding fragment, wherein the IgG4 has S228P and R409K substitutions (according to the EU numbering system).

[0030] The antibody or antigen-binding fragment has one or more of the following characteristics:

[0031] (i) Ability to cross-react with cyno OX40;

[0032] (ii) does not interfere with the OX40-OX40L interaction;

[0033] (iii) Capable of stimulating T cells, particularly at an EC concentration equal to or greater than 0.06 ng / ml 50 ;

[0034] (iv) Capable of activating CD4 + T cells, particularly as measured in a mixed lymphocyte reaction (MLR) assay;

[0035] (v) capable of mediating ADCC, particularly as measured in an ADCC assay based on lactate dehydrogenase (LDH) release;

[0036] (vi) Ability to deplete CD4 + Tregs;

[0037] (vii) Can increase CD8 + Teff / Treg ratio; or

[0038] (viii) Able to mediate partial tumor regression in animal tumor models.

[0039] A pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof, further comprising a pharmaceutically acceptable carrier.

[0040] A method for treating cancer, comprising administering an effective amount of the antibody or antigen-binding fragment to a patient in need thereof.

[0041] The method, wherein the cancer includes but is not limited to breast cancer, head and neck cancer, gastric cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, sarcoma, etc.

[0042] The method, wherein the antibody or the pharmaceutical composition is administered in combination with another therapeutic agent.

[0043] The method, wherein the therapeutic agent is paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, 5-azacytidine.

[0044] The method, wherein the therapeutic agent is paclitaxel, lenalidomide, or 5-azacytidine.

[0045] An isolated nucleic acid encoding the antibody or antigen-binding fragment.

[0046] A vector comprising the nucleic acid.

[0047] A host cell comprising the nucleic acid.

[0048] A method for producing an antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell and recovering the antibody or antigen-binding fragment from the culture.

[0049] The antibody or antigen-binding fragment thereof is used to treat breast cancer, head and neck cancer, gastric cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma and sarcoma or to reduce the likelihood of the cancer.

[0050] A diagnostic reagent comprises the labeled antibody or antigen-binding fragment thereof.

[0051] The diagnostic reagent, wherein the label is selected from the group consisting of a radioactive label, a fluorophore, a chromophore, an imaging agent and a metal ion.

[0052] In one embodiment, the antibody or antigen-binding fragment thereof comprises one or more complementarity determining regions (CDRs) having an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:24 and SEQ ID NO:25.

[0053] In another embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising one or more complementarity determining regions (HCDRs) having an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:24 and SEQ ID NO:5; and / or (b) a light chain variable region comprising one or more complementarity determining regions (LCDRs) having an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:25, SEQ ID NO:7, SEQ ID NO:19 and SEQ ID NO:8.

[0054] In another embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising three complementarity determining regions (HCDRs), wherein the three complementarity determining regions are HCDR1 having an amino acid sequence of SEQ ID NO:3; HCDR2 having an amino acid sequence of SEQ ID NO:4, SEQ ID NO:13, SEQ ID NO:18 or SEQ ID NO:24; and HCDR3 having an amino acid sequence of SEQ ID NO:5; and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs), wherein the three complementarity determining regions are LCDR1 having an amino acid sequence of SEQ ID NO:6 or SEQ ID NO:25; LCDR2 having an amino acid sequence of SEQ ID NO:7 or SEQ ID NO:19; and LCDR3 having an amino acid sequence of SEQ ID NO:8.

[0055] In another embodiment, the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising three complementarity determining regions (HCDRs), wherein the three complementarity determining regions are HCDR1 having an amino acid sequence of SEQ ID NO: 3, HCDR2 having an amino acid sequence of SEQ ID NO: 4, and HCDR3 having an amino acid sequence of SEQ ID NO: 5; or HCDR1 having an amino acid sequence of SEQ ID NO: 3, HCDR2 having an amino acid sequence of SEQ ID NO: 13, and HCDR3 having an amino acid sequence of SEQ ID NO: 5; or HCDR1 having an amino acid sequence of SEQ ID NO: 3, HCDR2 having an amino acid sequence of SEQ ID NO: 18, and HCDR3 having an amino acid sequence of SEQ ID NO: 5; or HCDR1 having an amino acid sequence of SEQ ID NO: 3, HCDR2 having an amino acid sequence of SEQ ID NO: 24, and HCDR3 having an amino acid sequence of SEQ ID NO: 5; and / or (b) a light chain variable region comprising three complementarity determining regions (LCDRs), wherein the three complementarity determining regions are HCDR1 having an amino acid sequence of SEQ ID NO: 3, HCDR2 having an amino acid sequence of SEQ ID NO: 13, and HCDR3 having an amino acid sequence of SEQ ID NO: 5. NO:6, LCDR1 having the amino acid sequence of SEQ ID NO:7, and LCDR3 having the amino acid sequence of SEQ ID NO:8; or LCDR1 having the amino acid sequence of SEQ ID NO:6, LCDR2 having the amino acid sequence of SEQ ID NO:19, and LCDR3 having the amino acid sequence of SEQ ID NO:8; or LCDR1 having the amino acid sequence of SEQ ID NO:25, LCDR2 having the amino acid sequence of SEQ ID NO:19, and LCDR3 having the amino acid sequence of SEQ ID NO:8.

[0056] In another embodiment, the antibody or antigen-binding fragment of the present disclosure comprises: a heavy chain variable region comprising a HCDR1 having an amino acid sequence of SEQ ID NO:3, a HCDR2 having an amino acid sequence of SEQ ID NO:4, and a HCDR3 having an amino acid sequence of SEQ ID NO:5; and a light chain variable region comprising a LCDR1 having an amino acid sequence of SEQ ID NO:6, a LCDR2 having an amino acid sequence of SEQ ID NO:7, and a LCDR3 having an amino acid sequence of SEQ ID NO:8.

[0057] In one embodiment, the antibody or antigen-binding fragment of the present disclosure comprises: a heavy chain variable region comprising a HCDR1 having an amino acid sequence of SEQ ID NO:3, a HCDR2 having an amino acid sequence of SEQ ID NO:13, and a HCDR3 having an amino acid sequence of SEQ ID NO:5; and a light chain variable region comprising a LCDR1 having an amino acid sequence of SEQ ID NO:6, a LCDR2 having an amino acid sequence of SEQ ID NO:7, and a LCDR3 having an amino acid sequence of SEQ ID NO:8.

[0058] In another embodiment, the antibody or antigen-binding fragment of the present disclosure comprises: a heavy chain variable region comprising a HCDR1 having an amino acid sequence of SEQ ID NO:3, a HCDR2 having an amino acid sequence of SEQ ID NO:18, and a HCDR3 having an amino acid sequence of SEQ ID NO:5; and a light chain variable region comprising a LCDR1 having an amino acid sequence of SEQ ID NO:6, a LCDR2 having an amino acid sequence of SEQ ID NO:19, and a LCDR3 having an amino acid sequence of SEQ ID NO:8.

[0059] In another embodiment, the antibody or antigen-binding fragment of the present disclosure comprises: a heavy chain variable region comprising a HCDR1 having an amino acid sequence of SEQ ID NO:3, a HCDR2 having an amino acid sequence of SEQ ID NO:24, and a HCDR3 having an amino acid sequence of SEQ ID NO:5; and a light chain variable region comprising a LCDR1 having an amino acid sequence of SEQ ID NO:25, a LCDR2 having an amino acid sequence of SEQ ID NO:19, and a LCDR3 having an amino acid sequence of SEQ ID NO:8.

[0060] In one embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain variable region having an amino acid sequence of SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:20 or SEQ ID NO:26, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:20 or SEQ ID NO:26; and / or (b) a light chain variable region having an amino acid sequence of SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:22 or SEQ ID NO:28, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:22 or SEQ ID NO:28.

[0061] In another embodiment, an antibody or antigen-binding fragment thereof of the present disclosure comprises: (a) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 20 or SEQ ID NO: 26, or an amino acid sequence having one, two or three amino acid substitutions in the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 20 or SEQ ID NO: 26; and / or (b) a light chain variable region having an amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 22 or SEQ ID NO: 28, or an amino acid sequence having one, two, three, four or five amino acid substitutions in the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 22 or SEQ ID NO: 28. In another embodiment, the amino acid substitutions are conservative amino acid substitutions.

[0062] In one embodiment, the antibody or antigen-binding fragment thereof of the present disclosure comprises:

[0063] (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9 and a light chain variable region having the amino acid sequence of SEQ ID NO: 11; or

[0064] (b) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14 and a light chain variable region having the amino acid sequence of SEQ ID NO: 16; or

[0065] (c) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 20 and a light chain variable region having the amino acid sequence of SEQ ID NO: 22; or

[0066] (d) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 28.

[0067] In one embodiment, the antibody of the present disclosure is of IgG1, IgG2, IgG3 or IgG4 isotype. In a more specific embodiment, the antibody of the present disclosure comprises the Fc domain of wild-type human IgG1 (also referred to as human IgG1wt or huIgG1) or IgG2. In another embodiment, the antibody of the present disclosure comprises the Fc domain of human IgG4 with S228P and / or R409K substitutions (according to the EU numbering system).

[0068] In one embodiment, the antibodies of the present disclosure are expressed at 1×10 -6 M to 1x10 -10 The binding affinity (K D ) binds to OX40. In another embodiment, the antibodies of the present disclosure are present at about 1x10 -6 M, about 1x10 -7 M, about 1x10 -8 M, about 1x10 -9 M or about 1x10 -10 The binding affinity (K D ) binds to OX40.

[0069] In another embodiment, the anti-human OX40 antibodies of the invention exhibit cross-species binding activity to cynomolgus monkey OX40.

[0070] In one embodiment, the anti-OX40 antibodies of the present disclosure bind to an epitope of human OX40 outside the OX40-OX40L interaction interface. In another embodiment, the anti-OX40 antibodies of the present disclosure do not compete with the OX40 ligand for binding to OX40. In yet another embodiment, the anti-OX40 antibodies of the present disclosure do not block the interaction between OX40 and its ligand OX40L.

[0071] The antibodies of the present disclosure are agonistic and significantly enhance immune responses. The present invention provides methods for testing the agonistic ability of anti-OX40 antibodies. In one embodiment, the antibodies of the present disclosure can significantly stimulate primary T cells to produce IL-2 in a mixed lymphocyte reaction (MLR) assay.

[0072] In one embodiment, the antibodies of the present disclosure have strong Fc-mediated effector functions. The antibodies mediate NK cell-mediated effects against OX40 Hi Antibody-dependent cellular cytotoxicity (ADCC) of target cells, such as regulatory T cells (Treg cells). In one aspect, the disclosure provides methods for evaluating anti-OX40 antibody-mediated in vitro depletion of specific T cell subsets based on different OX40 expression levels.

[0073] The antibodies or antigen-binding fragments of the present disclosure do not block OX40-OX40L interaction. In addition, the OX40 antibodies exhibit dose-dependent anti-tumor activity in vivo as shown in animal models. The dose-dependent activity is different from the activity spectrum of anti-OX40 antibodies that block OX40-OX40L interaction.

[0074] The present disclosure relates to an isolated nucleic acid comprising a nucleotide sequence encoding the amino acid sequence of the antibody or antigen-binding fragment. In one embodiment, the isolated nucleic acid comprises the VH nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 21 or SEQ ID NO: 27, or a nucleotide sequence having at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 21 or SEQ ID NO: 27, and encodes the VH region of the antibody or antigen-binding fragment of the present disclosure. Alternatively or additionally, the isolated nucleic acid comprises the VL nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 23 or SEQ ID NO: 29, or a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 23 or SEQ ID NO: 29, and encodes the VL region of the antibody or antigen-binding fragment of the present disclosure.

