EA046633B1 - METHODS FOR TREATING CANCER USING ANTIBODIES TO OX40 IN COMBINATION WITH ANTIBODIES TO TIGIT - Google Patents

Description

Field of technology

Disclosed herein is a method of treating cancer using a combination of antibodies or antigen-binding fragments thereof that bind to human OX40 and human TIGIT (T-cell immunoreceptor with Ig and ITIM domains).

Prior Art

OX40 (also known as ACT35, CD134 or TNFRSF4) is a type I transmembrane glycoprotein with a molecular mass of approximately 50 kDa and is a member of the tumor necrosis factor receptor super family (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 partial cysteine-rich domains (CRDs). The intracellular domain of OX40 contains one conserved QEE signal transduction motif that mediates binding to several TNFR (tumor necrosis factor receptor)-associated factors (TRAF-associated factor), including TRAF2, TRAF3 and TRAF5, allowing OX40 to bind with intracellular kinases (Arch and Thompson, 1998; Willoughby et al., 2017).

OX40 was initially discovered on activated rat CD4+ T cells, and then mouse and human homologues from T cells were cloned (al-Shamkhani et al., 1996; Calderhead et al., 1993). In addition to expression on activated CD4 T cells, including T helper (Th)1 cells, Th2 cells, IgR cells, and regulatory T cells (Treg), OX40 expression has also been detected on the surface of activated CD8 T cells , natural killer (NK) T cells, neutrophils and NK cells (Croft, 2010). In contrast, low OX40 expression is found on naive CD4 ⁺ and CD8 ⁺ T cells, as well as on most resting memory T cells (Croft, 2010; Soroosh et al., 2007). Surface expression of OX40 on naïve T cells is transient. Following TCR (T cell receptor) activation, OX40 expression on T cells increases significantly within 24 hours, peaking at approximately 2-3 days, persisting for approximately 5-6 days (Gramaglia et al., 1998).

OX40 ligand (OX40L, also known as gp34, CD252 or TNFSF4) is the only ligand for OX40. Like other members of the TNFSF (tumor necrosis factor superfamily), OX40L is a type II glycoprotein that contains 183 AA with an intracellular domain of 23 AA and an extracellular domain of 133 AA (Croft, 2010; Gough and Weinberg, 2009). OX40L naturally forms a homomeric trimeric complex on the cell surface. The ligand trimer interacts with three copies of OX40 at the ligand monomer-monomer interface mainly through the receptor regions CRD1, CRD2 and partly CRD3, but without the participation of CRD4 (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. al., 2004), macrophages (Weinberg et al., 1999) and Langerhans cells (Sato et al., 2002). Additionally, OX40L has been found to be expressed on other cell types such as NK cells, mast cells, subsets of activated T cells, as well as vascular endothelial cells and smooth muscle cells (Croft, 2010; Croft et al., 2009).

Trimerization of OX40 by ligation with trimeric OX40L or dimerization with agonistic antibodies promotes the recruitment and docking of the adapter molecules TRAF2, TRAF3 and/or TRAF5 to its intracellular QEE motif (Arch and Thompson, 1998; Willoughby et al., 2017). Recruitment and docking of TRAF2 and TRAF3 can further lead to activation of both canonical NF-kB1 and non-canonical NF-kB2 pathways, which play key roles in regulating T cell survival, differentiation, proliferation, cytokine production and effector functions (Croft, 2010; Gramaglia et al., 1998; Rogers et al., 2001; Song et al., 2005b;

In normal tissues, OX40 expression is low and occurs primarily on lymphocytes in lymphoid organs (Durkop et al., 1995). However, increased OX40 expression on immune cells has often been observed in both 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 cancers (Kjaergaard et al., 2000; Vetto et al., 1997; Weinberg et al., 2000). Notably, increased OX40 expression is associated with longer survival in patients with colorectal cancer and cutaneous melanoma, and inversely correlates with the occurrence of distant metastases and more advanced tumor features (Ladanyi et al., 2004; Petty et al., 2002; Sarff et al. ., 2008). It has also been shown that treatment with an anti-OX40 antibody can elicit antitumor efficacy in various mouse models (Aspeslagh et al., 2016), indicating the potential of OX40 as an immunotherapeutic target. In the first clinical trial in cancer patients, conducted by Curti et al., with an agonistic monoclonal antibody to OX40, evidence of antitumor efficacy and activation of tumor-specific T cells was observed, indicating that antibodies to OX40 are useful in enhancing antitumor T cell responses (Curti et al., 2013).

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The mechanism of action of agonistic antibodies to OX40 in mediating antitumor efficacy has been studied primarily in mouse tumor models (Weinberg et al., 2000). Until recently, the mechanism of action of agonistic antibodies to OX40 in tumors was explained by their ability to trigger a co-stimulatory signaling pathway in effector T cells, as well as inhibitory effects on Treg cell differentiation and function (Aspeslagh et al., 2016; Ito et al., 2006; St Rose et al., 2013; Voo et al., 2013). Recent studies have shown that in both animal models of tumors and cancer patients, tumor-infiltrating Treg cells express higher levels of OX40 than effector T cells (both CD4' and CD8+) and peripheral Treg cells (Lai et al ., 2016; Marabelle et al., 2013b; Montler et al., 2016; Therefore, the secondary effects by which anti-OX40 antibodies trigger antitumor responses depend on their Fc-mediated effector functions in depleting intratumoral OX40+ Treg cells through antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP) (Aspeslagh et al. , 2016; Marabelle et al., 2013a; Marabelle et al., 2013b; This work demonstrates that agonistic anti-OX40 antibodies with Fco-mediated effector function can preferentially deplete intratumoral Treg cells and improve ratios of CD8+ effector T cells to Treg cells in the tumor microenvironment (TME), leading to improved antitumor immune responses, enhanced tumor regression, and improved survival (Bulliard et al., 2014; Carboni et al., 2003; Jacquemin et al., 2015; Marabelle et al., 2013b). Based on these results, there is an unmet medical need for the development of OX40 agonist antibodies, with both agonist activity and Fco-mediated effector functions. To date, clinically used OX40 agonistic antibodies are mainly ligand-competitive antibodies that block the OX40-OX40L interaction (eg, WO2016196228A1). Because OX40OX40L interaction is required to enhance effective antitumor immunity, blockade of OX40-OX40L limits the effectiveness of these ligand-competitive antibodies. Therefore, OX40 agonistic antibodies, which specifically bind to OX40 without interfering with the interaction of OX40 with OX40L, are useful for the treatment of cancer and autoimmune disorders in both monotherapy and combination therapy. Increased TIGIT expression in tumor-infiltrating lymphocytes (TILs) and human peripheral blood mononuclear cells (PBMCs) has been reported in many cancers, such as lung cancer (Tassi, et al., Cancer Res. 2017 77:851-861), cancer esophageal (Xie J, et al., Oncotarget 2016 7: 63669-63678), breast cancer (Gil Del Alcazar CR, et al. 2017 Cancer Discov.), acute myeloid leukemia (AML) (Kong Y et al., Clin Cancer Res. 2016 22: 3057-66) and melanoma (Chauvin JM, et al., J Clin Invest. 2015 125: 2046-2058). Increased expression of TIGIT in AML is associated with poor patient survival prognosis (Kong Y et al., Clin Cancer Res. 2016 22: 3057-66). Upregulation of TIGIT signaling plays an important role not only in immune tolerance to cancer but also to chronic viral infection. During HIV infection, TIGIT expression on T cells was significantly higher and positively correlated with viral load and disease progression (Chew GM, et al., 2016 PLoS Pathog. 12: e1005349). In addition, blockade of the TIGIT receptor alone or in combination with another blockade can rescue functionally exhausted T cells both in vitro and in vivo (Chauvin JM, et al., J Clin Invest. 2015 125: 2046-2058; Chew GM, et al., 2016 PLoS Pathog. 12: e1005349; Johnston RJ, et al. Cancer Cell 2014 26: 923-937). In cases of cancer and viral infections, activation of TIGIT signaling promotes immune cell dysfunction, leading to cancer proliferation or prolonged viral infection. Inhibition of TIGIT-mediated inhibitory signaling by therapeutic agents can restore the functional activity of immune cells, including T cells, NK cells, and dendritic cells (DCs), thereby enhancing immunity against cancer or chronic viral infection.

The essence of the invention

The present invention is directed to a combination of agonistic antibodies to OX40 and antibodies to TIGIT and methods of using the combination of these antibodies in the treatment of cancer. In one embodiment, the present invention provides anti-OX40 antibodies in combination with anti-TIGIT antibodies. In one aspect, the OX40 agonistic antibody and antigen binding fragment of the present invention does not compete with OX40L or interfere with the binding of OX40 to its ligand OX40L. In one aspect, an anti-TIGIT antibody reduces TIGIT signaling.

The present invention covers the following embodiments.

A method of treating cancer comprising administering to a subject an effective amount of a non-competitive anti-OX40 antibody or an antigen-binding fragment thereof in combination with an anti-TIGIT antibody or an antigen-binding fragment thereof.

A method in which an anti-OX40 antibody specifically binds to human OX40 and contains:

(i) a heavy chain variable region that contains (a) HCDR (heavy chain complementarity 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 which contains: (d) LCDR (light chain complementarity determining region) 1 with SEQ ID NO: 25, (e) LCDR2 with SEQ ID NO: 19 and (f) LCDR3 with SEQ ID NO: 8 ;

- 2 046633 (ii) a heavy chain variable region which contains (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 18 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region a chain that contains: (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 which contains (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 13 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region which contains: (d) LCDR1 having SEQ ID NO: 6, (e) LCDR2 having SEQ ID NO: 7 and (f) LCDR3 having SEQ ID NO: 8; or (iv) a heavy chain variable region which comprises (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 4 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region, which contains: (d) LCDR1 of SEQ ID NO: 6, (e) LCDR2 of SEQ ID NO: 7 and (f) LCDR3 of SEQ ID NO: 8, in combination with an anti-TIGIT antibody or an antigen-binding fragment thereof.

A method in which the anti-OX40 antibody or antigen-binding antibody comprises:

(i) a heavy chain variable region (VH), which contains SEQ ID NO: 26, and a light chain variable region (VL), which contains SEQ ID NO: 28;

(ii) a heavy chain variable region (VH), which contains SEQ ID NO: 20, and a light chain variable region (VL), which contains SEQ ID NO: 22;

(iii) a heavy chain variable region (VH), which contains SEQ ID NO: 14, and a light chain variable region (VL), which contains SEQ ID NO: 16; or (iv) a heavy chain variable region (VH) that contains SEQ ID NO: 9, and a light chain variable region (VL) that contains SEQ ID NO: 11.

A method wherein an anti-TIGIT antibody or an antigen-binding fragment thereof comprises an antigen-binding domain of an antibody that specifically binds human TIGIT and comprises a heavy chain variable region comprising: HCDR1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33 and HCDR3 of SEQ ID NO: 33 ID NO: 34, and a light chain variable region comprising: LCDR1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37.

A method wherein the anti-TIGIT antibody comprises an antigen binding domain of an antibody that specifically binds human TIGIT and comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 41.

The method, wherein the anti-OX40 antibody or antigen binding fragment is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv and (Fab') ₂ fragments.

The method, wherein the anti-TIGIT antibody or antigen binding fragment is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv and (Fab')2 fragments.

The method in which the cancer is breast cancer, colon cancer, head and neck cancer, stomach cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma or sarcoma.

The method in which the breast cancer is metastatic breast cancer.

A method in which treatment results in a sustained anti-cancer response in a subject after treatment has ceased.

A method of enhancing, enhancing or stimulating an immune response or function, which includes administering to a subject an effective amount of a non-competitive anti-OX40 antibody or an antigen-binding fragment thereof in combination with an anti-TIGIT antibody or an antigen-binding fragment thereof.

A method in which an anti-OX40 antibody or antigen-binding fragment thereof specifically binds to human OX40 and comprises:

(i) a heavy chain variable region that contains (a) HCDR (heavy chain complementarity 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 which contains: (d) LCDR (light chain complementarity determining region) 1 with SEQ ID NO: 25, (e) LCDR2 with SEQ ID NO: 19 and (f) LCDR3 with SEQ ID NO: 8 ;

(ii) a heavy chain variable region which contains (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 18 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region which contains: (d) LCDR1 having SEQ ID NO: 6, (e) LCDR2 having SEQ ID NO: 19 and (f) LCDR3 having SEQ ID NO: 8;

(iii) a heavy chain variable region which contains (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 13 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region which contains: (d) LCDR1 having SEQ ID NO: 6, (e) LCDR2 having SEQ ID NO: 7 and (f) LCDR3 having SEQ ID NO: 8; or (iv) a heavy chain variable region which comprises (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 4 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region, which contains: (d) LCDR1 of SEQ ID NO: 6, (e) LCDR2 of SEQ ID NO: 7 and (f) LCDR3 of SEQ ID NO: 8, in combination with an anti-TIGIT antibody or an antigen-binding fragment thereof.