[0075] In another aspect, the present disclosure relates to a pharmaceutical composition comprising the OX40 antibody or antigen-binding fragment thereof and optionally a pharmaceutically acceptable excipient.

[0076] In yet another aspect, the disclosure relates to a method of treating a disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the OX40 antibody or antigen-binding fragment thereof or an OX40 antibody pharmaceutical composition. In another embodiment, the disease to be treated by the antibody or antigen-binding fragment is cancer or an autoimmune disease.

[0077] The present disclosure relates to the use of the antibody or antigen-binding fragment thereof or OX40 antibody pharmaceutical composition for treating diseases (such as cancer or autoimmune diseases). BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Schematic diagram of OX40-mIgG2a, OX40-huIgG1 and OX40-His constructs. OX40ECD: OX40 extracellular domain. N: N-terminus. C: C-terminus.

[0079] Figure 2 Affinity determination of purified chimeric (ch445) and humanized (445-1, 445-2, 445-3 and 445-3IgG4) anti-OX40 antibodies by surface plasmon resonance (SPR) is shown.

[0080] Figure 3 Determination of OX40 binding by flow cytometry is shown. OX40 positive HuT78 / OX40 cells were incubated with various anti-OX40 antibodies (antibodies ch445, 445-1, 445-2, 445-3 and 445-3IgG4) and subjected to FACS analysis. Results are shown with mean fluorescence intensity (MFI, Y axis).

[0081] Figure 4 Binding of OX40 antibodies by flow cytometry is shown. HuT78 / OX40 and HuT78 / cynoOX40 cells were stained with antibody 445-3 and mean fluorescence intensity (MFI, shown on the Y axis) was determined by flow cytometry.

[0082] Figure 5 Depicted is the affinity determination of 445-3 Fab for OX40 wild type and point mutants by surface plasmon resonance (SPR).

[0083] Figure 6 Detailed interactions between antibody 445-3 and its epitope on OX40 are shown. Antibody 445-3 and OX40 are depicted in light grey and black, respectively. Hydrogen bonds or salt bridges, π-π stacking, and van der Waals (VDW) interactions are indicated by dashed lines, double dashed lines, and solid lines, respectively.

[0084] Figure 7It was demonstrated that antibody 445-3 does not interfere with OX40L binding. OX40-mouse IgG2a (OX40-mIgG2a) fusion protein was pre-incubated with human IgG (+HuIgG), antibody 445-3 (+445-3) or antibody 1A7.gr1 (+1A7.gr1, see US 2015 / 0307617) at a molar ratio of 1:1 before staining HEK293 / OX40L cells. Binding of OX40L to the OX40-mIgG2a / anti-OX40 antibody complex was determined by co-incubation of HEK293 / OX40L cells with the OX40-mIgG2a / anti-OX40 antibody complex, followed by reaction with anti-mouse IgG secondary Ab and flow cytometry. Results are shown as mean ± SD of two replicates. Statistical significance: *: P < 0.05; **: P < 0.01.

[0085] Figure 8 Shown is a structural alignment of OX40 / 445-3 Fab with the reported OX40 / OX40L complex (PDB code: 2HEV). OX40L is shown in white, 445-3 Fab in grey, and OX40 in black.

[0086] Figures 9A to 9B show that anti-OX40 antibody 445-3 induces IL-2 production and TCR stimulation. OX40-positive HuT78 / OX40 cells (Figure 9A) were combined with an artificial antigen presenting cell (APC) line (HEK293 / OS8 低 -FcγRI) were co-cultured overnight in the presence of anti-OX40 antibody, and IL-2 production was used as a readout of T cell stimulation (Figure 9B). IL-2 in the culture supernatant was detected by ELISA. The results are shown as the mean ± SD of three replicates.

[0087] Fig.10 Anti-OX40 antibodies were shown to enhance MLR responses. + T cells were co-cultured in the presence of anti-OX40 antibody (0.1-10 μg / ml) for 2 days. IL-2 in the supernatant was detected by ELISA. All tests were performed in quadruplicate and the results are shown as mean ± SD. Statistical significance: *: P < 0.05; **: P < 0.01.

[0088] Fig.11Anti-OX40 antibody 445-3 was demonstrated to induce ADCC. ADCC assays were performed in the presence of anti-OX40 antibody (0.004-3 μg / ml) or control using NK92MI / CD16V cells as effector cells and HuT78 / OX40 cells as target cells. Equal numbers of effector and target cells were co-cultured for 5 hours before detecting lactate dehydrogenase (LDH) release. Percent cytotoxicity (Y axis) was calculated based on the manufacturer's protocol as described in Example 12. Results are shown as mean ± SD of three replicates.

[0089] Figures 12A to 12C show that the combination of anti-OX40 antibody 445-3 and NK cells increases CD8 + Effector T cells to Treg ratio. Human PBMCs were preactivated by PHA-L (1 μg / ml) and then co-cultured with NK92MI / CD16V cells in the presence of anti-OX40 antibody or control. The percentages of different T cell subsets were determined by flow cytometry. CD8 + Effector T cells to Treg ratio. Figure 12A shows the CD8+ / total T cell ratio. Figure 12B is the Treg / total T cell ratio. Figure 12C shows the CD8+ / Treg ratio. Data are shown as mean ± SD of two replicates. Statistical significance between 445-3 and 1A7.gr1 at the indicated concentrations is shown. *: P < 0.05; **: P < 0.01.

[0090] FIG. 13A to FIG. 13B The anti-OX40 antibody 445-3, but not 1A7.gr1, revealed dose-dependent antitumor activity in the MC38 colorectal cancer syngeneic model of OX40 humanized mice. MC38 mouse colon cancer cells (2×10 7 After randomization based on tumor volume, animals were injected intraperitoneally with anti-OX40 antibodies or isotype control once a week for three times as indicated. Fig.13A Increasing doses of the 445-3 antibody were compared to increasing doses of the 1A7.gr1 antibody and reductions in tumor growth. Fig. 13B Data for all mice treated with that particular dose are presented. Data are presented as mean tumor volume ± standard error of the mean (SEM), 6 mice per group. Statistical significance: *: P < 0.05 compared to isotype control.

[0091] FIG. 14A to FIG. 14B is a table of amino acid changes made in the OX40 antibodies.

[0092] definition

[0093] Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0094] As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0095] Unless the context clearly indicates otherwise, the term "or" is used to mean, and is used interchangeably with, the term "and / or."

[0096] As used herein, the term "anti-cancer agent" refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0097] The term "OX40" refers to a type I transmembrane glycoprotein of approximately 50 KD that is a member of the tumor necrosis factor receptor superfamily. OX40 is also known as ACT35, CD134, or TNFRSF4. The amino acid sequence of human OX40 (SEQ ID NO: 1) can also be found as accession number NP_003318, and the nucleotide sequence encoding the OX40 protein is accession number: X75962.1. The term "OX40 ligand" or "OX40L" refers to the only ligand of OX40 and is interchangeable with gp34, CD252, or TNFSF4.

[0098] When applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids, the terms "administration", "administering", treatment ("treating" and "treatment") herein refer to the contact of an exogenous agent, therapeutic agent, diagnostic agent or composition with an animal, a human, a subject, a cell, a tissue, an organ or a biological fluid. Cellular treatment includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. The terms "administering" and "treatment" also refer to in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnostic agent, a binding compound or by another cell. The term "subject" herein includes any organism, preferably an animal, more preferably a mammal (e.g., rats, mice, dogs, cats, rabbits) and most preferably a human. Treatment of any disease or disorder refers to improving a disease or disorder (i.e., slowing down or preventing or reducing the development of a disease or at least one clinical symptom thereof) in one aspect. In another aspect, "treatment" ("treat", "treating" or "treatment") refers to alleviating or improving at least one physical parameter, including those that the patient may not be able to discern. In yet another aspect, "treat," "treating," or "treatment" refers to regulating a disease or disorder physically (e.g., stabilizing a discernible symptom), physiologically (e.g., stabilizing a physical parameter), or both. In yet another aspect, "treat," "treating," or "treatment" refers to preventing or delaying the onset or development or progression of a disease or disorder.

[0099] In the context of this disclosure, the term "subject" is a mammal, such as a primate, preferably a higher primate, such as a human (eg, a patient suffering from or at risk of suffering from a disorder described herein).

[0100] The term "affinity" as used herein refers to the strength of the interaction between an antibody and an antigen. Within the antigen, the variable regions of the antibody "arms" interact with the antigen at many sites through non-covalent forces; the more interactions, the stronger the affinity.

[0101] The term "antibody" as used herein refers to a polypeptide of the immunoglobulin family that can non-covalently, reversibly and specifically bind to the corresponding antigen. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into regions with high variability, called complementary determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0102] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies. The antibody may be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0103] In some embodiments, the anti-OX40 antibody comprises at least one antigen binding site or at least one variable region. In some embodiments, the anti-OX40 antibody comprises an antigen binding fragment from an OX40 antibody described herein. In some embodiments, the anti-OX40 antibody is isolated or recombinant.

[0104] The term "monoclonal antibody" or "mAb" or "Mab" herein means a population of substantially homogeneous antibodies, i.e., except for possible naturally occurring mutations that can be present in small amounts, the amino acid sequences of the antibody molecules contained in the population are identical. In contrast, conventional (polyclonal) antibody preparations typically include a variety of different antibodies with different amino acid sequences in their variable domains (particularly their complementary determining regions (CDRs), which are typically specific for different epitopes). The modifier "monoclonal" indicates the characteristics of antibodies obtained from a substantially homogeneous antibody population and should not be construed as requiring antibodies to be produced by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, e.g., Kohler et al., Nature 1975 256:495-497; U.S. Pat. No. 4,376,110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Coldspring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein may belong to any immunoglobulin class, including IgG, IgM, IgD, IgE, IgA and any subclass thereof, such as IgG1, IgG2, IgG3, IgG4. Hybridomas producing monoclonal antibodies may be cultured in vitro or in vivo. High titers of monoclonal antibodies may be obtained by in vivo production, wherein cells from a single hybridoma are injected intraperitoneally into mice (e.g., originally initiated Balb / c mice) to produce ascites containing high concentrations of the desired antibodies. Monoclonal antibodies of the isotype IgM or IgG can be purified from such ascites fluid or from the culture supernatant using column chromatography methods well known to those skilled in the art.

[0105] In general, the basic antibody structural unit comprises a tetramer. Each tetramer comprises two pairs of identical polypeptide chains, each pair having a "light chain" (about 25 kDa) and a "heavy chain" (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that are primarily responsible for antigen recognition. The carboxyl terminal portion of the heavy chain can define a constant region that is primarily responsible for effector function. Typically, human light chains are classified into kappa and lambda light chains. In addition, human heavy chains are typically classified into alpha, delta, epsilon, gamma or mu, and the isotypes of antibodies are defined as IgA, IgD, IgE, IgG and IgM, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D" region of about 10 or more amino acids.

[0106] The variable regions of each light chain / heavy chain (VL / VH) pair form an antibody binding site. Therefore, in general, a complete antibody has two binding sites. Except for bifunctional or bispecific antibodies, the two binding sites are generally the same.

[0107] Typically, the variable domains of both the heavy and light chains contain three hypervariable regions, also referred to as "complementarity determining regions (CDRs)", which are located between relatively conserved framework regions (FRs). The CDRs are typically aligned by the framework regions to enable binding to specific epitopes. In general, from N-terminus to C-terminus, both the light and heavy chain variable domains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4). The positions of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, and AbM (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)). The definition of antigen binding sites is also described in Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, MP, Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., in Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). In the combined Kabat and Chothia numbering scheme, in some embodiments, a CDR corresponds to an amino acid residue that is part of a Kabat CDR, a Chothia CDR, or both.For example, the CDRs correspond to amino acid residues 26-35 (HC CDR1), 50-65 (HC CDR2), and 95-102 (HC CDR3) in a VH (e.g., a mammalian VH, e.g., a human VH); and amino acid residues 24-34 (LC CDR1), 50-56 (LC CDR2), and 89-97 (LC CDR3) in a VL (e.g., a mammalian VL, e.g., a human VL).

[0108] The term "hypervariable region" means the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from "CDR" (i.e., VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable domain and VH-CDR1, VH-CDR2 and VH-CDR3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Public Health Service, National Institutes of Health, Bethesda, Maryland (defining the CDR regions of antibodies according to sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of antibodies according to structure). The term "framework" or "FR" residues means those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0109] Unless otherwise indicated, "antigen-binding fragment" means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen bound by a full-length antibody, such as a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as single-chain Fv (ScFv); nanobodies, and multispecific antibodies formed from antibody fragments.

[0110] An antibody "specifically binds" to a target protein means that the antibody exhibits preferential binding to the target compared to other proteins, but such specificity does not require absolute binding specificity. An antibody is considered to be "specific" for its intended target if its binding determines the presence of the target protein in a sample without, for example, producing undesirable results (such as false positives). An antibody or antigen-binding fragment thereof useful in the present disclosure will bind to the target protein with an affinity that is at least 2 times greater, preferably at least 10 times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than the affinity for non-target proteins. An antibody herein is said to specifically bind to a polypeptide comprising a given amino acid sequence (e.g., the amino acid sequence of a human OX40 molecule) if it binds to a polypeptide comprising the sequence but not to a protein lacking the sequence.

[0111] The term "human antibody" herein means an antibody that comprises only human immunoglobulin protein sequences. If produced in mice, in mouse cells, or in hybridomas derived from mouse cells, human antibodies may contain mouse carbohydrate chains. Similarly, "mouse antibody" or "rat antibody" means an antibody that comprises only mouse or rat immunoglobulin protein sequences, respectively.

[0112] The term "humanized antibody" means an antibody form containing sequences from non-human (e.g., mouse) antibodies and human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Generally speaking, a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. When it is necessary to distinguish humanized antibodies from parent rodent antibodies, prefixes "hum", "hu", "Hu" or "h" are added to the antibody clone name. The humanized form of a rodent antibody will generally comprise the same CDR sequence of a parent rodent antibody, although some amino acid substitutions may be included to increase affinity, improve the stability of the humanized antibody, remove post-translational modifications or for other reasons.

[0113] The term "corresponding human germline sequence" refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that has the highest determined amino acid sequence identity to a reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. A corresponding human germline sequence may also refer to a human variable region amino acid sequence or subsequence that has the highest amino acid sequence identity to a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. A corresponding human germline sequence may be a framework region only, a complementary determining region only, a framework and complementary determining region, a variable segment (as defined above), or other combinations of sequences or subsequences comprising a variable region. Sequence identity may be determined using the methods described herein, for example, by aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference variable region nucleic acid or amino acid sequence.

[0114] The term "equilibrium dissociation constant (K D , M)” refers to the dissociation rate constant (kd, time-1 ) divided by the association rate constant (ka, time -1 , M -1 The equilibrium dissociation constant can be measured using any method known in the art. Antibodies of the present disclosure will generally have a dissociation constant of less than about 10 -7 or 10 -8 M (e.g., less than about 10 -9 M or 10 -10 M, in some aspects, is less than about 10 -11 M, 10 -12 M or 10 -13 M) equilibrium dissociation constant.

[0115] The term "cancer" or "tumor" herein has the broadest meaning as understood in the art, and refers to the physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of this disclosure, cancer is not limited to a certain type or location.

[0116] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat the therapeutic conditions or disorders described in this disclosure. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner. Such administration also includes co-administration with multiple or separate containers (e.g., capsules, powders, and liquids) for each active ingredient. Prior to administration, the powder and / or liquid may be reconstituted or diluted to the desired dose. In addition, such administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide the beneficial effects of the drug combination in treating the conditions or disorders described herein.

[0117] In the context of the present disclosure, when referring to an amino acid sequence, the term "conservative substitution" means replacing an original amino acid with a new amino acid that does not substantially change the chemical, physical and / or functional properties of the antibody or fragment, such as its binding affinity to OX40. Specifically, common conservative substitutions of amino acids are shown in the table below and are well known in the art.

[0118] Exemplary conservative amino acid substitutions

[0119]

[0120]

[0121] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet a certain positive threshold score T when aligned with a word of the same length in a database sequence. T is called the neighbor word score threshold. These initial neighbor word hits are used as values ​​to start the search to find longer HSPs containing them. Word hits are extended in both directions along each sequence for as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score drops by the amount X from its maximum achieved value; when the cumulative score becomes zero or lower due to the accumulation of one or more negative scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following as default parameters: word length (W) of 11, expectation (E) of 10, M=5, N=-4, and comparison of both chains. For amino acid sequences, the BLAST program uses as default parameters a wordlength of 3, an expectation (E) of 10, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0122] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One similarity measure provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0123] The percent identity between two amino acid sequences can also 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 a 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 using 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, using a BLOSUM62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[0124] The term "nucleic acid" is used interchangeably with the term "polynucleotide" herein and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or connections, which are synthetic, naturally occurring, and non-naturally occurring, have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs).

[0125] In the context of nucleic acids, the term "operably linked" refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship between a transcriptional regulatory sequence and a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system. Typically, a promoter transcriptional regulatory sequence operably linked to a transcribed sequence is physically continuous with the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences (such as enhancers) do not need to be physically continuous or located in close proximity to the coding sequence whose transcription they enhance.

[0126] In some aspects, the present disclosure provides compositions (e.g., pharmaceutically acceptable compositions) comprising an anti-OX40 antibody described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible, etc. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

[0127] Compositions disclosed herein can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. Suitable forms depend on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable or infusible solutions. A suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0128] As used herein, the term "therapeutically effective amount" refers to an amount of an antibody that, when administered to a subject for the treatment of a disease, or at least one clinical symptom of a disease or disorder, is sufficient to produce an effect on such treatment of the disease, disorder or symptom. The "therapeutically effective amount" may vary with: the antibody; the disease, disorder and / or symptoms of the disease or disorder; the severity of the disease, disorder and / or symptoms of the disease or disorder; the age of the subject to be treated and / or the weight of the subject to be treated. In any given example, the appropriate amount will be clear to those skilled in the art or can be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combined subject used to effectively treat the disease, disorder or condition. DETAILED DESCRIPTION

[0129] The present disclosure provides antibodies and antigen-binding fragments that specifically bind to human OX40. In addition, the present disclosure provides antibodies that have desirable pharmacokinetic characteristics and other desirable properties and can therefore be used to reduce the likelihood of cancer or treat cancer. The present disclosure also provides pharmaceutical compositions comprising the antibodies, and methods of preparing and using such pharmaceutical compositions to prevent and treat cancer and related disorders.

[0130] Anti-OX40 Antibodies

[0131] The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to OX40. The antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof generated as described below.

[0132] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to OX40, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VH domain having an amino acid sequence of SEQ ID NO: 14, 20 or 26 (Table 3). The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to OX40, wherein the antibodies or antigen-binding fragments comprise a VH CDR having an amino acid sequence of any one of the VH CDRs listed in Table 3. In one aspect, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to OX40, wherein the antibodies comprise (or alternatively consist of) one, two, three or more VH CDRs having an amino acid sequence of any of the VH CDRs listed in Table 3.

[0133] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to OX40, wherein the antibodies or antigen-binding fragments comprise a VL domain having an amino acid sequence of SEQ ID NO: 16, 22 or 28 (Table 3). The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to OX40, wherein the antibodies or antigen-binding fragments comprise a VL CDR having an amino acid sequence of any one of the VL CDRs listed in Table 3. In particular, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to OX40, wherein the antibodies or antigen-binding fragments comprise (or alternatively consist of) one, two, three or more VL CDRs having an amino acid sequence of any of the VL CDRs listed in Table 3.

[0134] Other antibodies or antigen-binding fragments thereof of the present disclosure include amino acids that have been mutated but have at least 60%, 70%, 80%, 90%, 95% or 99% identity percentage in the CDR regions with the CDR regions depicted in the sequences described in Table 3. In some aspects, it includes mutant amino acid sequences, wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the CDR regions when compared to the CDR regions depicted in the sequences described in Table 3.

[0135] Other antibodies of the present disclosure include those in which the amino acids or nucleic acids encoding the amino acids have been mutated, but have at least 60%, 70%, 80%, 90%, 95% or 99% identity to the sequences set forth in Table 3. In some aspects, it includes mutant amino acid sequences in which no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the variable region when compared to the variable region depicted in the sequences set forth in Table 3, but retain substantially the same therapeutic activity.

[0136] The disclosure also provides nucleic acid sequences encoding VH, VL, full-length heavy chain and full-length light chain of antibodies that specifically bind to OX40.Such nucleic acid sequences can be optimized for expression in mammalian cells.

[0137] Identification of epitopes and antibodies binding to the same epitopes

[0138] The present disclosure provides antibodies and antigen-binding fragments thereof that bind to an epitope of human OX40. In certain aspects, the antibodies and antigen-binding fragments may bind to the same epitope of OX40.

[0139] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-OX40 antibodies described in Table 3. Thus, additional antibodies and antigen-binding fragments thereof can be identified based on their ability to cross-compete with other antibodies in a binding assay (e.g., competitively inhibit binding with other antibodies in a statistically significant manner). The ability of a test antibody to inhibit binding of an antibody and antigen-binding fragment thereof of the present disclosure to OX40 demonstrates that the test antibody can compete with the antibody or antigen-binding fragment thereof for binding to OX40. Without being bound by any one theory, such an antibody can bind to an epitope on OX40 that is the same or related (e.g., structurally similar or spatially adjacent) to the antibody or antigen-binding fragment thereof that it competes with. In a certain aspect, an antibody that binds to the same epitope on OX40 as an antibody or antigen-binding fragment thereof of the present disclosure is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

[0140] Further changes to the Fc region framework

[0141] In yet other aspects, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues so that the antibody has a changed affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand to which its affinity is changed can be, for example, an Fc receptor or the C1 component of complement. This method is described in, for example, U.S. Patent Nos. 5,624,821 and 5,648,260, both of which are from Winter et al.

[0142] In another aspect, one or more amino acid residues can be replaced with one or more different amino acid residues such that the antibody has altered CIq binding and / or reduced or abolished complement dependent cytotoxicity (CDC).This approach is described in US Pat. No. 6,194,551 to Idusogie et al.

[0143] In yet another aspect, one or more amino acid residues are changed to change the ability of the antibody to fix complement. The method is described in, for example, PCT Publication WO 94 / 29351 by Bodmer et al. In a specific aspect, one or more amino acids of an antibody or its antigen-binding fragment of the present disclosure are replaced by one or more heterotypic amino acid residues of the IgG1 subclass and the κ isotype. The heterotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgG1, IgG2 and IgG3 subclasses and the constant region of the light chain of the κ isotype as described in Jefferis et al., MAbs.1:332-338 (2009).

[0144] In another aspect, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors by modifying one or more amino acids. This approach is described, for example, in PCT Publication WO 00 / 42072 by Presta. In addition, the binding sites for FcγRI, FcγRII, FcγRIII, and FcRn have been mapped on human IgG1, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276: 6591-6604, 2001).