A method in which the anti-OX40 antibody or antigen-binding antibody comprises:

(i) a heavy chain variable region (VH), which contains SEQ ID NO: 26, and a light chain variable region (VL), which contains SEQ ID NO: 28;

(ii) a heavy chain variable region (VH), which contains SEQ ID NO: 20, and a light chain variable region (VL), which contains SEQ ID NO: 22;

- 3 046633 (iii) a heavy chain variable region (VH), which contains SEQ ID NO: 14, and a light chain variable region (VL), which contains SEQ ID NO: 16; or (iv) a heavy chain variable region (VH) that contains SEQ ID NO: 9, and a light chain variable region (VL) that contains SEQ ID NO: 11.

A method wherein an anti-TIGIT antibody or an antigen-binding fragment thereof comprises an antigen-binding domain of an antibody that specifically binds human TIGIT and comprises a heavy chain variable region comprising: HCDR1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33 and HCDR3 of SEQ ID NO: 33 ID NO: 34, and a light chain variable region comprising: LCDR1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36, and LCDR3 of SEQ ID NO: 37.

The method, wherein the anti-TIGIT antibody or antigen-binding fragment thereof comprises an antigen-binding domain of the antibody that specifically binds human TIGIT, and comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 39, and a light chain variable region (VL), containing the amino acid sequence SEQ ID NO: 41.

The method, wherein the anti-OX40 antibody or antigen binding fragment thereof is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv and (Fab') ₂ fragments.

The method, wherein the anti-TIGIT antibody or antigen binding fragment thereof is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv and (Fab') ₂ fragments.

A method in which the stimulation of an immune response involves T cells or NK cells.

A method in which the stimulation of an immune response is characterized by increased sensitivity to antigenic stimulation.

A method in which T cells or NK cells have increased cytokine secretion, proliferation, or cytolytic activity.

The method in which the T cells are CD4+ and CD8+ T cells.

A method in which administration results in a sustained immune response in a subject after treatment has ceased.

In one embodiment, said antibody or antigen binding fragment thereof comprises one or more complementarity determining regions (CDRs) having an amino acid sequence selected from the group consisting of 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.

In another embodiment, said 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.

In another embodiment, said antibody or antigen binding fragment thereof comprises:

(a) a heavy chain variable region containing three complementarity determining regions (HCDRs), which are HCDR1 having the amino acid sequence of SEQ ID NO: 3, HCDR2 having the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 18 or SEQ ID NO: 24, and HCDR3 having the amino acid sequence of SEQ ID NO: 5; and/or (b) a light chain variable region comprising three complementarity determining regions (LCDRs), which are LCDR1 having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 25, LCDR2 having the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 19, and LCDR3 having the amino acid sequence of SEQ ID NO: 8.

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

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

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

In another embodiment, an antibody or antigen binding fragment of the present invention comprises: a heavy chain variable region comprising HCDR1 having the amino acid sequence of SEQ ID NO: 3, HCDR2 having the amino acid sequence of SEQ ID NO: 18, and HCDR3 having the amino acid sequence of SEQ ID NO: 18 : 5, and a light chain variable region comprising 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.

In another embodiment, an antibody or antigen binding fragment of the present invention comprises: a heavy chain variable region comprising HCDR1 having the amino acid sequence of SEQ ID NO: 3, HCDR2 having the amino acid sequence of SEQ ID NO: 24, and HCDR3 having the amino acid sequence of SEQ ID NO: 24 : 5, and a light chain variable region comprising 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.

In one embodiment, an antibody of the present invention or an antigen binding fragment thereof comprises: (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 20 or SEQ ID NO: 26 or amino acid a sequence at least 95%, 96%, 97%, 98%, or 99% identical to any 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 of at least 95%, 96% , 97%, 98%, or 99% identical to any of SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 22, or SEQ ID NO: 28.

In another embodiment, an antibody of the present invention or an antigen binding fragment thereof comprises: (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 20 or SEQ ID NO: 26 or the amino acid a 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. In one embodiment, an antibody of the present invention or an antigen binding fragment thereof comprises:

(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 (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 (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 (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.

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

In one embodiment, an antibody of the present invention binds to OX40 with a binding affinity ( _KD ) of 1x 10 ^-6 M to 1x 10 ^-10 M. In another embodiment, an antibody of the present invention binds to OX40 with a binding affinity ( _KD ) of about 1x10 ^-6 M, about 1x10 ^-7 M, about 1x10 ^-8 M, about 1x10 ^-9 M or about 1x10 ^-10 M.

In another embodiment, the anti-human OX40 antibody of the present invention exhibits cross-species binding activity to cynomolgus OX40. In one embodiment, the anti-OX40 antibody of the present invention binds to a human OX40 epitope beyond the OX40-OX40L interface. In another embodiment, the anti-OX40 antibody of the present invention does not compete with the OX40 ligand binding to OX40. In yet another embodiment, the anti-OX40 antibody of the present invention does not block the interaction between OX40 and its ligand OX40L.

The antibodies of the present invention are agonistic and significantly enhance the immune response. In an embodiment, the antibody of the present invention can significantly stimulate a primary T cell to produce IL-2 in a mixed lymphocyte culture (MSC) response assay.

In one embodiment, the antibodies of the present invention have potent Fc-mediated effector functions. Antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) against OX40 ^H1 target cells, such as regulatory T cells (Treg cells), via NK cells. In one aspect of the invention, a method is provided for assessing anti-OX40 antibody-mediated in vitro depletion of specific T cell subsets based on different levels of OX40 expression. Antibodies or antigen binding fragments of the present invention do not block the OX40-OX40L interaction. In addition, antibodies to OX40 exhibit dose-dependent antitumor activity in vivo, as shown in animal models. Dose-dependent activity is differentiated from the activity profile of antibodies to OX40, which block the OX40-OX40L interaction.

The present invention relates to isolated nucleic acids containing nucleotide sequences encoding the amino acid sequence of an antibody or antigen binding fragment. In one embodiment, the isolated nucleic acid contains the nucleotide sequence VH 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 with SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 21 or SEQ ID NO: 27, and encodes the VH region of an antibody or antigen binding fragment of the present invention. Alternatively or additionally, the isolated nucleic acid comprises a 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 having at least 95%, 96%, 97% , 98% or 99% identity with SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 23 or SEQ ID NO: 29, and encodes the VL region of an antibody or antigen binding fragment of the present invention.

In yet another aspect, the present invention provides a method of treating a disease in a subject which includes administering a therapeutically effective amount of an anti-OX40 antibody or an antigen binding fragment thereof or a pharmaceutical composition based on an anti-OX40 antibody to a subject in need thereof in combination with an anti-TIGIT antibody or an antigen binding fragment thereof. . In another embodiment, the disease to be treated with the anti-OX40 antibody in combination with the anti-TIGIT antibody is cancer or an autoimmune disease.

Brief description of graphic materials

Fig. 1 is a schematic diagram of the OX40-mIgG2a, OX40-huIgG1, and OX40-His constructs. OX40 ECD: extracellular domain of OX40. N: N-terminal. S: C-end.

In fig. Figure 2 shows the affinity determination of purified chimeric (ch445) and humanized (445-1, 445-2, 445-3 and 445-3 IgG4) anti-OX40 antibodies using surface plasmon resonance (SPR).

In fig. Figure 3 shows the determination of OX40 binding by flow cytometry. OX40-positive HuT78/OX40 cells were incubated with various OX40 antibodies (ch445, 445-1, 445-2, 445-3, and 445-3 IgG4 antibodies) and subjected to fluorescence-activated cell sorting (FACS). The result is illustrated using the mean fluorescence intensity (MFI, Y-axis).

In fig. Figure 4 shows the binding of anti-OX40 antibodies by flow cytometry. HuT78/OX40 and HuT78/cynoOX40 cells were stained with antibody 445-3, and the mean fluorescence intensity (MFI, shown on the Y axis) was determined by flow cytometry.

In fig. Figure 5 illustrates the determination of the affinity of Fab 445-3 for wild-type and spot OX40

- 6 046633 mutants using surface plasmon resonance (SPR).

In fig. 6 shows detailed interactions between antibody 445-3 and its epitopes on OX40. Antibodies 445-3 and OX40 are shown in pale gray and black, respectively. Hydrogen bonding or salt bridge, π-π interaction and van der Waals (VDW) interaction are indicated by dashed, double dashed and solid lines, respectively.

In fig. 7 demonstrates that antibody 445-3 does not interfere with binding to OX40L. Before staining HEK293/OX40L cells, OX40-mouse IgG2a fusion protein (OX40-mIgG2a) was preincubated with human IgG (+HuIgG), antibody 445-3 (+445-3) or antibody 1A7.gr1 (+1A7.gr1, see below). US 2015/0307617) in a molar ratio of 1 : 1. The binding of OX40L to the OX40mIgG2a/anti-OX40 antibody complex was determined by co-incubation of HEK293/OX40L cells and the OX40-mIgG2a/anti-OX40 antibody complex, followed by reaction with a secondary anti-mouse IgG antibody and flow cytometry . The results were presented as mean ± SD (standard deviation from the arithmetic mean) in two parallel measurements. Statistical significance: *: P less than 0.05; **: P less than 0.01.

In fig. Figure 8 shows a structural alignment of Fab OX40/445-3 with the indicated OX40/OX40L complex (PDB code: 2HEV). OX40L is shown in white, Fab 445-3 is shown in gray, and OX40 is shown in black.

In fig. 9A,B show that anti-OX40 antibody 445-3 induces IL-2 production in combination with TCR stimulation. OX40-positive HiT78/OX40 cells (Fig. 9A) were co-cultured with an artificial antigen presenting cell (APC) line (HEK293/OS8 ^Low -FcyRI) in the presence of anti-OX40 antibodies overnight, and IL-2 production was used as an indication of T stimulation -cells (Fig. 9B). IL-2 in the culture supernatant was detected using ELISA (enzyme-linked immunosorbent assay). Results are presented as mean ± SD of three parallel measurements.

Fig. 10 illustrates that antibodies to OX40 enhance SCL responses. In vitro differentiated dendritic cells (DCs) were cocultured with allogeneic CD4+ T cells in the presence of OX40 antibodies (0.1–10 μg/ml) for 2 days. IL-2 in the supernatant was detected using ELISA. All tests were performed in four parallel measurements, and the results are shown as mean ± SD. Statistical significance: *: P less than 0.05; **: P less than 0.01.

In fig. 11 demonstrates that anti-OX40 antibody 445-3 induces ADCC. ADCC assay was performed using NK92MI/CD16V cells as effector cells and HuT78/OX40 cells as target cells in the presence of anti-OX40 antibodies (0.004-3 μg/ml) or as controls. Equal numbers of effector cells and target cells were cocultured for 5 h until lactate dehydrogenase (LDH) release was detected. Percent cytotoxicity (Y-axis) was calculated based on the manufacturer's protocol as described in Example 12. Results are presented as mean ± SD of triplicate measurements.

In fig. 12A-C show that anti-OX40 antibody 445-3 in combination with NK cells increases the ratio of CD8+ effector T cells to Treg cells in activated PBMCs in vitro. Human PBMCs were preactivated with PHA-L (1 μg/ml) and then cocultured with NK92MI/CD16V cells in the presence of anti-OX40 antibodies or control. The percentages of different T cell subsets were determined using flow cytometry. Next, the ratio of CD8+ effector T cells to Treg cells was calculated. In fig. 12A shows the ratio of CD8+ to Total T cells. In fig. 12B illustrates the ratio of Treg cells to Total T cells. In fig. 12C shows the ratio of CD8+ to Treg cells. Data are presented as mean ± SD in two parallel measurements. Statistical significance between 445-3 and 1A7.gr1 at the indicated concentrations is shown. *: P less than 0.05; **: P less than 0.01.

In fig. 13A, B show that anti-OX40 antibody 445-3, but not 1A7.gr1, exhibits dose-dependent antitumor activity in the syngeneic MC38 colorectal cancer model in OX40 humanized mice. Mouse colon carcinoma cells line MC38 (2x10 ⁷ ) were implanted subcutaneously into female transgenic mice carrying human OX40. After randomization according to tumor volume, animals were treated by intraperitoneal injection with either anti-OX40 antibody or isotype control once weekly three times as indicated. In fig. 13A compares increasing doses of antibody 445-3 with increasing doses of antibody 1A7.gr1 and reducing tumor growth. In fig. 13B shows data for all mice treated with this specific dose. Data are presented as mean tumor volume ± standard error of the mean (SEM) from 6 mice per group. Statistical significance: *: P less than 0.05 compared with isotype control.

Fig. 14A,B is a table of amino acid changes that have been made in the anti-OX40 antibodies.

In fig. 15 shows the effectiveness of anti-OX40 antibodies in combination with anti-TIGIT antibodies in a mouse model of metastatic breast cancer.

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Definitions

Unless otherwise defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one skilled in the art.

As used herein, including the appended claims, words in the singular include their corresponding plurals unless the context clearly requires otherwise.

The term or is used to designate and is used interchangeably with the term and/or, unless the context clearly indicates otherwise.

The term anticancer agent as used herein 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 agents and radiotherapeutic agents, targeted anticancer agents and immunotherapeutic agents.