[0145] In still another aspect, the glycosylation of the modified antibody. For example, a non-glycosylated antibody (i.e., the antibody lacks glycosylation or has reduced glycosylation) can be prepared. Glycosylation can be changed, for example, to increase the affinity of the antibody to the "antigen". Such carbohydrate modifications can be achieved by, for example, changing one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made, which lead to the elimination of one or more variable region framework glycosylation sites, thereby eliminating the glycosylation of the site. This glycosylation can increase the affinity of the antibody to the antigen. This method is described in, for example, U.S. Patent Nos. 5,714,350 and 6,350,861 of Co et al.

[0146] Additionally or alternatively, antibodies with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells for expressing recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such a cell line exhibit low fucosylation. PCT Publication WO 03 / 035835 to Presta describes a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 to Umana et al. describes a cell line engineered to express a glycosyltransferase that modifies glycoproteins (e.g., β(1,4)-N-acetylglucosamine transferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0147] On the other hand, if it is desired to reduce ADCC, human antibody subclass IgG4 has been shown in many previous reports to have only moderate ADCC and almost no CDC effector function (Moore GL et al. 2010 MAbs, 2: 181-189). On the other hand, native IgG4 was found to be unstable under stress conditions (such as in acidic buffer or at elevated temperatures) (Angal, S. 1993 Mol Immunol, 30: 105-108; Dall'Acqua, W. et al. 1998 Biochemistry, 37: 9266-9273; Aalberse et al. 2002 Immunol, 105: 9-19). ADCC can be reduced by connecting the antibody to an IgG4 engineered with a combination of alterations that have reduced or ineffective FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector functions. Considering the physicochemical properties of antibodies as biopharmaceuticals, a less desirable intrinsic property of IgG4 is that its two heavy chains dynamically separate in solution to form half antibodies, which leads to the generation of bispecific antibodies in vivo via a process called "Fab arm exchange" (Van der Neut Kolfschoten M et al. 2007 Science, 317: 1554-157). The mutation of serine to proline at position 228 (EU numbering system) appears to inhibit the separation of IgG4 heavy chains (Angal, S. 1993 Mol Immunol, 30: 105-108; Aalberse et al. 2002 Immunol, 105: 9-19). It has been reported that some amino acid residues in the hinge and γFc region have an influence on the interaction of antibodies with Fcγ receptors (Chappel SM et al. 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, KL et al. 1999 Eur J Immunol, 29:2613-2624; Clynes, RA et al., 2000 Nature Medicine, 6:443-446; Arnold JN, 2007 Annu Rev immunol, 25:21-50). In addition, some IgG4 isotypes that rarely occur in the human population can also induce different physicochemical properties (Brusco, A. et al. 1998 Eur J Immunogenet, 25: 349-55; Aalberse et al. 2002 Immunol, 105: 9-19). In order to generate OX40 antibodies with low ADCC, CDC and instability, the hinge and Fc regions of human IgG4 can be modified and many changes can be introduced.These modified IgG4 Fc molecules can be found in SEQ ID NOs: 83-88 of U.S. Patent No. 8,735,553 to Li et al.

[0148] OX40 antibody generation

[0149] Anti-OX40 antibodies and antigen-binding fragments thereof can be produced by any means known in the art, including but not limited to recombinant expression, chemical synthesis and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any appropriate host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0150] The present disclosure also provides polynucleotides encoding antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or segments comprising complementary determining regions as described herein. In some aspects, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with a polynucleotide selected from SEQ ID NO: 15, 21 or 27. In some aspects, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with a polynucleotide selected from SEQ ID NO: 17, 23 or 29.

[0151] The polynucleotides of the present disclosure may encode variable region sequences of anti-OX40 antibodies. They may also encode both variable and constant regions of antibodies. Some polynucleotide sequences encode polypeptides comprising the variable regions of both the heavy and light chains of one of the exemplary anti-OX40 antibodies. Some other polynucleotides encode two polypeptide segments that are substantially identical to the variable regions of the heavy and light chains of one of the murine antibodies, respectively.

[0152] Also provided in the present disclosure are expression vectors and host cells for producing anti-OX40 antibodies. The choice of expression vector depends on the intended host cell of the vector to be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotide encoding the anti-OX40 antibody chain or antigen-binding fragment. In some aspects, inducible promoters are used to prevent the expression of the inserted sequence, except under the control of inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. The culture of the transformed organism can be amplified under non-inducing conditions without biasing the population toward the coding sequence, and the host cell better tolerates the expression product of the coding sequence. In addition to the promoter, other regulatory elements may also be needed or desired to effectively express the anti-OX40 antibody or antigen-binding fragment. These elements typically include an ATG start codon and an adjacent ribosome binding site or other sequence. In addition, expression efficiency can be increased by including an enhancer appropriate for the cell system in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

[0153] Host cells for containing and expressing anti-OX40 antibody chains can be prokaryotic or eukaryotic. Escherichia coli (E. coli) is a prokaryotic host that can be used to clone and express the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli (such as Bacillus subtilis) and other enterobacteriaceae (such as Salmonella, Serratia and various Pseudomonas species). In these prokaryotic hosts, expression vectors can also be prepared, which usually contain expression control sequences (e.g., replication origins) compatible with the host cells. In addition, there will be any number of various well-known promoters, such as lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems, or promoter systems from bacteriophage lambda. The promoter is usually optionally expressed with an operator sequence and has a ribosome binding site sequence, etc., for initiating and completing transcription and translation. Other microorganisms (such as yeast) can also be used to express anti-OX40 polypeptides. A combination of insect cells and baculovirus vectors can also be used.

[0154] In other aspects, mammalian host cells are used to express and produce the anti-OX40 polypeptides of the present disclosure. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines with exogenous expression vectors. These mammalian cell lines include any normally lethal or normal or abnormal immortal animal or human cells. For example, many suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell culture for expressing polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Publishers, New York City, New York, 1987. The expression vector of the mammalian host cell can include expression control sequences, such as replication origins, promoters and enhancers (see, for example, Queen et al., Immunol. Rev. 89: 49-68, 1986), as well as necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific and / or regulatable or adjustable. Useful promoters include but are not limited to metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline-inducible CMV promoters (such as human immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

[0155] Testing and diagnostic methods

[0156] The antibodies or antigen-binding fragments of the present disclosure can be used in a variety of applications, including but not limited to methods for detecting OX40. In one aspect, the antibodies or antigen-binding fragments can be used to detect the presence of OX40 in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In certain aspects, the biological sample includes cells or tissues. In other aspects, such tissues include normal and / or cancerous tissues that express OX40 at higher levels relative to other tissues.

[0157] In one aspect, the disclosure provides methods for detecting the presence of OX40 in a biological sample. In certain aspects, the method comprises contacting the biological sample with an anti-OX40 antibody under conditions that allow binding of the antibody to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample may include, but is not limited to, a urine or blood sample.

[0158] Also included are methods for diagnosing disorders associated with the expression of OX40. In certain aspects, the method comprises contacting a test cell with an anti-OX40 antibody; determining the expression level of OX40 in the test cell (quantitatively or qualitatively) by detecting the binding of the anti-OX40 antibody to the OX40 polypeptide; and comparing the expression level in the test cell with the expression level of OX40 in a control cell (e.g., a normal cell of the same tissue origin as the test cell or non-OX40 expressing cell), wherein a higher expression level of OX40 in the test cell compared to the control cell indicates the presence of a disorder associated with the expression of OX40.

[0159] Treatment

[0160] The antibodies or antigen-binding fragments of the present disclosure can be used in a variety of applications, including but not limited to methods of treating OX40-related disorders or diseases. In one aspect, the OX40-related disorder or disease is cancer.

[0161] In one aspect, the disclosure provides methods for treating cancer. In certain aspects, the methods comprise administering an effective amount of an anti-OX40 antibody or antigen-binding fragment to a patient in need thereof. Cancer may include, but is not limited to, breast cancer, head and neck cancer, gastric cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, and sarcoma.

[0162] Antibodies or antigen-binding fragments of the present invention can be given by any suitable means, including parenteral, intrapulmonary and intranasal, and if local treatment is required, can be intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, which depends in part on whether administration is short-lived or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations, push injections and pulse infusions at different time points.

[0163] The antibody or antigen-binding fragment of the present invention will be formulated, administered and given in a manner consistent with good medical practice. Factors considered in this context include the specific disorder treated, the specific mammal treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the medicament, the method of administration, the administration schedule, and other factors known to the practitioner. Antibodies are not necessary, but are optionally formulated with one or more medicaments currently used to prevent or treat the disorder in question. The effective amount of such other medicaments depends on the amount of the antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These medicaments are usually used with the same dosage and route of administration as described herein, or with about 1% to 99% of the dosage described herein, or with any dosage and any route determined to be appropriate based on experience / clinically.

[0164] For the prevention or treatment of disease, the appropriate dosage of the antibody or antigen-binding fragment of the present invention will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prevention or treatment purposes, previous therapy, the patient's clinical history and reaction to the antibody, and the judgment of the attending physician. The antibody is suitable for administration to the patient once or in a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 100 mg / kg can be an initial candidate dose for administration to the patient, whether, for example, by one or more separate administrations or by continuous infusion. Depending on the above factors, a typical daily dose range can be about 1 μg / kg to 100 mg / kg or more. For repeated administration for several days or longer, depending on the condition, treatment is usually continued until the desired disease symptoms are suppressed. Such dosages can be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives about two to about twenty or, for example, about six doses of the antibody). An initial higher loading dose can be given, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of this therapy can be easily monitored by conventional techniques and assays.

[0165] Combination therapy

[0166] In one aspect, the OX40 antibodies of the present disclosure may be used in combination with other therapeutic agents. Other therapeutic agents that may be used with the OX40 antibodies of the present disclosure include, but are not limited to, chemotherapeutic agents (e.g., paclitaxel or a paclitaxel agent; (e.g., ), docetaxel; carboplatin; topotecan; cisplatin; irinotecan, doxorubicin, lenalidomide, 5-azacytidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib), multikinase inhibitors (e.g., MGCD265, RGB-286638), CD-20 targeted agents (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeted agents (e.g., alemtuzumab), prednisolone, daclizumab Potitin alfa, lenalidomide, Bcl-2 inhibitors (e.g., oblimersen sodium), Aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD-19 targeted agents (e.g., MEDI-551, MOR208), MEK inhibitors (e.g., ABT-348), JAK-2 inhibitors (e.g., INCB018424), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR / ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), HGF / SF inhibitors (e.g., AMG 102), EGEN-001, Polo-like kinase 1 inhibitors (e.g., BI 672).

[0167] Pharmaceutical compositions and formulations

[0168] Compositions (including pharmaceutical formulations) comprising anti-OX40 antibodies or antigen-binding fragments or polynucleotides containing sequences encoding anti-OX40 antibodies or antigen-binding fragments are also provided. In certain embodiments, the compositions comprise one or more antibodies or antigen-binding fragments that bind to OX40, or one or more polynucleotides containing sequences encoding one or more antibodies or antigen-binding fragments that bind to OX40. These compositions may also comprise suitable carriers well known in the art, such as pharmaceutically acceptable excipients, including buffers.

[0169] Pharmaceutical formulations of OX40 antibodies or antigen-binding fragments as described herein are prepared by mixing such antibodies or antigen-binding fragments having the desired degree of purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Pat. Nos. 7,871,607 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0170] Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0171] Sustained release preparations can be prepared. Suitable forms of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg films, or microcapsules.

[0172] Formulations for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.

[0173] Example

[0174] Example 1: Generation of anti-OX40 monoclonal antibodies

[0175] Anti-OX40 monoclonal antibodies were generated based on conventional hybridoma fusion technology (de StGroth and Sheidegger, 1980 J Immunol Methods 35:1; Mechetner, 2007 Methods Mol Biol 378:1) with slight modifications. Antibodies with high binding activity in enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) assays were selected for further characterization.

[0176] OX40 recombinant protein for immunoassays and binding assays

[0177] The cDNA encoding full-length human OX40 (SEQ ID NO: 1) was synthesized by Sino Biological (Beijing, China) based on the GenBank sequence (Accession No.: X75962.1). The coding region of the signal peptide and the extracellular domain (ECD) consisting of amino acids (AA) 1-216 of OX-40 (SEQ ID NO: 2) was PCR amplified and cloned into an in-house developed expression vector, in which the C-terminus was fused to the Fc domain of mouse IgG2a, the Fc domain of human IgG1 wild-type heavy chain, or a His tag, which produced three recombinant fusion protein expression plasmids OX40-mIgG2a, OX40-huIgG1, and OX40-His, respectively. The schematic diagram of the OX40 fusion protein is shown in Figure 1 In. For the production of recombinant fusion proteins, OX40-mIgG2a, OX40-huIgG1 and OX40-His expression plasmids were transiently transfected into 293G cells and cultured in a CO2 incubator equipped with a rotary shaker for 7 days. The supernatant containing the recombinant protein was collected and clarified by centrifugation. OX40-mIgG2a and OX40-huIgG1 were purified using a protein A column (catalog number: 17-5438-02, GE Life Sciences). OX40-His was purified using a Ni agarose column (catalog number: 17-5318-02, GE LifeScience). OX40-mIgG2a, OX40-huIgG and OX40-His proteins were dialyzed with phosphate buffered saline (PBS) and stored in a -80°C refrigerator in small aliquots.

[0178] Stable expression cell lines

[0179] To generate stable cell lines expressing full-length human OX40 (OX40) or cynomolgus monkey OX40 (cynoOX40), these genes were cloned into the retroviral vector pFB-Neo (Cat. No. 217561, Agilent, USA). Retroviral transduction was performed based on a previously described protocol (Zhang et al., 2005). HuT78 and HEK293 cells were retrovirally transduced with viruses containing human OX40 or cynoOX40, respectively, to generate HuT78 / OX40, HEK293 / OX40, and HuT78 / cynoOX40 cell lines.

[0180] Immunization, hybridoma fusion and cloning

[0181] Eight to twelve week old Balb / c mice (from HFKBIOSCIENCE CO., LTD, Beijing, China) were immunized intraperitoneally with 200 μL antigen mixture containing 10 μg OX40-mIgG2a and Quick-Antibody immune adjuvant (Catalog No.: KX0210041, KangBiQuan, Beijing, China). The process was repeated within three weeks. Two weeks after the second immunization, the OX40 binding of mouse serum was evaluated by ELISA and FACS. Ten days after serum screening, mice with the highest anti-OX40 antibody serum titer were boosted via intraperitoneal injection of 10 μg OX40-mIgG2a. Three days after boosting immunization, spleen cells were separated and fused with mouse myeloma cell line SP2 / 0 cells (ATCC, Manassas, Virginia) using standard techniques (Somat Cell Genet, 1977 3:231).

[0182] Evaluation of OX40 binding activity of antibodies by ELISA and FACS

[0183] The supernatants of hybridoma clones were initially screened by ELISA with some modifications as described in (Methods in Molecular Biology (2007) 378: 33-52). Briefly, OX40-His protein was coated overnight at 4°C in 96-well plates. After washing with PBS / 0.05% Tween-20, the plates were blocked with PBS / 3% BSA for 2 hours at room temperature. Subsequently, the plates were washed with PBS / 0.05% Tween-20 and incubated with cell supernatants for 1 hour at room temperature. HRP-linked anti-mouse IgG antibody (Cat. No. 115035-008, Jackson ImmunoResearch Inc, peroxidase AffiniPure goat anti-mouse IgG, Fcγ fragment specific) and substrate (Cat. No. 00-4201-56, eBioscience, USA) were used to visualize the colored absorbance signal at a wavelength of 450 nm, which was measured by using a plate reader (SpectraMax Paradigm, Molecular Devices / PHERAstar, BMG LABTECH). Positive parental clones were selected from the fusion screening by indirect ELISA. ELISA positive clones were further verified by FACS using the above-mentioned HuT78 / OX40 and HuT78 / cynoOX40 cells. Cells expressing OX40 (10 5 cells / well) were incubated with ELISA-positive hybridoma supernatants and then with anti-mouse IgG The cells were then bound to the 660 antibody (Catalog No. 50-4010-82, eBioscience, USA). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte 8HT, Merck-Millipore, USA).

[0184] Functional assays were performed on conditioned media from hybridomas that showed positive signals in both ELISA and FACS screening to identify antibodies with good functional activity in human immune cell-based assays (see the following section). Antibodies with the desired functional activity were further subcloned and characterized.

[0185] Subcloning of hybridomas and adaptation to serum-free or low-serum media

[0186] After initial screening by ELISA, FACS and functional assays as described above, positive hybridoma clones were subcloned by limiting dilution to ensure clonality. The best antibody subclones were verified by functional assays and adapted for growth in CDM4 MAb medium (Catalog No.: SH30801.02, Hyclone, USA) containing 3% FBS.

[0187] Monoclonal antibody expression and purification

[0188] Hybridoma cells expressing the best antibody clones were cultured in CDM4 MAb medium (Catalog No.: SH30801.02, Hyclone) and incubated in a CO2 incubator at 37°C for 5 to 7 days. The conditioned medium was collected by centrifugation and filtered through a 0.22 μm membrane before purification. The mouse antibodies in the supernatant were applied to and bound to a protein A column (Catalog No.: 17-5438-02, GE Life Sciences) according to the manufacturer's instructions. The procedure typically produces antibodies with a purity greater than 90%. Protein A affinity-purified antibodies were dialyzed with PBS or, if necessary, further purified using a HiLoad 16 / 60 Superdex200 column (Catalog No.: 28-9893-35, GE Life Sciences) to remove aggregates. The protein concentration was determined by measuring the absorbance at 280 nm. The final antibody preparation was stored in aliquots in a -80°C refrigerator.

[0189] Example 2: Cloning and sequence analysis of anti-OX40 antibodies

[0190] Harvest mouse hybridoma clones, using Ultrapure RNA kit (catalog number: 74104, QIAGEN, Germany) to prepare total cell RNA based on the manufacturer's protocol. Use the cDNA synthesis kit (catalog number: 18080-051) from Invitrogen to synthesize the first chain cDNA, and use PCR kit (catalog number: CW0686, CWBio, Beijing, China) to carry out PCR amplification of VH and VL of hybridoma antibody. Oligonucleotide primers for antibody cDNA cloning of heavy chain variable region (VH) and light chain variable region (VL) are synthesized by Invitrogen (Beijing, China) based on previously reported sequences (Brocks et al. 2001MolMed 7: 461). The PCR product is directly used for sequencing or subcloned into pEASY-Blunt cloning vector (catalog number: CB101 TransGen, China), and then sequenced by Genewiz (Beijing, China). The amino acid sequences of VH and VL regions are derived from the DNA sequencing results.

[0191] Based on the Kabat (Wu and Kabat 1970 J. Exp. Med. 132: 211-250) system, the complementary determining regions (CDRs) of murine antibodies were defined by sequence annotation and by sequence analysis using computer programs. The amino acid sequences of the representative best clone Mu445 (VH and VL) are listed in Table 1 (SEQ ID NOs. 9 and 11). The CDR sequences of Mu445 are listed in Table 2 (SEQ ID NOs. 3-8).

[0192] Table 1. Amino acid sequences of Mu445 VH and VL regions

[0193]

[0194] Table 2. CDR sequences (amino acids) of the VH and VL regions of mouse monoclonal antibody Mu445

[0195]

[0196]

[0197] Example 3: Humanization of mouse anti-human OX40 antibody 445

[0198] Antibody Humanization and Engineering

[0199] For the humanization of Mu445, sequences with high homology to the cDNA sequence of the Mu445 variable region were searched in the human germline IgG gene by sequence comparison against the human immunoglobulin gene database in IMGT. Human IGHV and IGKV genes that are present at high frequencies in the human antibody library (Glanville et al., 2009 PNAS 106: 20216-20221) and are highly homologous to Mu445 were selected as templates for humanization.

[0200] Humanization (Methods in Molecular Biology, Antibody Engineering, Methods and Protocols, Vol. 248: Humana Press) was performed by CDR transplantation, and the humanized antibody was engineered into a human IgG1 wild-type form by using an expression vector developed internally. In the first round of humanization, the sudden change from mouse to human amino acid residues in the framework region was guided by simulated 3D structural analysis, and the mouse framework residues with structural importance for maintaining the typical structure of CDR were retained in the first form of humanized antibody 445 (see 445-1, Table 3). The six CDRs of 445-1 have HCDR1 (SEQ ID NO: 3), HCDR2 (SEQ ID NO: 13), HCDR3 (SEQ ID NO: 5) and LCDR1 (SEQ ID NO: 6), LCDR2 (SEQ ID NO: 7) and LCDR3 (SEQ ID NO: 8) amino acid sequences. The heavy chain variable region of Mu445-1 has an amino acid sequence of (VH) SEQ ID NO: 14 encoded by a nucleotide sequence of SEQ ID NO: 15, and the light chain variable region has an amino acid sequence of (VL) SEQ ID NO: 16 encoded by a nucleotide sequence of SEQ ID NO: 17. Specifically, the LCDRs (SEQ ID NOs: 6-8) of Mu445 were transplanted into the framework of the human germline variable gene IGVK1-39, wherein two mouse framework residues (I 44 and Y 71 ) (SEQ ID NO: 16). HCDR1 (SEQ ID NO: 3), HCDR2 (SEQ ID NO: 13) and HCDR3 (SEQ ID NO: 5) were transplanted into the framework of the human germline variable gene IGHV1-69, retaining two mouse framework residues (L 70 and S 72 )(SEQ ID NO: 14). In the 445 humanized variant (445-1), only the N-terminal half of the Kabat HCDR2 was grafted, because based on the modeled 3D structure, only the N-terminal half was predicted to be important for antigen binding.

[0201] 445-1 was constructed into a humanized full-length antibody using an expression vector developed in-house containing the constant regions of human wild-type IgG1 (IgG1wt) and κ chains and a subcloning site that was easily adapted. The 445-1 antibody was expressed by co-transfecting the above two constructs into 293G cells and purified using a protein A column (Catalog No.: 17-5438-02, GE Life Sciences). The purified antibody was concentrated to 0.5-10 mg / mL in PBS and stored in aliquots at -80°C.

[0202] Using the 445-1 antibody, several single amino acid changes were made to convert the mouse residues retained in the framework regions of VH and VL to the corresponding human germline residues, such as I44P and Y71F in VL and L70I and S72A in VH. In addition, several single amino acid changes were made in the CDRs to reduce the potential risk of isomerization and to improve the level of humanization. For example, changes of T51A and D50E were made in LCDR2, and changes of D56E, G57A and N61A were made in HCDR2. All humanization changes were made using primers containing mutations at specific positions and a site-directed mutagenesis kit (Catalog No.: AP231-11, TransGen, Beijing, China). The desired changes were verified by sequencing.

[0203] The amino acid changes in the 445-1 antibody were evaluated for its binding to OX40 and thermal stability. Antibody 445-2 (see Table 3), comprising HCDR1 of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 18, HCDR3 of SEQ ID NO: 5, LCDR1 of SEQ ID NO: 6, LCDR2 of SEQ ID NO: 19, and LCDR3 of SEQ ID NO: 8, was constructed from the combination of the specific changes described above. When comparing the two antibodies, the results showed that both antibodies 445-2 and 445-1 exhibited comparable binding affinity (see Tables 4 and 5 below).