The term OX40 refers to a type I transmembrane glycoprotein with a molecular mass of approximately 50 kDa, a member of the tumor necrosis factor receptor superfamily.

OX40 is also known as AST35, CD134 or TNFRSF4. The amino acid sequence of human OX40 (SEQ ID NO: 1) can also be found under the accession number NP_003318, and the accession number of the nucleotide sequence encoding the OX40 protein: X75962.1. The term OX40 ligand or OX40L refers to a single OX40 ligand and is interchangeable with gp34, CD252, or TNFSF4.

As used herein, the terms administration, administration, treatment and treatment, when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, mean contact of an exogenous pharmaceutical, therapeutic, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid. Treatment of a cell includes contact of the reagent with the cell, as well as contact of the reagent with a liquid, the liquid being in contact with the cell. The term administration and treatment also means in vitro and ex vivo treatment of, for example, a cell, reagent, diagnostic, binding compound or other cell. The term subject as used herein includes any organism, preferably an animal, more preferably a mammal (eg, rat, mouse, dog, cat, rabbit), and most preferably human. Treatment of any disease or disorder refers in one aspect to reducing the intensity of the disease or disorder (ie, slowing or stopping or reducing the progression of the disease or at least one of its clinical symptoms). In another aspect, the term treat, treatment, or treatment refers to alleviating or decreasing the intensity of at least one physical parameter, including those that may be indiscernible to the patient himself. In yet another aspect, treat, treatment, or treatment refers to modulating a disease or disorder, either physically (eg, by stabilizing a noticeable symptom), or physiologically (eg, by stabilizing a physical parameter), or both. In yet another aspect, treat, treatment or treatment refers to preventing or delaying the onset or development or progression of a disease or disorder.

The term subject in the context of the present invention is a mammal, such as a primate, preferably a great ape, such as a human (eg, a patient who has or is at risk for a disorder described herein).

As used herein, the term affinity refers to the strength of interaction between an antibody and an antigen. Within an antigen, the variable arm of an antibody interacts through noncovalent interactions with the antigen at multiple sites; the more interactions, the stronger the affinity. As used herein, the term antibody refers to a polypeptide of the immunoglobulin family that can bind a corresponding antigen non-covalently, reversibly and specifically. For example, a naturally occurring IgG antibody is a tetramer containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2 and CH3. Each light chain contains 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 of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved 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 heavy and light chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C'1c[) of the classical complement system. The term antibody includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chi

- 8 046633 dimensional antibodies and anti-idiotypic (anti-Id) antibodies. Antibodies can be of any isotype/class (eg, IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

In some embodiments, anti-OX40 antibodies comprise at least one antigen binding site or at least a variable region. In some embodiments, anti-OX40 antibodies comprise an antigen binding fragment from an anti-OX40 antibody described herein. In some embodiments, the anti-OX40 antibody is isolated or recombinant. The term monoclonal antibody or mAb or Mab as used herein means a population of essentially homogeneous antibodies, i.e. The antibody molecules contained in the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minute quantities. In contrast, traditional (polyclonal) antibody preparations typically contain many different antibodies having different amino acid sequences in their variable domains, in particular their complementarity determining regions (CDRs), which are often specific for different epitopes. The definition of monoclonal indicates that the antibody is derived from an essentially homogeneous population of antibodies and should not be interpreted as requiring production of the antibody through any particular method. Monoclonal antibodies (mAbs) can be produced by methods known to those skilled in the art. See, for example, Kohler et al., Nature 1975 256:495-497; US Patent No. 4376110; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory 1988; and Colligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The antibodies disclosed herein can be of any class of immunoglobulins, including IgG, IgM, IgD, IgE, IgA, and any subclass thereof such as IgG1, IgG2, IgG3, IgG4. The hybridoma producing the monoclonal antibody can be cultured in vitro or in vivo. High titers of monoclonal antibodies can be obtained by in vivo production, where cells from individual hybridomas are administered by intraperitoneal injection into mice, such as Balb/c mice, to produce ascites fluid containing high concentrations of the desired antibodies. Monoclonal antibodies of the IgM or IgG isotype can be purified from such ascites fluids or from cell culture supernatants using column chromatography techniques well known to those skilled in the art.

In general, the basic structural unit of an antibody contains a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each having one light chain (about 25 kDa) and one heavy chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal fragment of the heavy chain may define a constant region primarily responsible for effector function. Generally, human light chains are classified as kappa and lambda light chains. In addition, human heavy chains are generally classified as α, δ, ε, γ, or μ, and they define antibody isotypes as IgA, IgD, IgE, IgG, and IgM, respectively. In the light and heavy chains, the variable and constant regions are connected by a J region of about 12 or more amino acids, with the heavy chain also containing a D region of about 10 or more amino acids.

The variable regions of each light/heavy chain (VL/VH) pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. With the exception of bifunctional or bispecific antibodies, the two binding sites are generally the same.

Typically, the variable domains of both the heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), which are located between relatively conserved framework regions (FRs). CDRs are typically aligned with framework regions to allow binding to a specific epitope. Typically, from N-terminus to C-terminus, the variable domains of both the light and heavy chains 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 the CDRs and framework regions can be determined using various definitions well known in the art, for example, Cabot, Chotia and AbM (see, for example, 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); :799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)). Definitions of antigen binding sites are also described in the following works: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M. P., 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 M. J. E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). In some embodiments, in a combined Kabat and Chotia numbering scheme, CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chotia CDR, or both. For example, CDRs correspond to amino acid residues 26-35 (HC CDR1), 50-65 (HC CDR2) and 95-102 (HC CDR3) in VH, eg, mammalian VH, eg, human VH; and amino acid residues 24-34 (LC CDR1), 50-56 (LC CDR2) and 89-97 (LC CDR3) in VL, eg mammalian VL, eg human VL.

- 9 046633

The term hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region contains amino acid residues from the CDRs (ie, VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region and VH-CDR1, VH-CDR2 and VHCDR3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of an antibody by sequence); also see Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of an antibody by structure). The term framework or FR residues means those variable domain residues that differ from the hypervariable region residues defined herein as CDR residues.

Unless otherwise specified, antigen binding fragment means antigen binding fragments of antibodies, i.e. antibody fragments that retain the ability to specifically bind to an antigen bound by a full-length antibody, for example, fragments that retain 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, for example single chain Fv (ScFv); nanobodies and polyspecific antibodies formed from antibody fragments.

An antibody specifically binds to a target protein means that the antibody exhibits preferential binding to that target over other proteins, but this specificity does not require absolute binding specificity. An antibody is considered specific for its intended target if its binding is determinative of the presence of the target protein in the sample, for example, without producing undesirable results such as false positives. Antibodies or antigen-binding fragments thereof used herein will bind to the target protein with an affinity that is at least two 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. As used herein, an antibody is considered to specifically bind to a polypeptide containing a given amino acid sequence, for example, the amino acid sequence of the human OX40 molecule, if it binds to polypeptides containing that sequence, but does not bind to proteins lacking that sequence.

The term human antibody as used herein means an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain murine carbohydrate chains if it is produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Likewise, mouse antibody or rat antibody means an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively. The term humanized antibody refers to antibody forms that contain non-human (eg, murine) antibody sequences as well as human antibodies. Such antibodies contain a minimal sequence derived from non-human immunoglobulin. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions correspond to framework regions of a human immunoglobulin sequence. The humanized antibody will also not necessarily contain at least an immunoglobulin constant region (Fc) fragment, typically from human immunoglobulin. The prefix hum, hu, Hu, or h is added to antibody clone designations when it is necessary to distinguish humanized antibodies from the rodent parent antibodies. Humanized forms of rodent antibodies generally contain the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, remove post-translational modification, or for other reasons.

As used herein, the term noncompetitive means that binding of the antibody occurs and does not interfere with the binding of the ligand to the receptor. 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 the reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by the sequences human germline immunoglobulin variable region. The corresponding human germline sequence may also refer to the human variable region amino acid sequence or subsequence with the highest amino acid sequence identity to the reference variable region amino acid sequence or subsequence compared to all other variable region amino acid sequences evaluated. The corresponding human germline sequence may be only framework regions, only complementarity-determining regions, framework and complementarity-determining regions.

- 10 046633 regions, variable segment (as defined above) or other combinations of sequences or subsequences that contain a variable region. Sequence identity can be determined using methods described herein, for example, aligning two sequences using BLAST (Base Local Alignment Search Tool), ALIGN, or other alignment algorithm known in the art. The corresponding human germline nucleic acid or amino acid sequence may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with reference nucleotide or amino acid sequence of the variable region. The term equilibrium dissociation constant (KD, M) refers to the dissociation rate constant (kd, time ^-1 ) divided by the association rate constant (ka, time ^-1 , M ^-1 ). Equilibrium dissociation constants can be measured using any method known in the art. Antibodies of the present invention generally have an equilibrium dissociation constant of less than about 10 ^-7 or 10 ^-8 M, for example, less than about 10 ^-9 M or 10 ^-10 M, in some aspects less than about 10 ^-11 M, 10 ^-12 M or 10 ^-13 M.

The terms cancer or tumor as used herein have the broadest meaning as understood in the art and refer to a physiological condition in mammals that is typically characterized by unregulated cell growth. In the context of the present invention, cancer is not limited to a specific type or location.

The term combination therapy refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present invention. Such administration includes co-administration of these therapeutic agents substantially simultaneously. Such administration also includes co-administration in multiple or separate containers (eg, capsules, powders and liquids) for each active ingredient. Powders and/or liquids may be reconstituted or diluted to the desired dose before administration. In addition, such administration also includes sequential application of each type of therapeutic agent, either 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. In the context of the present invention, when reference is made to an amino acid sequence, the term conservative substitution means the replacement of the original amino acid with a new amino acid that does not significantly change the chemical, physical and/or functional properties of the antibody or fragment, for example, its binding affinity for OX40. In particular, common conservative amino acid substitutions are presented in the following table and are well known in the art.

Illustrative Conservative Amino Acid Substitutions

Original amino acid remainder One-letter and three-letter codes Conservative replacement Alanin A or Ala Gly; Ser Arginine R or Arg LYS; His Asparagine N or Asn Gin; His Aspartic acid D or Asp Gin; Asn Cysteine C or Cys Ser; Ala

- 11 046633

Glutamine Q or Gin Asn Glutamic acid E or Glu Asp; Gin Glycine G or Gly Ala Histidine H or His Asn; Gin Isoleucine I or Not Leu; Vai Leucine L or Leu He; val Lysine K or Lys Arg; His Methionine M or Met Leu; He; Tyr Phenylalanine F or Phe Tyr; Met; Leu Proline P or Pro Ala Serin S or Ser Thr Threonine T or Thr Ser Tryptophan W or Tgr Tyr; Phe Tyrosine Y or Tug Trp; Phe Valin V or Vai He; Leu

Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST algorithms 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 analyzes is publicly available from the National Center for Biotechnology Information (NCBI). This algorithm involves, first, identifying maximum similarity sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some threshold T with a positive score when matching a word of the same length in the base sequence data. T is called the threshold indicator of similarity of neighboring words. These initial matches of neighboring words act as values for initial searches of longer HSPs containing them. Word matches are extended in both directions along each sequence as much as the cumulative alignment scores can be extended. Aggregate scores are calculated for nucleotide sequences using the parameters M (reward score for pairs with matching residues, always greater than 0) and N (penalty score for mismatched residues, always less than 0). For amino acid sequences, the substitution matrix is used to calculate the cumulative score. Extension of word matches in each direction stops when: the cumulative alignment score falls below the X value of its obtained maximum value; the cumulative score tends to zero or lower due to the accumulation of one or more residual alignments with a negative score; or the end of any sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, M of 5, N of minus 4, and a comparison of both regions as default values. For amino acid sequences, BLAST defaults to a word length of 3 and an expected value (E) of 10, and the alignment (B) of the BLOSUM62 substitution matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915, 1989) 50, expected value (E) 10, M equal to 5, N equal to minus 4 and comparison of both sites.

The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the lowest cumulative probability (P(N)), which provides a measure of the probability by which 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 lowest overall probability when comparing 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.

The percentage identity between two amino acid sequences can also be determined using an algorithm by E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11-17, (1988), which was included in the ALIGN program (version 2.0), using the PAM120 weight table, penalty for

- 12 046633 gap extension 12 and gap penalty 4. Additionally, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol, Biol. Biol. 48: 444-453, (1970), which was included in the GAP program in the GCG software package using either the BLOSUM62 matrix or the PAM250 matrix, and a gap penalty of 16, 14, 12, 10, 8, 6 or 4 and a penalty for extending the gap 1, 2, 3, 4, 5 or 6. The term nucleic acid is used interchangeably herein with the term polynucleotide and refers to deoxyribonucleotides or ribonucleotides and their polymers in either single- or double-stranded form. The term covers nucleic acids containing known nucleotide analogs or modified backbone or linkage residues that are synthetic, natural or non-natural, that have similar binding properties as the reference nucleic acid, and that are metabolized in a manner similar to the reference nucleotides. Examples of such analogues include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNAs).