[0204] Starting from the 445-2 antibody, several additional amino acid changes were made in the framework region of VL to further improve binding affinity / kinetics, such as changes to amino acids G41D and K42G. In addition, several single amino acid changes were made in the CDRs of both VH and VL to reduce the risk of immunogenicity and improve thermal stability, such as S24R in LCDR1 and A61N in HCDR2. The resulting changes showed improved binding activity or thermal stability compared to 445-2.

[0205] The humanized 445 antibody was further engineered by introducing specific amino acid changes in the CDR and framework regions to improve the molecular and biophysical properties for human therapeutic use. Considerations included removal of deleterious post-translational modifications, improved thermal stability (T m ), surface hydrophobicity and isoelectronic point (pI), while maintaining binding activity.

[0206] The humanized monoclonal antibody 445-3 (see Table 3) comprising HCDR1 of SEQ ID NO:3, HCDR2 of SEQ ID NO:24, HCDR 3 of SEQ ID NO:5, LCDR1 of SEQ ID NO:25, LCDR2 of SEQ ID NO:19 and LCDR3 of SEQ ID NO:8 was constructed by the above-mentioned maturation process and characterized in detail. Antibody 445-3 was also made into an IgG2 form (445-3IgG2) comprising the Fc domain of the wild-type heavy chain of human IgG2, and an IgG4 form (445-3IgG4) comprising the Fc domain of human IgG4 with S228P and R409K mutations. The results showed that 445-3 and 445-2 exhibited comparable binding affinities (see Tables 4 and 5).

[0207] Table 3.445 Antibody Sequences

[0208]

[0209]

[0210]

[0211] Example 4: Binding kinetics and affinity determination of anti-OX40 antibodies by SPR

[0212] Using BIAcore TMT-200 (GE Life Sciences) was used to characterize the binding kinetics and affinity of anti-OX40 antibodies by SPR assay. In short, anti-human IgG antibodies were immobilized on activated CM5 biosensor chips (Catalog No.: BR100530, GE Life Sciences). Antibodies with human IgG Fc regions were flowed over the chip surface and captured by anti-human IgG antibodies. Serial dilutions of recombinant OX40 proteins with His tags (Catalog No.: 10481-H08H, SinoBiological) were then flowed over the chip surface, and changes in the surface plasmon resonance signal were analyzed to calculate the association rate (ka) and dissociation rate (kd) by using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). The equilibrium dissociation constant (K D ) was calculated as the ratio kd / ka. The results of the SPR assay for the binding profiles of anti-OX40 antibodies are summarized in Figure 2 and Table 4. The average K with antibody 445-3 (9.47 nM) D The binding profile of ch445-3 was slightly better than antibodies 445-2 (13.5 nM) and 445-1 (17.1 nM), and similar to ch445. The binding profile of 445-3 IgG4 was similar to 445-3 (with IgG1 Fc), indicating that the change in Fc between IgG4 and IgG1 does not alter the specific binding of the 445-3 antibody.

[0213] Table 4. Binding affinity of anti-OX40 antibodies obtained by SPR

[0214]

[0215] *ch445 consists of the Mu445 variable domain fused to the human IgG1wt / κ constant region

[0216] Example 5: Determination of the binding affinity of anti-OX40 antibodies to OX40 expressed on HuT78 cells

[0217] To evaluate the binding activity of anti-OX40 antibodies to OX40 expressed on the surface of live cells, HuT78 cells were transfected with human OX40 as described in Example 1 to generate a line expressing OX40. Live HuT78 / OX40 cells were seeded in 96-well plates and incubated with serial dilutions of various anti-OX40 antibodies. Goat anti-human IgG-FITC (Catalog No.: A0556, Beyotime) was used as a secondary antibody to detect antibody binding to the cell surface. The EC values ​​for dose-dependent binding to human OX40 were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism.50 Value. Figure 3 As shown in Table 5, the OX40 antibody has a high affinity for OX40. The OX40 antibody of the present disclosure was also found to have a relatively high maximum fluorescence intensity level measured by flow cytometry (see the last column of Table 5), indicating that the antibody dissociates slowly from OX40, which is a more ideal binding profile.

[0218] Table 5. EC values ​​for dose-dependent binding of humanized 445 variants to OX40 50

[0219]

[0220] Example 6: Determination of cross-reactivity of anti-OX40 antibodies

[0221] To evaluate the cross-reactivity of antibody 445-3 with human and cynomolgus monkey (cyno) OX40, cells expressing human OX40 (HuT78 / OX40) and cyno OX40 (HuT78 / cynoOX40) were seeded in 96-well plates and incubated with a dilution series of OX40 antibodies. Goat anti-human IgG-FITC (Catalog No.: A0556, Beyotime) was used as a secondary antibody for detection. The EC values ​​for dose-dependent binding to human and cynomolgus monkey native OX40 were determined by fitting the dose-response data to a four-parameter logistic model using GraphPad Prism. 50 The results are shown below Figure 4 and Table 6. Antibody 445-3 binds to both human and cynomolgus monkey OX40 with similar EC values ​​as shown below 50 Value cross-reaction.

[0222] Table 6. EC binding of antibody 445-3 to human and cynomolgus monkey OX40 50

[0223]

[0224]

[0225] Example 7: Co-crystallization and structure determination of OX40 and 445-3 Fab

[0226] In order to understand the binding mechanism of OX40 to the antibodies of the present disclosure, the co-crystal structure of the Fab of OX40 and 445-3 was resolved. Mutations were introduced at residues T148 and N160 to block the glycosylation of OX40 and improve the homogeneity of the protein. The DNA encoding the mutant human OX40 (residues M1-D170, with two mutation sites T148A and N160A) was cloned into an expression vector containing a six-His tag, and the construct was transiently transfected into 293G cells at 37°C for protein expression for 7 days. The cells were harvested, the supernatant was collected and incubated with the His tag affinity resin at 4°C for 1 hour. The resin was washed three times with a buffer containing 20mMTris (pH 8.0), 300mM NaCl and 30mM imidazole. The OX40 protein was then eluted with a buffer containing 20 mM Tris (pH 8.0), 300 mM NaCl, and 250 mM imidazole, and then further purified using Superdex 200 (GE Healthcare) in a buffer containing 20 mM Tris (pH 8.0), 100 mM NaCl.

[0227] The coding sequences of the heavy and light chains of 445-3Fab were cloned into expression vectors containing a hexa-His tag at the C-terminus of the heavy chain, and these expression vectors were transiently co-transfected into 293G cells for protein expression at 37°C for 7 days. The purification steps of 445-3Fab were the same as those used for the mutant OX40 proteins described above.

[0228] Purified OX40 and 445-3 Fab were mixed at a 1:1 molar ratio and incubated on ice for 30 minutes, and then further purified using Superdex 200 (GE Healthcare) in a buffer containing 20 mM Tris (pH 8.0), 100 mM NaCl. The complex peak was collected and concentrated to approximately 30 mg / ml.

[0229] Co-crystal screening was performed by mixing the protein complex with a reservoir solution at a volume ratio of 1: 1. Co-crystals were obtained from hanging drops incubated by vapor diffusion at 20°C with a reservoir solution containing 0.1 M HEPES (pH 7.0), 1% PEG 2,000 MME and 0.95 M sodium succinate.

[0230] Co-crystals were harvested using a nylon loop and immersed in a reservoir solution supplemented with 20% glycerol for 10 seconds. Diffraction data were collected on the BL17U1 of the Shanghai Synchrotron Radiation Facility and processed with the XDS program. The structure of IgG Fab (chains C and D of the PDB: 5CZX) and the structure of OX40 (chain R of the PDB: 2HEV) were used as molecular replacement search models and the phases were analyzed using the program PHASER. The Phenix.refine graphical interface was used to perform rigid body, TLS and restricted refinement for X-ray data, then adjusted with the COOT program and further refined in the Phenix.refine program. Table 7 summarizes X-ray data collection and refinement statistics.

[0231] Table 7. Data collection and refinement statistics

[0232]

[0233] The values ​​in brackets refer to the highest resolution slot (shell).

[0234] a R 合并 =∑∑ i |I(h) i -<I(h)> | / ∑∑ i |I(h) i |, where<I(h)> is the average intensity of the equivalents.

[0235] b R 工作 =∑|Fo-Fc| / ∑|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.

[0236] c R 自由 =∑|Fo-Fc| / ∑|Fo|, calculated using a test data set (randomly selected from 5% of the total data of observed reflections).

[0237] Example 8: Epitope Identification of Antibody 445-3 by SPR

[0238] Guided by the co-crystal structure of OX40 and antibody 445-3Fab, we selected and generated a series of single mutations in the human OX40 protein to further identify the key epitopes of the anti-OX40 antibodies of the present disclosure. Single-point mutations were performed on the human OX40 / IgG1 fusion construct using a site-directed mutagenesis kit (Catalog No.: AP231-11, TransGen). The desired mutations were verified by sequencing. Expression and preparation of OX40 mutants were achieved by transfection into 293G cells and purified using a protein A column (Catalog No.: 17-5438-02, GE Life Sciences).

[0239] The binding affinity of OX40 point mutants to 445-3Fab was characterized by SPR assay using BIAcore 8K (GE Life Sciences). In brief, OX40 mutants and wild-type OX40 were immobilized on a CM5 biosensor chip (Catalog Number: BR100530, GE Life Sciences) using EDC and NHS. Serial dilutions of 445-3Fab in HBS-EP+ buffer (Catalog Number: BR-1008-26, GE Life Sciences) were then flowed over the chip surface at 30 μl / min using a contact time of 180 s and a dissociation time of 600 s. The changes in the surface plasmon resonance signal were analyzed using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences) to calculate the association rate (ka) and dissociation rate (kd). The equilibrium dissociation constant (K D ) was calculated as the ratio kd / ka. The K of the mutant D The shift fold was calculated as the ratio mutant K D / WT K D An overview of epitope identification by SPR assay is summarized in Figure 5 and Table 8. The results indicated that mutation of residues H153, I165 and E167 in OX40 to alanine significantly reduced the binding of antibody 445-3 to OX40, and mutation of residues T154 and D170 to alanine moderately reduced the binding of antibody 445-3 to OX40.

[0240] The detailed interactions of antibody 445-3 with residues H153, T154, I165, E167, and D170 of OX40 are shown in Figure 6 The side chain of H153 on OX40 is surrounded by a small pocket of 445-3 on the interaction interface, thus 重 S31 and 重 G102 forms hydrogen bonds with 重 Y101 forms a π-π stack. The side chain of E167重 Y50 and 重 N52 forms a hydrogen bond, while D170 forms a 重 S31 and 重 K28 forms hydrogen bonds and salt bridges, which can further stabilize the complex. 重 Y105, I165 and 重 The van der Waals (VDW) interaction between R59 and 445-3 contributes to the high affinity of antibody 445-3 for OX40.

[0241] In summary, residues H153, I165, and E167 of OX40 were identified as important residues for interaction with antibody 445-3. In addition, amino acids T154 and D170 of OX40 were also important contact residues for antibody 445-3. This data indicates that the epitopes of antibody 445-3 are residues H153, T154, I165, E167, and D170 of OX40. These epitopes are present in the sequence The important contact residues are indicated in bold and underlined.

[0242] Table 8. Epitope identification of antibody 445-3 by SPR assay

[0243] Mutants <![CDATA[Mutant K D / WT K D > H153A No binding detected T154A 8 Q156A 1.9 S161A 1.1 S162A 0.6 I165A 28 E167A 135 D170A 8

[0244] Significant effect: No binding detected, or mutant K D / WT K D The value is greater than 10. Moderate effect: mutant K D / WTK D The value is between 5 and 10. Non-significant effect: mutant K D / WT K D The value of is less than 5.

[0245] Example 9: Anti-OX40 antibody 445-3 does not block OX40-OX40L interaction.

[0246] To determine whether antibody 445-3 interferes with OX40-OX40L interaction, a cell-based flow cytometry assay was established. In this assay, antibody 445-3, reference antibody 1A7.gr1, control huIgG, or culture medium alone were pre-incubated with a fusion protein of human OX40 and mouse IgG2a Fc (OX40-mIgG2a). The antibody and fusion protein complex were then added to HEK293 cells expressing OX40L. If the OX40 antibody does not interfere with the OX40-OX40L interaction, the OX40 antibody-OX40 mIgG2a complex will still bind to surface OX40L, and this interaction can be detected using an anti-mouse Fc secondary antibody.

[0247] like Figure 7 As shown, antibody 445-3 does not reduce the binding of OX40 to OX40L even at high concentrations, indicating that 445-3 does not interfere with the OX40-OX40L interaction. This indicates that 445-3 does not bind at the OX40L binding site or does not bind close enough to sterically hinder OX40L binding. In contrast, the positive control antibody 1A7.gr1 completely blocked the binding of OX40 to OX40L, as shown in Figure 7 shown.

[0248] In addition, the co-crystal structure of OX40 and 445-3Fab in complex was solved and aligned with the OX40 / OX40L complex (PDB code: 2HEV), as shown in Figure 8 As shown. The OX40 ligand trimer interacts with OX40 mainly through the CRD1 (cysteine-rich domain), CRD2 and part of the CRD3 region of OX40 (Compaan and Hymowitz, 2006), while the antibody 445-3 interacts with OX40 only through the CRD4 region. In short, the 445-3 antibody and the OX40L trimer bind to different regions of OX40 and the antibody 445-3 does not interfere with the OX40 / OX40L interaction. This result is related to the epitope mapping data described in the above examples. The CRD4 of OX40 is located at amino acids 127-167, and the epitope of antibody 445-3 partially overlaps with this region. The sequence of OX40 CRD4 (amino acids 127-167) is shown below, and the partial overlap of the 445-3 epitope is indicated by bold and underline:

[0249]

[0250] Example 10: Agonist activity of anti-OX40 antibody 445-3

[0251] To investigate the agonistic function of antibody 445-3, the OX40-positive T cell line HuT78 / OX40 was combined with an artificial antigen presenting cell (APC) line (HEK293 / OS8 低 -FcγRI) were co-cultured overnight in the presence or absence of 445-3 or 1A7.gr1, and IL-2 production was used as a readout for T cell stimulation. 低 In HEK293 cells, genes encoding membrane-bound anti-CD3 antibody OKT3 (OS8) (as disclosed in U.S. Pat. No. 8,735,553) and human FcγRI (CD64) were stably co-transduced into HEK293 cells. Since immune activation induced by anti-OX40 antibodies depends on antibody cross-linking (Voo et al., 2013), HEK293 / OS8 低-FcγRI on FcγRI provides the basis for the cross-linking of OX40 mediated by the anti-OX40 antibody after dual engagement of the anti-OX40 antibody with both OX40 and FcγRI. As shown in FIG9 , the anti-OX40 antibody 445-3 inhibited the cross-linking of OX40 in a dose-dependent manner at an EC of 0.06 ng / ml. 50 Highly effective in enhancing TCR signaling. Slightly weaker activity was also observed for the reference Ab 1A7.gr1. In contrast, control human IgG (10 μg / mL) or blank showed no effect on IL-2 production.

[0252] Example 11: Anti-OX40 antibody 445-3 promotes immune response in mixed lymphocyte reaction (MLR) assay

[0253] To determine whether antibody 445-3 can stimulate T cell activation, a mixed lymphocyte reaction (MLR) assay was established as previously described (Tourkova et al., 2001). Briefly, CD14 T cells derived from human PBMCs were cultured with GM-CSF and IL-4 followed by LPS stimulation. + Next, DCs treated with mitomycin C were incubated with allogeneic CD4 + T cells were co-cultured for 2 days. IL-2 production in the co-cultures was measured by ELISA as a readout of the MLR response.

[0254] like Fig.10 As shown, antibody 445-3 significantly promoted IL-2 production, indicating that 445-3 activated CD4 + In contrast, the reference antibody 1A7.gr1 showed significantly (P < 0.05) weaker activity in the MLR assay.

[0255] Example 12: Anti-OX40 antibody 445-3 exhibits ADCC activity

[0256] An ADCC assay based on lactate dehydrogenase (LDH) release was established to investigate whether antibody 445-3 could kill cells expressing OX40. Hi The NK92MI / CD16V cell line was generated as effector cells by co-transducing CD16v158 (V158 allele) and FcRγ genes into the NK cell line NK92MI (ATCC, Manassas, VA). The T cell line HuT78 / OX40 expressing OX40 was used as target cells. The same number (3x10 4) target cells and effector cells were co-cultured for 5 hours in the presence of anti-OX40 antibody (0.004-3 μg / ml) or control Ab. Cytotoxicity was evaluated by LDH release using the CytoTox 96 non-radioactive cytotoxicity assay kit (Promega, Madison, Wisconsin). Specific lysis was calculated by the formula shown below.

[0257]

[0258] like Fig.11 As shown, antibody 445-3 showed a dose-dependent effect (EC 50 : 0.027 μg / mL) kills OX40 via ADCC Hi The ADCC effect of antibody 445-3 was similar to that of the 1A7.grl control antibody. In contrast, 445-3 in the IgG4 Fc format with S228P and R409K mutations (445-3-IgG4) did not show any significant ADCC effect compared to control human IgG or blank. The results are consistent with previous findings that ADCC of IgG4 Fc is weak or silent (An Z et al. mAbs 2009).

[0259] Example 13: Anti-OX40 antibody 445-3 preferentially depletes CD4 in vitro + Treg and increase CD8 + Teff / Treg ratio

[0260] It has been shown in several animal tumor models that anti-OX40 antibodies can deplete tumor-infiltrating OX40 Hi Treg and increase CD8 + The ratio of T cells to Tregs is increased (Bulliard et al., 2014; Carboni et al., 2003; Jacquemin et al., 2015; Marabelle et al., 2013b). As a result, the immune response is enhanced, leading to tumor regression and prolonged survival.

[0261] Considering in vitro activated or intratumoral CD4 + Foxp3 + Given the fact that Tregs preferentially express OX40 over other T cell subsets (Lai et al., 2016; Marabelle et al., 2013b; Montler et al., 2016; Soroosh et al., 2007; Timperi et al., 2016), a human PBMC-based assay was established to study the killing of OX40 by antibody 445-3. HiIn brief, PBMCs were pre-activated for 1 day by PHA-L (1 μg / mL) to induce OX40 expression and used as target cells. Then effector NK92MI / CD16V cells (5×10 4 ) were co-cultured with an equal number of target cells in the presence of anti-OX40 antibody (0.001-10 μg / mL) or placebo overnight. The percentage of each T cell subset was determined by flow cytometry. As shown in Figures 12A and 12B, treatment with antibody 445-3 induced CD8 + Increased T cell percentage and CD4 + Foxp3 + The decrease in the percentage of Tregs. + The ratio of T cells to Tregs was greatly improved (Figure 12C). Weaker results were obtained with 1A7.gr1 treatment. This result demonstrates that 445-3 enhances CD8 + T cell function induces anti-tumor immunity but limits the therapeutic application of Treg-mediated immune tolerance.

[0262] Example 14: Anti-OX40 antibody 445-3 exerts dose-dependent anti-tumor activity in a mouse tumor model

[0263] The efficacy of the anti-OX40 antibody 445-3 was shown in a mouse tumor model. Murine MC38 colon tumor cells were subcutaneously implanted into C57 mice transgenic for human OX40 (Biocytogen, Beijing, China). After implantation of tumor cells, tumor volume was measured twice a week and expressed in mm using the following formula: 3 Calculate as a unit: V = 0.5 (axb 2 ), where a and b are the long and short diameters of the tumor, respectively. When the tumor size reaches approximately 190 mm 3 When the mean volume of 1A7.gr1 was 0.5, the mice were randomly divided into 7 groups and injected intraperitoneally with 445-3 or 1A7.gr1 antibody once a week for three weeks. Human IgG was given as an isotype control. Partial regression (PR) was defined as a tumor volume less than 50% of the starting tumor volume on the first day of dosing in three consecutive measurements. Tumor growth inhibition (TGI) was calculated using the following formula:

[0264]

[0265] Treatment t = tumor volume treated at time t

[0266] Treatment t0 = tumor volume treated at time 0

[0267] Placebo t = tumor volume of placebo at time t

[0268] Placebo t0 = tumor volume of placebo at time 0

[0269] The results demonstrated that 445-3 had a dose-dependent antitumor efficacy at doses of 0.4 mg / kg, 2 mg / kg, and 10 mg / kg by intraperitoneal injection. Administration of 445-3 resulted in 53% (0.4 mg / kg), 69% (2 mg / kg), and 94% (10 mg / kg) tumor growth inhibition, and resulted in partial regressions from baseline of 0% (0.4 mg / kg), 17% (2 mg / kg), and 33% (10 mg / kg). In contrast, no partial regressions were observed with antibody 1A7.gr1. In vivo data indicate that the ligand non-blocking antibody 445-3 is more suitable for antitumor therapy than the OX40-OX40L blocking antibody 1A7.gr1 ( Fig.13A and Fig. 13B , Table 9).

[0270] Table 9. Efficacy of 445-3 and 1A7.gr1 in the murine MC38 colon tumor mouse model

[0271]

[0272] Example 15: Amino Acid Alterations of Anti-OX40 Antibodies

[0273] Several amino acid changes were selected for improvement of OX40 antibodies. Amino acid changes were made to improve affinity or increase humanization. PCR primer sets were designed for the appropriate amino acid changes, synthesized and used to modify anti-OX40 antibodies. For example, changes of K28T in the heavy chain and S24R in the light chain resulted in an EC of 1.3949 as determined by FACS. 50 The changes of Y27G in the heavy chain and S24R in the light chain resulted in a K increase of 1.7-fold compared to the original 445-2 antibody. D The increase was 1.7 times compared to the original 445-2 antibody. These changes are summarized in FIG. 14A to FIG. 14B middle.