The term operably linked in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (eg, DNA) segments. Typically the term refers to the functional interaction of a transcriptional regulatory sequence with a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates transcription of the coding sequence in a corresponding host cell or other expression system. Typically, promoter transcriptional control sequences that are operably linked to a transcribed sequence are physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, do not necessarily need to be physically adjacent or in close proximity to the coding sequences whose transcription they enhance.

In some aspects, the present invention provides compositions, for example, pharmaceutically acceptable compositions, which include an anti-OX40 antibody described herein formulated together with at least one pharmaceutically acceptable excipient. As used herein, the term pharmaceutically acceptable excipient includes any and all solvents, dispersion media, isotonic and absorption retarding agents, etc., that are physiologically compatible. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (eg, by injection or infusion).

The compositions disclosed herein can be in various forms. These include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (eg injections and infusions), dispersions or suspensions, liposomes and suppositories. The appropriate form depends on the intended route of administration and therapeutic use. Typical suitable compositions are in the form of injections or infusion solutions. One suitable route of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In some embodiments, the antibody is administered by intramuscular or subcutaneous injection. As used herein, the term therapeutically effective amount refers to an amount of antibody that, when administered to a subject to treat a disease or at least one of the clinical symptoms of a disease or disorder, is sufficient to effect such treatment of the disease, disorder or symptom. The therapeutically effective amount may vary depending on 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 being treated, and/or the weight of the subject being treated. The appropriate amount in any particular case may be obvious to those skilled in the art or can be determined through routine experimentation. In the case of combination therapy, the term therapeutically effective amount refers to the total amount of the combination entities to effectively treat a disease, disorder or condition.

As used herein, the phrase in combination with means that the anti-OX40 antibody or binding fragment is administered to a subject simultaneously with, before, or after administration of the anti-TIGIT antibody or binding fragment. In some embodiments, the anti-OX40 antibody or binding fragment is co-formulated with the anti-TIGIT antibody or binding fragment.

Detailed Description of the Invention

Antibodies to TIGIT.

The present invention provides antibodies, antigen-binding fragments that specifically bind human TIGIT, also known as VSIG9 and VSTM3 (see GenBank accession number: NM_173799). Moreover, the present invention provides antibodies that have the same

- 13 046633 late pharmacokinetic characteristics and other desirable characteristics, and therefore can be used to reduce the likelihood of cancer or treat cancer. The present invention further provides pharmaceutical compositions for the prevention and treatment of cancer and related disorders.

The anti-TIGIT antibodies of the present invention can be found in WO 2019/129261. Also provided herein are antibodies to TIGIT, comprising an antigen binding domain of an antibody that specifically binds human TIGIT, and comprising a heavy chain variable region (VH) containing complementarity determining regions (CDRs): HCDR1, comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence presented in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence presented in SEQ ID NO: 34; and a light chain variable region (VL) comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 35, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 36, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: : 37. In another embodiment, an anti-TIGIT antibody comprises an antigen binding domain of an antibody that specifically binds human TIGIT, and comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 39, and a light chain variable region (VL) comprising amino acid sequence SEQ ID NO: 41.

Antibodies to OX40.

The present invention provides antibodies, antigen binding fragments, that specifically bind human OX40. In addition, the present invention provides antibodies that have desirable pharmacokinetic characteristics and other desirable features, and thus can be used to reduce the likelihood of cancer or treat cancer. The present invention further provides pharmaceutical compositions containing antibodies and methods for preparing and using such pharmaceutical compositions for the prevention and treatment of cancer and related disorders.

The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to OX40. The antibodies or antigen binding fragments of the present invention include, but are not limited to, antibodies or antigen binding fragments thereof prepared as described below.

The present invention provides antibodies or antigen binding fragments that specifically bind to OX40, wherein said antibodies or antibody fragments (eg, antigen binding fragments) contain a VH domain having the amino acid sequence of SEQ ID NO: 14, 20 or 26 (Table 3). The present invention also provides antibodies or antigen-binding fragments that specifically bind OX40, said antibodies or antigen-binding fragments comprising a VH CDR having the amino acid sequence of any of the VH CDRs listed in Table 1. 3. In one aspect, the present invention provides antibodies or antigen binding fragments that specifically bind to OX40, wherein said antibodies comprise (or alternatively consist of) one, two, three or more VH CDRs having the amino acid sequence of any of the VH CDRs, listed in table. 3.

The present invention provides antibodies or antigen binding fragments that specifically bind to OX40, wherein said antibodies or antigen binding fragments comprise a VL domain having the amino acid sequence of SEQ ID NO: 16, 22 or 28 (Table 3). The present invention also provides antibodies or antigen-binding fragments that specifically bind to OX40, said antibodies or antigen-binding fragments comprising a VL CDR having the amino acid sequence of any of the VL CDRs listed in Table 1. 3. In particular, the invention provides antibodies or antigen-binding fragments that specifically bind to OX40, wherein said antibodies or antigen-binding fragments contain (or, alternatively, consist of) one, two, three or more VL CDRs having the amino acid sequence of any of the CDRs VL listed in table. 3.

Other antibodies or antigen binding fragments thereof of the present invention include amino acids that have been mutated but have at least 60%, 70%, 80%, 90%, 95%, or 99% percent identity 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 in which no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the CDR regions compared to the CDR regions depicted in the sequence described in table. 3.

Other antibodies of the present invention include those antibodies in which amino acids or nucleic acids encoding amino acids have been mutated; but still have at least 60%, 70%, 80%, 90%, 95% or 99% percent identity with the sequences described 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 regions compared to the variable regions depicted in the sequence described in table. 3, when saving by

- 14 046633 essentially the same therapeutic activity.

The present invention also provides nucleic acid sequences that encode VH, VL, full length heavy chain and full length light chain antibodies that specifically bind to OX40. Such nucleotide sequences can be optimized for expression in mammalian cells.

Identification of Epitopes and Antibodies that Bind to the Same Epitope The present invention provides antibodies and antigen binding fragments thereof that bind to the human OX40 epitope. In some aspects, antibodies and antigen binding fragments can bind to the same OX40 epitope. The present invention also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as the anti-OX40 antibodies described in Table 1. 3. Thus, additional antibodies and antigen binding fragments thereof can be identified based on their ability to cross-compete (eg, competitively inhibit binding, in a statistically significant manner) with other antibodies in binding assays. The ability of a test antibody to inhibit the binding of antibodies and antigen binding fragments thereof of the present invention to OX40 demonstrates that the test antibody can compete with the antibody or antigen binding fragments thereof for binding to OX40. Such an antibody may, without being bound by any theory, bind to the same or related (eg, structurally similar or spatially proximal) epitope on OX40 as the antibody or antigen-binding fragments thereof with which it competes. In some aspect, an antibody that binds to the same epitope on OX40 as the antibodies or antigen binding fragments thereof of the present invention is a human or humanized monoclonal antibody. Such human or humanized monoclonal antibodies can be prepared and isolated as described herein.

Further modification of the Fc region framework.

In yet other aspects, the Fc region is modified by replacing at least one amino acid residue with another amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids may be replaced by another amino acid residue such that the antibody has altered affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which the affinity is changed may, for example, be an Fc receptor or a complement component C1. This approach is described, for example, in US patent No. 5624821 and 5648260 Winter et al.

In another aspect, one or more amino acid residues may be replaced by one or more different amino acid residues such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described, for example, in US Patent No. 6,194,551 to Idusogie et al.

In yet another aspect, one or more amino acid residues are altered, thereby altering the ability of the antibody to fix complement. This approach is described, for example, in PCT publication WO 94/29351 Bodmer et al. In a particular aspect, one or more amino acids of the antibody or antigen binding fragment thereof of the present invention are replaced by one or more allotypic amino acid residues for the IgG1 subclass and kappa isotype. Allotypic amino acid residues also include, but are not limited to, the heavy chain constant region of the IgG1, IgG2 and IgG3 subclasses, as well as the kappa isotype light chain constant region, as described in Jefferis et al., MAbs. 1:332-338 (2009).

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 the Fcy receptor by modifying one or more amino acids. This approach is described, for example, in PCT publication WO 00/42072 Presta. In addition, the binding sites of FcyRI, FcyRII, FcyRIII and FcRn on human IgG1 have been mapped and variants with improved binding have been described (see Shields, R.L. et al., 2001 J. Biol. Chem. 276:6591-6604, 2001).

In yet another aspect, the glycosylation of the antibody is modified. For example, an antibody may be aglycosylated (ie, the antibody has no or reduced glycosylation). Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be made, for example, by changing one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions may be made that result in the elimination of one or more glycosylation sites of the variable region framework, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. This approach is described, for example, in US patent No. 5714350 and 6350861 Co et al.

Additionally or alternatively, an antibody can be produced that has an altered glycosylation pattern, such as a hypofucosylated antibody having reduced amounts of fucosyl residues, or an antibody having increased GlcNac bisection structures. It was prod

- 15 046633 demonstrated that such altered glycosylation patterns increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant antibodies are expressed to thereby produce antibodies with altered glycosylation. For example, in EP 1176195 Hang et al. describes a cell line with a functionally disrupted FUT8 gene that encodes a fucosyltransferase such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT publication WO 03/035835 Presta describes a variant of the CHO (Chinese hamster ovary) cell line, Lecl3 cells, with a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also leads to hypofucosylation of antibodies expressed in this host cell ( see also Shields et al. (2002) J. Chem. 277: 26733-26740). In PCT publication WO 99/54342, Umana et al. describe cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased GlcNac bisection structures, resulting in increasing ADCC activity in antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

In another aspect, if reduction in ADCC is desired, many previous reports have shown that the human IgG4 antibody subclass has only moderate ADCC and little to no CDC effector function (Moore G L, et al. 2010 MAbs, 2:181-189). On the other hand, native IgG4 has been found to be less stable under stress conditions such as acidic buffer or elevated temperature (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). Reduced ADCC can be achieved by functionally binding an antibody to IgG4 engineered with combinations of changes to have reduced or null FcyR binding or C1q binding activities, thereby reducing or eliminating ADCC and CDC effector functions. Considering the physicochemical properties of an antibody as a biological drug, one of the less desirable properties inherent to IgG4 is the dynamic separation of its two heavy chains in solution to form a half-antibody, which leads to bispecific antibodies produced in vivo through a process called Fab exchange ( Van der Neut Kolfschoten M, et al. 2007 Science, 317:1554-157). The serine to proline mutation at position 228 (EU numbering system) was found to be inhibitory to IgG4 heavy chain separation (Angal, S. 1993 Mol Immunol, 30:105-108; Aalberse et al. 2002 Immunol, 105:9-19). Some of the amino acid residues in the hinge region and yFc region have been reported to influence the interaction of antibodies with Fcy receptors (Chappel S M, et al. 1991 Proc. Natl. Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J, 9:115-119; Armour, K. L. et al. 1999 Eur J Immunol, 29: 2613-2624; Clynes, R. A. et al, 2000 Nature Medicine, 6: 443-446; 2007 Annu Rev immunol, 25:21-50). In addition, some IgG4 isoforms, which are rare in the human population, may also exhibit different physicochemical properties (Brusco, A. et al. 1998 Eur J Immunogenet, 25:349-55; Aalberse et al. 2002 Immunol, 105:9-19 ). To obtain OX40 antibodies with low ADCC, CDC and instability, the hinge region and Fc region of human IgG4 can be modified and a number of changes can be made. These modified IgG4 Fc molecules can be found in SEQ ID NO: 83-88, US Patent No. 8735553 to Li et al.

Production of antibodies to OX40.

Antibodies to OX40 and antigen-binding fragments thereof can be produced by any methods known in the art, including, but not limited to, recombinant expression, chemical synthesis and enzymatic cleavage of antibody tetramers, while full-length monoclonal antibodies can be produced, for example, by hybridoma or recombinant production. Recombinant expression can occur from any suitable host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

The present invention further provides polynucleotides encoding the antibodies described herein, for example, polynucleotides encoding heavy or light chain variable regions or segments containing complementarity determining regions, as described herein. In some aspects, the polynucleotide encoding heavy chain variable regions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 15, 21, or 27. In some aspects, the polynucleotide encoding light chain variable regions has at least 85%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 17, 23, or 29. The polynucleotides of the present invention may encode the variable region sequence of an anti-OX40 antibody. They can also code

- 16 046633 both the variable region and the constant region of the antibody. Some of the polynucleotide sequences encode a polypeptide that contains variable regions of both the heavy chain and the light chain of one of the typical antibodies to OX40. Some other polynucleotides encode two polypeptide segments that are, respectively, substantially identical to the heavy chain and light chain variable regions of one of the murine antibodies.

The present invention also provides expression vectors and host cells for producing antibodies to OX40. The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (eg, enhancers) that are operably linked to polynucleotides encoding an anti-OX40 antibody chain or antigen binding fragment. In some aspects, an inducible promoter is used to prevent expression of the inserted sequences except under inducing conditions. Inducible promoters include, for example, arabinose lacZ, metallothionein promoter or heat shock promoter. Cultures of transformed organisms can be expanded under non-inducing conditions without population bias for coding sequences whose expression products are better tolerated by host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of an anti-OX40 antibody or antigen binding fragment. These elements typically include an ATG start codon (the ATG sequence in eukaryotic DNA) and an adjacent ribosome binding site or other sequences. In addition, the efficiency of expression can be increased by including enhancers suitable for the cell system being used (see, for example, 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 enhance expression in mammalian host cells.

The host cells for collecting and expressing anti-OX40 antibody chains can be either prokaryotic or eukaryotic. E. coli is one of the prokaryotic hosts suitable for cloning and expression of the polynucleotides of the present invention. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis and other enterobacteria such as Salmonella, Serratia and various Pseudomonas species. Expression vectors can also be created in these prokaryotic hosts, which typically contain expression control sequences compatible with the host cell (eg, origin of replication). In addition, any number of different well known promoters may be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the lambda phage promoter system. Promoters typically control expression, optionally through an operator sequence, and have ribosome binding site sequences, etc., to initiate and terminate transcription and translation. Other microbes, such as yeast, can also be used to express anti-OX40 polypeptides. Insect cells can also be used in combination with baculovirus vectors.

In other aspects, mammalian host cells are used to express and produce the anti-OX40 polypeptides of the present invention. For example, they may be either a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell line containing an exogenous expression vector. These include any normal mortal or normal or abnormal immortal cells of an animal or human. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS (monkey kidney) cell lines, HEK 293 (human embryonic kidney) cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell culture for the expression of polypeptides has been discussed generally in, for example, Winnacker, From Genes to Clones, VCH Publishers, NY, N.Y., 1987. Expression vectors for mammalian host cells may include expression control sequences such as origins of replication, promoter and enhancer (see, for example, Queen et al., Immunol. Rev. 89: 49-68, 1986), and necessary information processing sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences . These expression vectors typically contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type specific, stage specific, and/or modulated or regulated. Suitable promoters include, but are not limited to, metallothionein promoter, constitutive adenovirus major late promoter, dexamethasone-inducible MMTV (mouse mammary tumor virus) promoter, SV40 (polyoma virus) promoter, MRP polIII promoter, constitutive MPSV (polysaccharide meningococcal virus) promoter. vaccine), the tetracycline-inducible CMV (cytamegalovirus) promoter (such as the human CMV immediate early promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

Methods of detection and diagnosis.

The antibodies or antigen binding fragments of the present invention are suitable for various

- 17 046633 applications, including, but not limited to, methods for detecting OX40. In one aspect, antibodies or antigen binding fragments are useful for detecting 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 comprises a cell or tissue. In other aspects, such tissues include normal and/or cancerous tissues that express OX40 at higher levels compared to other tissues.

In one aspect of the present invention, a method is provided for detecting the presence of OX40 in a biological sample. In certain aspects, the method includes contacting a biological sample with an anti-OX40 antibody under conditions that permit binding of the antibody to an antigen, and detecting the formation of a complex between the antibody and the antigen. The biological sample may include, but is not limited to, urine or blood samples. Also included is a method for diagnosing a disorder associated with OX40 expression. In certain aspects, the method includes contacting the test cell with an anti-OX40 antibody; determining the level of expression (either quantitatively or qualitatively) of OX40 in a test cell by detecting the binding of an anti-OX40 antibody to an 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 a cell that does not express OX40), with a higher level of OX40 expression in the test cell compared to the control cell indicates the presence of a disorder associated with OX40 expression.

Methods of treatment.

The antibodies or antigen binding fragments of the present invention are suitable for a variety of uses, including, but not limited to, methods of treating a disease or disorder associated with OX40. In one aspect, the OX40-related disease or disorder is cancer.

In one aspect of the present invention, a method of treating cancer is provided. In certain aspects, the method comprises administering to a patient in need thereof an effective amount of an anti-OX40 antibody or antigen binding fragment. Cancer may include, without limitation, breast cancer, head and neck cancer, stomach cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma or sarcoma. The antibody or antigen binding fragment of the present invention can be administered by any suitable route, including parenteral, intrapulmonary and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosage may be administered by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. Various dosage regimens are discussed herein, including, but not limited to, single or multiple administrations at different times, bolus administration, and pulsatile infusion.

The antibodies or antigen binding fragments of the present invention will be formulated, dosed and administered in accordance with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the route of administration, the schedule of administration and other factors known to practitioners. The antibody need not be, but may be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors described above. Typically, these other agents are administered at the same dosages and routes of administration as described herein, or at doses that are about 1-99% of the doses described herein, or at any dose and route. administrations empirically/clinically considered appropriate. For the prevention or treatment of a disease, the appropriate dosage of an antibody or antigen binding fragment of the present invention will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, administration of the antibody for prophylactic or therapeutic purposes, prior therapy, the patient's medical history and response to the antibody. and at the discretion of the attending physician. The antibody is suitably administered to the patient either once or over a series of treatments. Depending on the type and severity of the disease, the initial intended dosage to be administered to a patient, for example, by one or more separate administrations or by continuous infusion, is from about 1 μg/kg to 100 mg/kg of antibody. One typical daily dose may range from about 1 mcg/kg to 100 mg/kg or more depending on the above factors. With repeated administrations over several days or longer, depending on the condition, treatment is usually continued until the desired suppression of disease symptoms. Such doses may be administered intermittently, for example, every week or every three weeks (eg, such that the patient receives from about two to about twenty doses, or, for example, about six doses of the antibody). On

- 18 046633 an initial higher loading dose may be administered followed by one or more lower doses. However, other treatment regimens may be suitable. The progress of such treatment can be monitored using traditional methods and tests.

Combination therapy.

In one aspect, the anti-OX40 antibodies of the present invention can be used in combination with other therapeutic agents, for example, an anti-TIGIT antibody. Other therapeutic agents that may be used with the anti-OX40 antibodies of the present invention include, but are not limited to: chemotherapy agent (eg, paclitaxel or paclitaxel agent; (eg, Abraxane®), 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, tyrosine kinase inhibitor (eg, EGFR inhibitor (eg, Erlotini) b) , multikinase inhibitor (eg, MGCD265, RGB-2866), CD-20 targeting agent (eg, rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeting agent (eg, alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bc1 inhibitor -2 (eg, oblimersen sodium), aurora kinase inhibitor (eg, MLN8237, TAK-901), proteasome inhibitor (eg, bortezomib), CD-19 targeting agent (eg, MEDI-551, MOR208), MEK inhibitor (eg , ABT-348), JAK-2 inhibitor (eg, INCB018424), mTOR kinase inhibitor (eg, temsirolimus, everolimus), BCR/ABL inhibitor (eg, imatinib), ET-A receptor antagonist (eg, ZD4054), TRAIL agonist receptor 2 (TR-2) (eg, CS-1008), HGF/SF inhibitor (eg, AmG 102), EGEN-001, Polo-like kinase 1 inhibitor (eg, BI 672).

An anti-OX40 antibody in combination with an anti-TIGIT antibody as disclosed herein can be administered by various known routes, for example, orally, topically, rectally, parenterally, by inhalation spray, or via an implanted reservoir, although the most appropriate route in any particular case will be depend on the specific host and the nature and severity of the conditions for which the active ingredient is administered. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The combination of an anti-OX40 antibody and an anti-TIGIT antibody can be administered in various ways. Each antibody can be administered parenterally, for example, subcutaneously, intradermally, intravenously or intraperitoneally, independently of the other antibody. In one embodiment, the anti-OX40 antibody or anti-TIGIT antibody is administered once daily (once daily, QID), twice daily (bid, twice daily), three times daily, four once a day or five times a day depending on the patient's needs.

Pharmaceutical compositions and formulations.

Also provided are compositions, including pharmaceutical compositions, containing an anti-OX40 antibody or an antigen-binding fragment, or polynucleotides containing sequences encoding an anti-OX40 antibody or an antigen-binding fragment. In certain embodiments, the compositions contain 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 further contain suitable carriers, such as pharmaceutically acceptable excipients, including buffers, which are well known in the art. Pharmaceutical formulations of an anti-OX40 antibody or antigen binding fragment as described herein are prepared by admixing such antibody or antigen binding fragment having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to: buffers, for example, phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (eg, octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (containing less than 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA (ethylenediaminetetraacetic acid); sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG). As used herein, examples of pharmaceutically acceptable carriers further include agents for dispersing the drug in the intercellular space, for example, soluble

- 19 046633 neutral active hyaluronidase glycoproteins (sHASEGP), e.g. soluble human hyaluronidase glycoproteins PH-20, e.g. rHuPH20 (HYLENEX®, Baxter International, Inc.). Some exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Pat. Nos. 7,871,607 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

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 WO2006/044908, the latter formulations containing a histidine acetate buffer.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of molded articles such as films or microcapsules.

Formulations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterilizing filtration membranes.

Examples

Example 1. Preparation of monoclonal antibody to OX40.

Monoclonal antibodies to OX40 were generated using traditional hybridoma fusion technology (de StGroth and Sheidegger, 1980 J Immunol Methods 35:1; Mechetner, 2007 Methods Mol Biol 378:1) with minor modifications. Antibodies with high binding activity in enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) were selected for further characterization.

Recombinant OX40 proteins for immunization and binding assays The cDNA encoding full-length human OX40 (SEQ ID NO: 1) was synthesized by Sino Biological (Beijing, China) based on the GenBank sequence (accession number: X75962.1). The signal peptide coding region and extracellular domain (ECD) consisting of amino acids (AA) 1-216 from OX-40 (SEQ ID NO: 2) were amplified by PCR (polymerase chain reaction) and cloned into self-developed expression vectors with C -end fused to the mouse IgG2a Fc domain, the human wild-type IgG1 heavy chain Fc domain, or a His tag, resulting in three recombinant fusion protein expression plasmids, OX40-mIgG2a, OX40-huIgG1, and OX40-His, respectively. A schematic representation of the OX40 fusion proteins is shown in FIG. 1. To obtain the recombinant fusion protein, the expression plasmids OX40-mIgG2a, OX40-huIgG1 and OX40-His were transiently transfected into 293G cells and cultured for 7 days in a CO ₂ incubator equipped with a rotating shaker. The supernatant containing the recombinant protein was collected and purified by centrifugation. OX40mIgG2a and OX40-huIgG1 were purified using a protein A column (Cat: 17-5438-02, GE Life Sciences). OX40-His was purified using a Ni Sepharose column (Cat: 17-5318-02, GE Life Science). OX40mIgG2a, OX40-huIgG, and OX40-His proteins were dialyzed to remove phosphate-buffered saline (PBS) and stored in a freezer at −80°C in small aliquots.

Cell lines with stable expression.

To obtain stable cell lines that express full-length human OX40 (OX40) or cynox40 OX40, these genes were cloned into the retroviral vector pFB-Neo (Cat: 217561, Agilent, USA). Retroviral transduction was performed based on the protocol described previously (Zhang et al., 2005). HuT78 and HEK293 cells were retrovirally transduced with virus containing human OX40 or CynoOX40, respectively, to generate the HuT78/OX40, HEK293/OX40, and HuT78/CynoOX40 cell lines.

Immunization, hybridoma fusion and cloning.

Eight to twelve week old Balb/c mice (from HFK BIOSCIENCE CO., LTD, Beijing, China) were immunized intraperitoneally with 200 μl of an antigen mixture containing 10 μg of OX40-mIgG2a and Quick-Antibody immunoadjuvant (Cat: KX0210041, KangBiQuan, Beijing, China ). The procedure was repeated after three weeks. Two weeks after the 2nd immunization, mouse sera were assessed for OX40 binding by ELISA and FACS. Ten days after serum screening, mice with the highest serum OX40 antibody titers were boosted by intraperitoneal injection of 10 μg of OX40-mIgG2a. Three days after the boost, splenocytes were isolated and fused with the murine myeloma cell line, SP2/0 cells (ATCC, Manassas VA) using standard methods (Somat Cell Genet, 1977 3:231). Evaluation of OX40 Antibody Binding Activity by ELISA and FACS Supernatants of hybridoma clones were initially screened by ELISA as described in (Methods in Molecular Biology (2007) 378:33-52) with some modifications. Briefly, OX40-His protein was coated into 96-well plates at 4°C overnight. After washing with PBS/0.05% Tween-20, the plates were blocked with PBS/3% BSA (bovine serum albumin) for 2 h at room temperature. The plates were then washed with PBS/0.05% Tween-20 and incubated with cell supernatants at room temperature for 1 hour. Anti-mouse IgG HRP (horseradish peroxidase)-coupled antibody (Cat: 115035-008, Jackson ImmunoResearch Inc, AffiniPure, Goat anti-IgG antibodies

- 20 046633 mouse horseradish peroxidase conjugated specific Fcy fragment) and substrate (Cat: 00-4201-56, eBioscience, USA) were used to obtain a color absorption signal at 450 nm, which was measured using a plate reader (SpectraMax Paradigm , Molecular Devices/PHERAstar, BMG LABTECH). Positive parental clones were selected by fusion screening using indirect ELISA. ELISA-positive clones were further checked by FACS using HuT78/OX40 and HuT78/cynoOX40 cells described above. OX40-expressing cells (105 cells/well) were incubated with ELISA-positive hybridoma supernatants, followed by binding with eFluor® 660 anti-mouse IgG antibodies (Cat: 50-4010-82, eBioscience, USA). Cellular fluorescence was quantified using a flow cytometer (Guava easyCyte 8HT, Merck-Millipore, USA).