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[0325] Zhang, T., Lemoi, BA, and Sentman, CL (2005). Chimeric NK-receptor-bearing T cells mediate antitumor immunotherapy. Blood 106, 1544-1551. Sequence Listing <110> BeiGene, Ltd. Liu, Ye Zhang, Tong Wang, Zuobai Li, Kang <120> Anti-OX40 antibodies and methods of use <130> F19W0526PCT <160> 31 <170> PatentIn Version 3.5 <210> 1 <211> 277 <212> PRT <213> Homo sapiens <400> 1 Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro 35 40 45 Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys 85 90 95 Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140 Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn 145 150 155 160 Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175 Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr 180 185 190 Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200 205 Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val 210 215 220 Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu 225 230 235 240 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 245 250 255 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 260 265 270 Thr Leu Ala Lys Ile 275 <210> 2 <211> 216 <212> PRT <213> Homo sapiens <400> 2 Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu 1 5 10 15 Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val 20 25 30 Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro 35 40 45 Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys 50 55 60 Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro 65 70 75 80 Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys 85 90 95 Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly 100 105 110 Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys 115 120 125 Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp 130 135 140 Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn 145 150 155 160 Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro 165 170 175 Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr 180 185 190 Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu 195 200 205 Val Pro Gly Gly Arg Ala Val Ala 210 215 <210> 3 <211> 5 <212> PRT <213> House mouse <400> 3 Ser Tyr Ile Ile His 1 5 <210> 4 <211> 17 <212> PRT <213> House mouse <400> 4 Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Arg Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 5 <211> 11 <212> PRT <213> House mouse <400> 5 Gly Tyr Tyr Gly Ser Ser Tyr Ala Met Asp Tyr 1 5 10 <210> 6 <211> 11 <212> PRT <213> House mouse <400> 6 Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 7 <211> 7 <212> PRT <213> House mouse <400> 7 Asp Thr Ser Thr Leu Tyr Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Mus musculus <400> 8 Gln Gln Tyr Ser Lys Leu Pro Tyr Thr 1 5 <210> 9 <211> 120 <212> PRT <213> Mus musculus <400> 9 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Ile Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Arg Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Tyr Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Gly Ser Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 10 <211> 360 <212> DNA <213> Mus musculus <400> 10 gaggtccagc tgcagcagtc tggacctgaa ctggtaaagc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggata taaattcact agctatatta tacactgggt gaagcagaag 120 cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg tactaggtac 180 aatgagaagt tcaaaggcaa ggccacactg acttcagaca aatcctccag cacagcctac 240 atggagtaca gcagcctgac ctctgaggac tctgcggtct attactgtgc aaggggttac 300 tacggtagta gctatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360 <210> 11 <211> 107 <212> PRT <213> Mus musculus <400> 11 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Ile Lys Leu Leu Ile 35 40 45 Tyr Asp Thr Ser Thr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Phe Leu Thr Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Lys Lys 100 105 <210> 12 <211> 321 <212> DNA <213> Mus musculus <400> 12 gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca gtgcaagtca gggcattagc aattatttaa actggtatca gcagaaacca 120 gatggaacta ttaaactcct gatctatgac acatcaacct tatactcagg agtcccatca 180 aggttcagtg gcagtgggtc tgggacagat tattttctca ccatcagcaa cctggaacct 240 gaagatattg ccacttacta ttgtcagcag tatagtaagc ttccgtacac gttcggaggg 300 gggaccaagc tggaaaaaaa a 321 <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 445-1 HCDR2 <400> 13 Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Arg Tyr Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 14 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 445-1 VH pro <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Gly Ser Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 15 <211> 360 <212> DNA <213> Artificial sequence <220> <223> 445-1 VH DNA <400> 15 caggtgcagc tggtgcagtc tggagcagag gtgaagaagc caggcagctc cgtgaaggtg 60 tcctgcaagg cctctggcta caagttcacc tcctatatca tccactgggt gcggcaggca 120 ccaggacagg gactggagtg gatgggctac atcaaccctt ataatgacgg cacacggtac 180 aaccagaagt ttcagggcag agtgaccctg acaagcgata agtctaccag cacagcctat 240 atggagctgt ctagcctgag gtccgaggac accgccgtgt actattgtgc cagaggctac 300 tatggctcct cttacgccat ggattattgg ggccagggca ccacagtgac agtgagctcc 360 <210> 16 <211> 107 <212> PRT <213> Artificial sequence <220> <223> 445-1 VK pro <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Ile Lys Leu Leu Ile 35 40 45 Tyr Asp Thr Ser Thr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 445-1 VK DNA <400> 17 gacatccaga tgacccagtc tcccagctcc ctgtccgcct ctgtgggcga tagggtgacc 60 atcacatgca gcgcctccca gggcatctcc aactacctga attggtatca gcagaagcca 120 ggcaaggcca tcaagctgct gatctacgac acctctacac tgtatagcgg cgtgccctcc 180 agattctctg gcagcggctc cggaaccgac tacaccctga caatctctag cctgcagccc 240 gaggatttcg ccacatacta ttgtcagcag tacagcaagc tgccttatac ctttggcggc 300 ggcacaaagg tggagatcaa g 321 <210> 18 <211> 17 <212> PRT <213> Synthetic sequence <220> <223> 445-2 HCDR2 <400> 18 Tyr Ile Asn Pro Tyr Asn Glu Gly Thr Arg Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 19 <211> 7 <212> PRT <213> Synthetic sequence <220> <223> 445-2 LCDR2 <400> 19 Asp Ala Ser Thr Leu Tyr Ser 1 5 <210> 20 <211> 120 <212> PRT <213> Synthetic sequence <220> <223> 445-2 VH pro <400> 20 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Glu Gly Thr Arg Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Gly Ser Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 21 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> 445-2 VH DNA <400> 21 caggtgcagc tggtgcagtc tggagcagag gtgaagaagc caggcagctc cgtgaaggtg 60 tcctgcaagg cctctggcta caagttcacc tcctatatca tccactgggt gcggcaggca 120 ccaggacagg gactggagtg gatgggctac atcaaccctt ataatgaggg cacacggtac 180 gcccagaagt ttcagggcag agtgaccctg acagccgata agtctaccag cacagcctat 240 atggagctgt ctagcctgag gtccgaggac accgccgtgt actattgtgc cagaggctac 300 tatggctcct cttacgccat ggattattgg ggccagggca ccacagtgac agtgagctcc 360 <210> 22 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 445-2 VK pro <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Ile Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Thr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 23 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 445-2 VK DNA <400> 23 gacatccaga tgacccagtc tcccagctcc ctgtccgcct ctgtgggcga tagggtgacc 60 atcacatgca gcgcctccca gggcatctcc aactacctga attggtatca gcagaagcca 120 ggcaaggcca tcaagctgct gatctacgac gcctctacac tgtatagcgg cgtgccctcc 180 agattctctg gcagcggctc cggaaccgac ttcaccctga caatctctag cctgcagccc 240 gaggatttcg ccacatacta ttgtcagcag tacagcaagc tgccttatac ctttggcggc 300 ggcacaaagg tggagatcaa g 321 <210> 24 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 445-3 HCDR2 <400> 24 Tyr Ile Asn Pro Tyr Asn Glu Gly Thr Arg Tyr Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 25 <211> 11 <212> PRT <213> artificial sequence <220> <223> 445-3 LCDR1 <400> 25 Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 26 <211> 120 <212> PRT <213> artificial sequence <220> <223> 445-3 VH pro <400> 26 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Lys Phe Thr Ser Tyr 20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Glu Gly Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Gly Ser Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 27 <211> 360 <212> DNA <213> Artificial sequence <220> <223> 445-3 VH DNA <400> 27 caggtgcagc tggtgcagtc tggagcagag gtgaagaagc caggcagctc cgtgaaggtg 60 tcctgcaagg cctctggcta caagttcacc tcctatatca tccactgggt gcggcaggca 120 ccaggacagg gactggagtg gatgggctac atcaaccctt ataatgaggg cacacggtac 180 aaccagaagt ttcagggcag agtgaccctg acagccgata agtctaccag cacagcctat 240 atggagctgt ctagcctgag gtccgaggac accgccgtgt actattgtgc cagaggctac 300 tatggctcct cttacgccat ggattattgg ggccagggca ccacagtgac agtgagctcc 360 <210> 28 <211> 107 <212> PRT <213> Artificial sequence <220> <223> 445-3 VK pro <400> 28 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Ala Ile Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Thr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 29 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 445-3 VK DNA <400> 29 gacatccaga tgacccagtc tcccagctcc ctgtccgcct ctgtgggcga tagggtgacc 60 atcacatgcc gggcctccca gggcatctcc aactacctga attggtatca gcagaagcca 120 gacggcgcca tcaagctgct gatctacgac gcctctacac tgtatagcgg cgtgccctcc 180 agattctctg gcagcggctc cggaaccgac ttcaccctga caatctctag cctgcagccc 240 gaggatttcg ccacatacta ttgtcagcag tacagcaagc tgccttatac ctttggcggc 300 ggcacaaagg tggagatcaa g 321 <210> 30 <211> 18 <212> PRT <213> Homo sapiens <400> 30 His Thr Leu Gln Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp 1 5 10 15 Arg Asp <210> 31 <211> 41 <212> PRT <213> Homo sapiens <400> 31 Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys 1 5 10 15 Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala 20 25 30 Ser Asn Ser Ser Asp Ala Ile Cys Glu 35 40

Claims

1. An antibody or an antigen-binding fragment thereof, which specifically binds to human OX40 and comprises: A heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: (a) HCDR (heavy chain complementarity determining region) 1 as shown in SEQ ID NO:3, (b) HCDR2 as shown in SEQ ID NO:24 and (c) HCDR3 as shown in SEQ ID NO:5; the light chain variable region comprising: (d) LCDR (light chain complementarity determining region) 1 as shown in SEQ ID NO:25, (e) LCDR2 as shown in SEQ ID NO:19 and (f) LCDR3 as shown in SEQ ID NO:

8.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: A heavy chain variable region (VH) comprising an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:26, and a light chain variable region (VL) comprising an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

28.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein one, two, three, four or five amino acids within SEQ ID NO: 26 and / or 28 have been inserted, deleted or substituted.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: The heavy chain variable region (VH) comprises SEQ ID NO:26 and the light chain variable region (VL) comprises SEQ ID NO:

28.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab' fragment or a F(ab')2 fragment.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which has OX40 agonist activity.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which binds to human OX40 at an epitope comprising one or more amino acid residues selected from H153 to D170 of human OX40.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which binds to human OX40 at an epitope comprising one or more amino acid residues selected from H153, T154, I165, E167 and D170 of human OX40.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which binds to human OX40 at an epitope comprising one or more amino acid residues selected from H153, I165 and E167 of human OX40.

10. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, which binds to human OX40 at or within SEQ ID NO:

30.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

12. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof has reduced glycosylation or is aglycosylated or is hypofucosylated.

13. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof comprises an added bisecting GlcNac structure.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the Fc domain of the antibody belongs to IgG1.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the Fc domain of the antibody belongs to IgG4.

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the IgG4 has a S228P substitution (according to the EU numbering system).

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the IgG4 has S228P and R409K substitutions (according to the EU numbering system).

18. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, having one or more of the following properties: (i) Ability to cross-react with cyno OX40; (ii) does not interfere with the OX40-OX40L interaction; (iii) can costimulate T cells to produce IL-2; (iv) Ability to co-activate CD4 + T cells, as measured in a mixed lymphocyte reaction (MLR) assay; (v) capable of mediating ADCC as measured in an ADCC assay based on lactate dehydrogenase (LDH) release; (vi) Ability to deplete CD4 + Tregs; (vii) Can increase CD8 + Teff / Treg ratio; or (viii) Able to mediate partial regression of OX40-related tumors in animal tumor models.

19. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

20. Use of an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 in the preparation of a medicament for treating or reducing the likelihood of an OX40-related cancer in a patient in need thereof, wherein the OX40-related cancer is breast cancer, head and neck cancer, gastric cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma or sarcoma.

21. The use of claim 20, wherein the antibody or antigen-binding fragment thereof is administered in combination with another therapeutic agent.

22. The use of claim 21, wherein the therapeutic agent is paclitaxel or a paclitaxel agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide or 5-azacytidine.

23. The use of claim 22, wherein the therapeutic agent is paclitaxel, lenalidomide or 5-azacytidine.

24. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 18.

25. A vector comprising the nucleic acid of claim 24.

26. A host cell comprising the nucleic acid of claim 24 or the vector of claim 25.

27. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell of claim 26 and recovering the antibody or the antigen-binding fragment thereof.

28. A diagnostic reagent comprising a labeled antibody or antigen-binding fragment thereof according to any one of claims 1 to 18.

29. The diagnostic agent of claim 28, wherein the label is selected from the group consisting of a radiolabel, a fluorophore, a chromophore, an imaging agent, and a metal ion.

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