Conditioned media from hybridomas that showed positive signals in both the ELISA and FACS screens were subjected to functional assays to identify antibodies with good functional activity in human immune cell-based assays (see following sections). Antibodies with the desired functional activity were further subcloned and characterized. Subcloning and adaptation of hybridomas to serum-free or low-serum media 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 validated by functional assays and adapted for growth in CDM4MAb medium (Cat: SH30801.02, Hyclone, USA) with 3% FBS (fetal bovine serum). Expression and purification of monoclonal antibodies

Hybridoma cells expressing the best antibody clones were cultured in CDM4MAb medium (Cat: SH30801.02, Hyclone) and incubated in a CO ₂ incubator for 5-7 days at 37°C. The conditioned medium was collected by centrifugation and filtered before purification through a 0.22 μm membrane. Mouse antibodies in the supernatants were applied and coupled to a protein A column (Cat: 17-5438-02, GE Life Sciences) according to the manufacturer's manual. The procedure typically produced antibodies with a purity greater than 90%. Protein A affinity purified antibodies were either dialyzed from PBS or, if necessary, further purified using a HiLoad 16/60 Superdex 200 column (Cat: 28-9893-35, GE Life Sciences) to remove aggregates. Protein concentrations were determined by measuring absorbance at 280 nm. The final antibody preparations were stored in aliquots in a freezer at minus 80°C.

Example 2. Cloning and sequence analysis of antibodies to OX40.

Mouse hybridoma clones were collected to obtain total cellular RNAs using the Ultrapure RNA kit (Cat: 74104, QIAGEN, Germany) based on the manufacturer's protocol. First-strand cDNA was synthesized using a cDNA synthesis kit from Invitrogen (Cat: 18080-051), and PNR amplification of VH and VL hybridoma antibodies was performed using a PNR kit (Cat: CW0686, CWBio, Beijing, China). Oligoprimers used for cloning variable heavy chain (VH) and variable light chain (VL) antibody cDNAs were synthesized using Invitrogen (Beijing, China) based on the sequences described previously (Brocks et al. 2001 Mol Med 7: 461). PNR products were used directly for sequencing or subcloned into the pEASY-Blunt cloning vector (Cat: CB101 TransGen, China) followed by sequencing using Genewiz (Beijing, China). The amino acid sequences of the VH and VL regions were deduced from DNA sequencing results.

The complementarity determining regions (CDRs) of murine antibodies were determined based on the Kabat system (Wu and Kabat 1970 J. Exp. Med. 132:211-250) through sequence annotation and using a computer sequence analysis program. The amino acid sequences of the representative best clone Mu445 (VH and VL) are given in Table. 1 (SEQ ID NO: 9 and 11). Mu445 CDR sequences are given in Table. 2 (SEQ ID NO: 3-8).

Table 1

Amino acid sequences of the VH and VL regions of Mi445

Mu445 VH SEQ GO NO: 9 EVQLQQSGPELVKPGASVKMSCKASGYKFTSYIIHWV KQKPGQGLEWIGYINPYNDGTRYNEKFKGKATLTSD KS S ST AYME YS SLT SED S AVYYC ARGYYGS S YAMD Y WGQGTSVTVSS Mu445 VL SEQIDNO: 11 DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQ KPDGTIKLLIYDTSTLYSGVPSRFSGSGSGTDYFLTISN LEPEDIATYYCQQYSKLPYTFGGGTKLEKK

- 21 046633

table 2

CDR (amino acid) sequences of the VH and VL regions of the murine monoclonal antibody Mu445

Antibody SEQ Ш NO CDR Subsequence Mu445 SEQ Ш NO: 3 HCDR1 (according to Kabat) SYIIH SEQ Ш NO: 4 HCDR2 (according to Kabat) YINPYNDGTRYNEKFKG SEQ Ш NO: 5 HCDR3 (according to Kabat) GYYGSSYAMDY SEQ Ш NO: 6 LCDR1 (according to Kabat) SASQGISNYLN SEQ Ш NO: 7 LCDR2 (according to Kabat) DTSTLYS SEQ Ш NO: 8 LCDR3 (according to Kabat) QQYSKLPYT

Example 3. Humanization of mouse anti-human OX40 antibody 445. Humanization and construction of the antibody.

To humanize Mu445 in human germline IgG genes, sequences that have a high degree of homology to Mu445 variable region cDNA sequences were searched for by comparing the sequences with the human immunoglobulin gene database in the IMGT (immunoglobulin gene database). The human IGHV and IGKV genes, which are present in the human antibody repertoire with high frequency (Glanville et al., 2009 PNAS 106:20216-20221) and are highly homologous to Mu445, were chosen as templates for humanization.

Humanization was performed using CDR grafting (Methods in Molecular Biology, Antibody Engineering, Methods and Protocols, Vol 248: Humana Press), and humanized antibodies were constructed in human wild-type IgG1 format using a self-developed expression vector. At the initial stage of humanization, mutations from mouse to human amino acid residues in the framework regions were determined using simulated 3D structural analysis, and mouse framework residues with structural significance for maintaining canonical CDR structures were retained in the first version of the humanized antibody 445 (see 445-1, table .3). Six CDRs of 445-1 have the amino acid sequences 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). The heavy chain variable region of 445-1 has the amino acid sequence (VH) of SEQ ID NO: 14, which is encoded by the nucleotide sequence of SEQ ID NO: 15, and the light chain variable region has the amino acid sequence (VL) of SEQ ID NO: 16, which is encoded by the nucleotide sequence SEQ ID NO: 17. Specifically, LCDR Mu445 (SEQ ID NO: 6-8) was grafted into the human germline variable gene backbone IGVK1-39, retaining two murine backbone 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 grafted into the human germline variable gene IGHV1-69 scaffold, retaining two murine scaffold residues ( _L70 and _S72 ) (SEQ ID NO: 14). In the humanization variants, antibody 445 (445-1) grafted only the N-terminal half of Kabat's HCDR2 because only the N-terminal half is predicted to be important for antigen binding according to the simulated 3D structure. Antibody 445-1 was constructed as a humanized full-length antibody using in-house developed expression vectors that contain the human IgG1 wild-type (IgG1wt) and kappa chain constant regions, respectively, with easy adaptation subcloning sites. Antibody 445-1 was expressed by co-transfecting the above two constructs into 293G cells and purified using a Protein A column (Cat: 17-5438-02, GE Life Sciences). The purified antibody was concentrated to 0.5-10 mg/ml in PBS and stored in aliquots in a freezer at minus 80°C.

Using antibody 445-1, several single amino acid changes were made converting mouse retained residues in the VH and VL framework 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 to the CDR to reduce the potential risk of isomerization and increase the level of humanization. For example, changes T51A and D50E were made in LCDR2, and changes D56E, G57A and N61A were made in HCDR2. All humanization changes were performed using primers containing mutations at specific positions and a site-directed mutagenesis kit (Cat: AP231-11, TransGen, Beijing, China). Desired changes were verified by sequencing.

Amino acid changes in antibody 445-1 were assessed by their binding to OX40 and thermal stability. Antibody 445-2 containing HCDR1 from SEQ ID NO: 3, HCDR2 from SEQ ID NO: 18, HCDR3 from SEQ ID NO: 5, LCDR1 from SEQ ID NO: 6, LCDR2 from SEQ ID NO: 19 and LCDR3 from SEQ ID NO:8 (see Table 3) was constructed from a combination of the specific changes described above. When comparing the two antibodies, the results showed that both antibodies 445-2 and 445-1 showed comparable binding affinities (see Table 4 and Table 5 below).

- 22 046633

Beginning with antibody 445-2, several additional amino acid changes in the VL framework region were made to further improve binding affinity/kinetics, such as amino acid changes G41D and K42G. Additionally, several single amino acid changes in the CDRs of both VH and VL were made with to reduce the risk of immunogenicity and increase thermal stability, for example, S24R in LCDR1 and A61N in HCDR2. The resulting changes showed either improved binding activity or thermal stability compared to 445-2.

The 445 humanized antibodies were further engineered by introducing specific amino acid changes into the CDR and framework regions to improve molecular and biophysical properties for therapeutic use in humans. Factors to consider included removal of deleterious post-translational modifications, improved thermal stability (Tm), surface hydrophobicity, and isoelectronic points (pI).

Humanized monoclonal antibody 445-3 comprising HCDR1 from SEQ ID NO: 3, HCDR2 from SEQ ID NO: 24, HCDR 3 from SEQ ID NO: 5, LCDR1 from SEQ ID NO: 25, LCDR2 from SEQ ID NO: 19 and LCDR3 from SEQ ID NO: 8 (see Table 3), was constructed through the maturation process described above and is described in detail. Antibody 445-3 was also produced in an IgG2 version (445-3 IgG2) containing the Fc domain of the wild-type human IgG2 heavy chain, and an IgG4 version containing the Fc domain of human IgG4 with the S228P and R409K27 mutations (445-3 IgG4). The results showed that antibodies 445-3 and 445-2 showed comparable binding affinities (see Table 4 and Table 5).

Table 3

Antibody 445 sequences

Antibody SEQ GO NO Subsequence 445-1 SEQ GO NO: 3 HCDR1 (according to Kabat) SYIIH SEQIDNO: 13 HCDR2 (according to Kabat) YINPYNDGTRYNQKFQG SEQ GO NO: 5 HCDR3 (according to Kabat) GYYGSSYAMDY SEQ GO NO: 6 LCDR1 (according to Kabat) SASQGISNYLN SEQ GO NO: 7 LCDR2 (according to Kabat) DTSTLYS SEQ GO NO: 8 LCDR3 (according to Kabat) QQYSKLPYT SEQ GO NO: 14 VH QVQLVQSGAEVKKPGSSVKVSCKASGYKF TSYIIHWVRQAPGQGLEWMGYINPYNDGT RYNQKFQGRVTLTSDKSTSTAYMELSSLRS EDT AVYYC ARGYYGS S YAMD YWGQGTT V SS

- 23 046633

SEQIDNO: 16 VL DIQMTQSPSSLSASVGDRVTITCSASQGISN YLNWYQQKPGKAIKLLIYDTSTLYSGVPSR FSGSGSGTDYTLTISSLQPEDFATYYCQQYS KLPYTFGGGTKVEIK 445-2 SEQ GO NO: 3 HCDR1 (no Kabatu) SYIIH SEQIDNO: 18 HCDR2 (no Kabatu) YINPYNEGTRYAQKFQG SEQ ID NO: 5 HCDR3 (no Kabatu) GYYGSSYAMDY SEQ ID NO: 6 LCDR1 (no Kabatu) SASQGISNYLN SEQIDNO: 19 LCDR2 (no Kabatu) DASTLYS SEQ ID NO: 8 LCDR3 (no Kabatu) QQYSKLPYT SEQ ID NO: 20 VH QVQLVQSGAEVKKPGSSVKVSCKASGYKF TSYIIHWVRQAPGQGLEWMGYINPYNEGT RYAQKFQGRVTLTADKSTSTAYMELSSLRS EDT AVYYC ARGYYGS S YAMD YWGQGTT V TVSS SEQ ID NO: 22 VL DIQMTQSPSSLSASVGDRVTITCSASQGISN YLNWYQQKPGKAIKLLIYDASTLYSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQYS KLPYTFGGGTKVEIK 445-3 SEQ ID NO: 3 HCDR1 (no Kabatu) SYIIH SEQ ID NO: 24 HCDR2 (no Kabatu) YINPYNEGTRYNQKFQG SEQ ID NO: 5 HCDR3 (no Kabatu) GYYGSSYAMDY SEQ ID NO: 25 LCDR1 (no Kabatu) RASQGISNYLN SEQIDNO: 19 LCDR2 (no DASTLYS

- 24 046633

Kabatu) SEQ GO NO: 8 LCDR3 (according to Kabat) QQYSKLPYT SEQ GO NO: 26 VH QVQLVQSGAEVKKPGSSVKVSCKASGYKF TSYIIHWVRQAPGQGLEWMGYINPYNEGT RYNQKFQGRVTLTADKSTSTAYMELSSLRS EDTAVYYC ARGY YYGS S YAMD YWGQGTT VTVSS SEQ GO NO: 28 VL DIQMTQSPSSLSASVGDRVTITCRASQGISN YLNWYQQKPDGAIKLLIYDASTLYSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYYCQQY SKLPYTFGGGTKVEIK

Example 4. Determination of the kinetics and binding affinity of antibodies to OX40 using SPR (Surface Plasmon Resonance).

Antibodies to OX40 were characterized for their kinetics and binding affinity using SPR assays using BIAcore™ T-200 (GE Life Sciences). Briefly, anti-human IgG antibody was immobilized on an activated CM5 biosensor chip (Cat: BR100530, GE Life Sciences). An antibody with the F region of human IgG was passed over the surface of the chip and captured by an antibody to human IgG. Then, serially diluted His-tagged recombinant OX40 protein (Cat: 10481-H08H, Sino Biological) was passed over the chip surface and changes in surface plasmon resonance signals were analyzed to calculate association rates (ka) and dissociation rates (kd) using the binding model Langmuir one-to-one (BIA Evaluation Software, GE Life Sciences). The equilibrium dissociation constant (KD) was calculated as the ratio of kd to ka. The results of the anti-OX40 antibody binding profiles determined by SPR are summarized in FIG. 2 and in table. 4. The binding profile with average KD value of antibody 445-3 (9.47 nM) was slightly better than that of antibody 445-2 (13.5 nM) and 445-1 (17.1 nM), and similar to that of at ch445. The binding profile of 445-3 IgG4 was similar to that of 445-3 (with IgG1 Fc), indicating that the change in Fc between IgG4 and IgG1 did not alter the specific binding of antibody 445-3.

Table 4

Binding affinities of antibodies to OX40 using SPR

Test takers ch445* 445-1 445-2 445-3 445-3 IgG4 options Test 1 ka (M'V) 1.74 x 10 ⁵ 1.56 x 10 ⁵ 2.76 x 10 ⁵ 1.82 x 10 ⁵ 1.61 x 10 ⁵ kd (with ¹ ) 1.43 x IO' ³ 2.77 x IO' ³ 3.90 x IO' ³ 1.67 x IO' ³ 1.61 x IO' ³ K _D (nM) 8.26 17.8 14.2 9.16 10.0 K _A (Μ' ¹ ) 1.22 x ^10s 0.56 x ^10s 0.71 x ^10s 1.09 x ^10s 1.00 x 10 ^s Test 2 ka (M'V) 2.65 x 10 ⁵ 2.37 x 10 ⁵ 2.06 x 10 ⁵ 1.63 x 10 ⁵ — kd (c ¹ ) 1.67 x IO' ³ 3.89 x IO' ³ 2.64 x IO' ³ 1.59 x IO' ³ — K _D (nM) 6.3 16.4 12.8 9.77 — K _A (Μ' ¹ ) 1.59 x ^10s 0.61 x ^10s 0.78 x ^10s 1.03 x ^10s — Average _Kd (hM) 7.28 17.1 13.5 9.47 10.0 K _a (Μ' ¹ ) 1.41 x ^10s 0.59 x ^10s 0.75 x 10 ^s 1.06 x ^10s 1.00 x 10 ^s

*ch445 consists of Mu445 variable domains fused to human IgG1wt/kappa constant regions

Example 5. Determination of the binding affinity of antibodies to OX40 with OX40 expressed on HuT78 cells.

To evaluate the binding activity of anti-OX40 antibodies to OX40 expressed on the surface of living cells, HuT78 cells were transfected with human OX40 as described in Example 1 to generate an OX40 expression line. Live HuT78/OX40 cells were seeded in a 96-well plate and incubated with serially diluted various anti-OX40 antibodies. Goat anti-human IgG-FITC antibody

- 25 046633 (Cat: A0556, Beyotime) was used as a secondary antibody to detect antibody binding to the cell surface. _EC50 (half maximal effective concentration) values for dose-dependent binding to human OX40 were determined by fitting dose-response data to a four-parameter logistic model using GraphPad Prism. As shown in FIG. 3 and in table. 5, anti-OX40 antibodies had high affinity for OX40. It was also found that the anti-OX40 antibodies of the present invention had a relatively higher upper level of fluorescence intensity measured by flow cytometry (see last column in Table 5), indicating a slower dissociation of the antibody from OX40, which is more desirable binding profile.

Table 5

EC ₅₀ dose-dependent binding of humanized variants 445 to OX40

Antibody EC50 (µg/ml) Best (MFI)

Test 1 Test 2 Average Average ch445 0.321 0.277 0.299 725 445-1 0.293 0.278 0.285 525 445-2 0.323 0.363 0.343 620 445-3 0.337 0.319 0.328 910 445-3 0.263 N/A (No data available) 0.263 892

Example 6. Determination of cross-reactivity of antibodies to OX40.

To assess the cross-reactivity of the 445-3 antibody to human and cynomolgus OX40, cells expressing human OX40 (HuT78/OX40) and cynomolgus OX40 (HuT78/CynoOX40) were seeded in 96-well plates and incubated with a series of dilutions of anti-OX40 antibodies. Goat anti-human IgG-FITC antibody (Cat: A0556, Beyotime) was used as a secondary detection antibody. _EC50 values for dose-dependent binding to native human and cynomolgus OX40 were determined by fitting dose-response data to a four-parameter logistic model using GraphPad Prism. The result is shown in Fig. 4 and below in table. 6. Antibody 445-3 cross-reacts with both human and cynomolgus OX40 with similar EC ₅₀ values as shown below.

Table 6

EC50 binding of antibody 445-3 to human and cynomolgus monkey OX40

Cell line EC50 (µg/ml) 445-3 Best (MFI) NiT78/OX40 0.174 575 HuT78/CynoOX40 0.171 594

Example 7. Cocrystallization and structural determination of OX40 with Fab 445-3.

To understand the binding mechanism of OX40 to the antibodies of the present invention, the cocrystal structure of OX40 and Fab 445-3 was solved. Mutations at residues T148 and N160 were introduced to block OX40 glycosylation and improve protein homogeneity. DNA encoding mutant human OX40 (M1-D170 residues with two mutated sites, T148A and N160A) was cloned into an expression vector incorporating a hexa-His tag, and the construct was transiently transfected into 293G cells for protein expression at 37°C for 7 days. Cells and supernatant were collected and incubated with His-tagged affinity resin at 4°C for 1 hour. The resin was washed three times with a buffer containing 20 mM Tris, pH 8.0, 300 mM NaCl, and 30 mM imidazole. The OX40 protein was then eluted with a buffer containing 20 mM Tris, pH 8.0, 300 mM NaCl, and 250 mM imidazole, followed by further purification with Superdex 200 (GE Healthcare) in a buffer containing 20 mM Tris, pH 8.0, 100 mM NaCl .

The heavy chain and light chain coding sequences of Fab 445-3 were cloned into an expression vector incorporating a hexa-His tag at the C terminus of the heavy chain, and they were transiently cotransfected into 293G cells for protein expression at 37°C for 7 days. The purification steps for Fab 445-3 were the same as for the OX40 mutant protein above. Purified OX40 and Fab 445-3 were mixed in a 1:1 molar ratio and incubated for 30 min on ice, followed by additional purification with Superdex 200 (GE Healthcare) in a buffer containing 20 mM Tris, pH 8.0, 100 mM NaCl. The peak of the complex was collected and concentrated to approximately 30 mg/ml.

Cocrystal screening was performed by mixing the protein complex with the reservoir solution in a 1:1 volume ratio. Cocrystals were prepared from hanging drops cultured at 20°C by vapor diffusion with a reservoir solution containing 0.1 M HEPES (4-(2-hydroxyethyl)-1

- 26 046633 piperazine ethanesulfonic acid), pH 7.0, 1% PEG 2000 MME and 0.95 M sodium succinate.

Nylon loops were used to collect cocrystals, and the crystals were immersed in a reservoir solution supplemented with 20% glycerol for 10 s. Diffraction data were collected at the BL17U1 synchrotron at the Shanghai Synchrotron Radiation Facility and processed using the XDS program. The phase was solved in PHASER using the Fab IgG structure (C and D chains in PDB: 5CZX) and the OX40 structure (R chain in PDB: 2HEV) as search models for molecular replacement. The Phenix.refme GUI was used for rigid body, TLS (translational/librational/helical) refinement, and limited X-ray refinement, followed by COOT correction and further refinement in Phenix.refine. Statistics on the collection and refinement of X-ray data are summarized in Table. 7.

Table 7

Statistics of data collection and clarification

Data collection Radiation source BL17U1,SSRF Space group R 31 2 1 Cell dimensions (A) a = 183.96 b = 183.96 c = 79.09 Angles (°) a = 90.00 β = 90.00 γ = 120.00 Resolution (A) 159.3-2.55 (2.63-2.55) Total number of reflexes 988771(81305) Number of unique reflexes 50306 (4625) Completeness (%) 99.9 (99.9) Repeatability 19.7(17.6) Merge ^a 0.059 (0.962) I/sigma (I) 29.4 (3.5) Wilson's ratio B (A) 73.9 Clarification Resolution (A) 60.22-2.55 Number of reflexes 50008 rmsd (standard deviation) of bond lengths (A) 0.010 rmsd of brace angles (°) 0.856 Rwork ^b (%) 19.27 Rfree ^C (%) 21.60 Average protein B factors 97.10 Ramachandran Map (%) Appropriate 96.34 Acceptable 3.48 Outliers 0.17

Values in parentheses refer to the highest resolution shell. ^a Rmerge = ΣΣϊΙ-ΠΝί where - represents the average intensity of the equivalent. ^b Rwork = ΣΙΤο - Fel/ΣΙΤοΙ

- 27 046633 where Fo and Fc represent the observed and calculated structure factor amplitudes, respectively.

^C Rfree = ΣίΓο -Fc|/2|Fo| calculated using a test data set, with 5% of the total data randomly selected from the observed reflexes.

Example 8. Identification of the epitope of antibody 445-3 using SPR.

Guided by the co-crystal structure of OX40 and the Fab 445-3 antibody, we selected and generated a series of single mutations in the human OX40 protein to further identify key epitopes of the OX40 antibodies of the present invention. Single-point mutations were made in the human OX40/IgG1 fusion construct using a site-directed mutagenesis kit (Cat: AP231-11, TransGen). Desirable mutations were verified by sequencing. Expression and production of OX40 mutants was achieved by transfection into 293G cells and purified using a protein A column (Cat: 17-5438-02, GE Life Sciences).

The binding affinity of OX40 point mutants to Fab 445-3 was characterized by NIR assays using BIAcore 8K (GE Life Sciences). Briefly, OX40 mutants and wild-type OX40 were immobilized on a CM5 biosensor chip (Cat: BR100530, GE Life Sciences) using EDC (1-Ethyl-3(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide). Then, serially diluted Fab 445-3 in HBS-EP+ buffer (Cat: BR-1008-26, GE Life Sciences) was passed over the chip surface with a contact time of 180 s and a dissociation time of 600 s at 30 μL/min. Changes in surface plasmon resonance signals were analyzed to calculate association rates (ka) and dissociation rates (kd) using a one-to-one Langmuir binding model (BIA Evaluation Software, GE Life Sciences). The equilibrium dissociation constant ( _KD ) was calculated as the ratio of kd to ka. The KD fold shift for the mutant was calculated as the ratio of the KD of the mutant to the KD of the WT (wild type). The epitope identification profiles determined by NIR are summarized in FIG. 5 and in table. 8. The results showed that mutation of residues H153, 1165 and E167 to alanine in OX40 significantly reduced the binding of antibody 445-3 to OX40, and mutation of residues T154 and D170 to alanine moderately reduced binding of antibody 445-3 to OX40.

Detailed interactions between antibody 445-3 and residues H153, T154, 1165, E167, and D170 in OX40 are shown in FIG. 6. The H153 side chain on OX40 was surrounded by a small 445-3 pocket on the interaction surface, forming hydrogen bonds with heavy S31 and heavy G102 and π-π interaction with heavy Y101. The side chain of E167 forms hydrogen bonds with heavy Y50 and heavy N2, while D170 forms a hydrogen bond and salt bridge with heavy S31 and heavy K28, respectively, which may further stabilize the complex. Van der Waals (VDW) interactions between T154 and heavy Y105, I165 and heavy R59 contributed to the high affinity of antibody 445-3 for OX40. In conclusion, residues H153, I165 and E167 in OX40 were identified as important residues for interaction with antibody 445-3. In addition, amino acids T154 and D170 in OX40 are also important contact residues for antibody 445-3. These data indicate that the epitopes of antibody 445-3 are residues H153, T154, I165, E167, and D170 in OX40. These epitopes are in the sequence HTLQPASNSSDAICEDRD (SEQ ID NO: 30) with important contact residues in bold and underlined.

Table 8

Identification of the epitope of antibody 445-3 determined using SPR

Mutants KD Mutant / KD WT H153A No binding detected T154A 8 Q156A 1.9 S161A 1.1 S162A 0.6 I165A 28 E167A 135 D170A 8

Significant impact: no binding detected, or Mutant KD/WT KD value was greater than 10.

Moderate Impact: Mutant KD/WT KD was rated between 5 and 10.

Minor impact: Mutant KD/WT KD value was less than 5.

Example 9 Antibody 445-3 to OX40 does not block the OX40-OX40L interaction.

- 28 046633

To determine whether antibody 445-3 interferes with the OX40-OX40L interaction, cell-based flow cytometry analysis was performed. In this assay, antibody 445-3, reference antibody 1A7.gr1, control huIgG, or medium alone were preincubated with human OX40-mouse IgG2a Fc fusion protein (OX40-mIgG2a). The antibody-fusion protein complex was then added to HEK293 cells expressing OX40L. If anti-OX40 antibody does not interfere with the OX40-OX40L interaction, then the anti-OX40-OX40-mIgG2a complex will still bind to the surface of OX40L, and this interaction is detected by a secondary anti-mouse Fc antibody. As shown in FIG. 7, antibody 445-3, even at high concentration, does not reduce the binding of OX40 to OX40L, 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 binds close enough to sterically interfere with OX40L binding. In contrast, the positive control antibody 1A7.gr1 completely blocks OX40 binding to OX40L, as shown in FIG. 7.

In addition, the cocrystal structure of OX40 in complex with Fab 445-3 was solved and aligned with the OX40/OX40L complex (PDB code: 2HEV), as shown in Fig. 8. The OX40 ligand trimer interacts with OX40 primarily through CRD1 (cysteine-rich domain), CRD2, and partial CRD3 regions of OX40 (Compaan and Hymowitz, 2006), while antibody 445-3 interacts with OX40 only through the CRD4 region. Thus, antibody 445-3 and the OX40L trimer bind at different respective regions of OX40, and antibody 445-3 does not interfere with the OX40/OX40L interaction. This result correlates with the epitope mapping data described in the Examples above. CRD4 in OX40 is located at amino acids 127-167, and the epitope of antibody 445-3 partially overlaps this region. The OX40 sequence of CRD4 (amino acids 127-167) is shown below, with the partial overlap of the 445-3 epitope in bold and underlined: PCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICE (SEQ ID NO: 31).

Example 10. Agonistic activity of antibody 445-3 to OX40.

To investigate the agonistic functions of antibody 445-3, an OX40-positive T cell line (HuT78/OX40) was co-cultured with an artificial antigen presenting cell (APC) line (HEK293/OS81ow-FcyRI) in the presence or absence of 445-3 or 1A7.gr1 overnight, and IL-2 production was used as an indication for T cell stimulation. In HEK293/OS8 ^Low -FcyRI genes encoding membrane-bound anti-CD3 antibody OKT3 (OS8) (as disclosed in US Pat. No. 8,735,553) and human FcyRI (CD64) were stably co-transduced into HEK293 cells. Since anti-OX40 antibody-induced immune activation is dependent on antibody cross-linking (Voo et al., 2013), FcyRI on HEK293/OS8 ^Low -FcyRI provides the basis for anti-OX40 antibody-mediated cross-linking of OX40 by dual interaction of anti-OX40 antibody with both OX40 and FcyRI. As shown in FIG. 9, anti-OX40 antibody 445-3 was highly effective in enhancing TCR signaling in a dose-dependent manner with an EC50 of 0.06 ng/mL. Slightly weaker activities of the reference antibody 1A7.gr1 were also observed. In contrast, human IgG control (10 μg/ml) or blank control had no effect on IL-2 production.

Example 11 Antibody 445-3 to OX40 stimulated immune responses in a mixed lymphocyte culture (MSC) assay.

To determine whether antibody 445-3 could stimulate T cell activation, a mixed culture lymphocyte (MLC) assay was performed as described previously (Tourkova et al., 2001). Briefly, mature DCs were induced from human PBMC-derived CD14+ myeloid cells by culture with GM-CSF and IL-4 followed by LPS (liposaccharide) stimulation. Mitomycin C-treated DCs were then cocultured with allogeneic CD4+ T cells in the presence of anti-OX40 antibody 445-3 (0.1-10 μg/ml) for 2 days. IL-2 production in co-culture was detected by ELISA as an indication of the SCL response.

As shown in FIG. 10, antibody 445-3 significantly stimulated IL-2 production, indicating the ability of 445-3 to activate CD4+ T cells. In contrast, the reference antibody 1A7.gr1 showed significantly (P less than 0.05) weaker activity in the SCL assay.

Example 12 Anti-OX40 antibody 445-3 demonstrated ADCC activity.

To investigate whether antibody 445-3 could kill target cells expressing OX40 ¹¹ ', an ADCC assay based on lactate dehydrogenase (LDH) release was performed. The NK92MI/CD16V cell line was generated as effector cells by co-transducing the CD16v158 (V158 allele) and FcRy genes into the NK cell line, NK92MI (ATCC, Manassas VA). The T cell line expressing OX40, HuT78/OX40, was used as target cells. Equal numbers (3 x 10 ⁴ ) of target and effector cells were cocultured for 5 h in the presence of anti-OX40 antibody (0.004-3 μg/ml) or control antibodies. Cytotoxicity was assessed by LDH release using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI). Specific lysis was calculated using the formula below.

I Experimental lysis - Spontaneous lysis of Effector - Spontaneous lysis of Target % of Specific lysis = ----------------------------------- ------------------------------------------- x 100

Maximum Target Lysis - Spontaneous Target Lysis

- 29 046633

As shown in FIG. 11, antibody 445-3 demonstrated high efficiency in killing OX40 ^I1 targets by ADCC in a dose-dependent manner ( _EC50 : 0.027 μg/ml). The ADCC effect of antibody 445-3 was similar to that of the control antibody 1A7.gr1. In contrast, 445-3 with Fc format IgG4 with S228P and R409K mutations (445-3-IgG4) did not show any significant ADCC effects compared to human IgG control or blank control. The results are consistent with previous findings that IgG4 Fc is weak or asymptomatic for ADCC (An Z, et al. mAb 2009).

Example 13 Anti-OX40 antibody 445-3 preferentially depletes CD4+ Treg cells and increases the ratio between CD8+ Teff and Treg cells In vitro.

It has been shown in several animal tumor models that anti-OX40 antibodies can deplete tumor-infiltrating OX40 ^Hi Treg cells and increase the ratio between CD8+ T cells and Treg cells (Bulliard et al., 2014; Carboni et al., 2003; Jacquemin et al. ., 2015; Marabelle et al., 2013b). Consequently, the immune response is enhanced, leading to tumor regression and improved survival.

Considering the fact that in vitro activated or intratumoral CD4+ Foxp3 ⁺ Treg cells preferentially express OX40, in contrast to 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 performed to investigate the ability of antibody 445-3 to kill OX40 ^Hi cells, in particular Treg cells. Briefly, PBMCs were preactivated for 1 day with PHA-L (1 μg/ml) to induce OX40 expression and used as target cells. NK92MI/CD16V effector cells (as described in Example 12, 5 x 10 ⁴ ) were then co-cultured with an equal number of target cells in the presence of anti-OX40 antibodies (0.001-10 μg/ml) or placebo overnight. The percentage of each T cell subset was determined using flow cytometry. As shown in Figures 12A and 12B, treatment with antibody 445-3 induced an increase in the percentage of CD8 T cells and a decrease in the percentage of CD4 Foxp3 Treg cells in a dose-dependent manner. As a result, the ratios between CD8 ⁺ T cells and Treg cells were significantly improved (Figure 12C). Poorer results were obtained with 1A7.gr1 treatment. This result demonstrates the therapeutic utility of 445-3 in inducing antitumor immunity by enhancing CD8 ⁺ T cell functions but limiting Treg-mediated immune tolerance.

Example 14 Antibody 445-3 to OX40 exhibits dose-dependent antitumor activity in a mouse tumor model.

The effectiveness of the OX40 antibody 445-3 was demonstrated in a mouse tumor model. MC38 murine colon tumor cells were subcutaneously implanted into C57 transgenic mice carrying human OX40 (Biocytogen, Beijing, China). After implantation of tumor cells, tumor volumes were measured twice a week and calculated in mm ³ using the formula: V = 0.5 (axo), where a and b represent the dimensions along the long and short axes of the tumor, respectively. When tumors reached a mean volume of approximately 190 mm ³ , mice were randomized into 7 groups and given intraperitoneal injection of either antibody 445-3 or antibody 1A7.gr1 once weekly for three weeks. Human IgG was administered as an isotype control. Partial regression (PR) was defined as tumor volume less than 50% of initial tumor volume on the first day of dosing in three consecutive measurements. Tumor growth inhibition (TGR) was calculated using the following formula:

II /(processed volume t) - (processed volume ϋθ)λ \% growth inhibition = 100 X 1 - -----——---------— ---------- --------\ y (placebo volume t) - (placebo volume t0) J / treated volume t represents the treated tumor volume at time t, treated volume t ₀ represents the treated tumor volume at time 0, placebo volume t represents the placebo tumor volume at time t, placebo volume t ₀ represents the placebo tumor volume at time 0.

The results showed that 445-3 had dose-dependent antitumor efficacy when administered as an intraperitoneal injection at doses of 0.4 mg/kg, 2 mg/kg, and 10 mg/kg. Administration of 445-3 resulted in inhibition of tumor growth by 53% (0.4 mg/kg), 69% (2 mg/kg) and 94% (10 mg/kg) and led to partial regression by 0% (0.4 mg/kg), 17% (2 mg/kg) and 33% (10 mg/kg) relative to baseline. In contrast, no partial regression was observed with the 1A7.gr1 antibody. In vivo data indicate that the ligand non-blocking antibody 445-3 is better suited for antitumor therapy than the OX40-OX40L blocking antibody 1A7.gr1 (FIGS. 13A and 13B, Table 9).

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

Treatment Dose once a week (mg/kg) N Partial regression rate Average tumor volume on day 21 ( ^mm3 ) TRO on the 21st day (%)

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445-3 0.4 6 0% 953 53 2 6 17% 696 69 10 6 33% 280 94 lA7.grl 0.4 6 0% 886 57 2 6 0% 1163 41 10 6 0% 1030 49

Example 15. Amino acid changes in antibodies to OX40.

Several amino acids were selected for modification to improve OX40 antibodies. Amino acid changes have been made to improve affinity or to increase humanization. PCR primer sets were designed for the corresponding amino acid changes, synthesized, and used to modify the OX40 antibodies. For example, changing K28T in the heavy chain and S24R in the light chain resulted in a 1.7-fold increase in EC ₅₀ determined by FACS compared to the parent antibody 445-2. The change to Y27G in the heavy chain and S24R in the light chain resulted in a 1.7-fold increase in KD determined by Biacore compared to the parent antibody 445-2. These changes are summarized in Figures 14A-14B.

Example 16 Anti-OX40 Antibodies in Combination with Anti-TIGIT Antibodies in the Syngeneic MMTV-PyMT Mouse Model MMTV-PyMT is a murine model of breast cancer metastasis where MMTV-LTR is used to overexpress polyomavirus middle T antigen in the mammary gland. Mice develop highly metastatic tumors, and this model is commonly used to study the progression of breast cancer. Female FVB/N mice were implanted with 1 x ¹⁰⁶ intracerebral MMTV-PyMT tumor cells derived from spontaneously developed tumors in MMTV-PyMT transgenic mice. After inoculation for 8 days, the animals were randomized into 4 groups of 15 animals in each group. One group of mice was treated with vehicle (PBS) as a control.

OX86 is a rat anti-mouse OX40 antibody previously disclosed in WO2016/057667 that was further engineered with mouse IgG2a constant regions to reduce its immunogenicity as well as preserve its Fc-mediated functions in mouse studies. The VH and VL regions of the OX86 are shown below. As previously reported in the scientific literature, OX86 has a mechanism of action similar to antibody 445-3 in that it does not block the interaction between OX40 and OX40 ligand (al-Shamkhani Al, et al., Euro J. Immunol (1996) 26 (8); 1695-9, Zhang, P. et al. Cell Reports 27, 3117-3123). As monotherapy, OX86 was administered at a dose of 0.4 mg/kg once weekly (^./wk) by intraperitoneal (i.p.) injection.

OX86VH SEQ Ш NO: 43 QVQLKESGPGLVQPSQTLSLTCTVSGFSLTGYNLHWVRQPP GKGLEWMGR MRYDGDTYYNSVLKSRLSISRDTSKNQVFLKMNSLQTDDT AIYYCTRDGRG DSFDYWGQGVMVTVSS OX86VL SEQ Ш NO: 44 DIVMTQGALPNPVPSGESASITCRSSQSLVYKDGQTYLNWFL QRPGQSPQLLT YWMSTRASGVSDRFSGSGGTYFTLKISRVRAEDAGVYYCQ QVREYPFTFGS GTKLEIK

Mouse specific anti-TIGIT antibody (muTIGIT) was produced independently and administered at a dose of 5 mg/kg once a week by intraperitoneal injection. OX86 antibody in combination with muTIGIT was administered at the same dose for each individual antibody as previously described for monotherapy. Tumor volume and body weight were determined twice a week in two dimensions using a caliper and expressed in mm using the formula: V = 0.5 (ax^), where a and b represent the long and short axis dimensions of the tumor, respectively. Data are presented as mean tumor volume ± standard error of the mean (SEM). Tumor growth inhibition (TGR) was calculated using the following formula:

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I/ /(processed volume t) - (processed volume ϋθ)λ \% growth inhibition = 100 X 1 - -----—--------------------- -------\ y (placebo volume t) - (placebo volume GO) j 1 treated volume t represents the treated tumor volume at time t, treated volume t ₀ represents the treated tumor volume at time 0, volume placebo t represents the placebo tumor volume at time t, placebo volume t ₀ represents the placebo tumor volume at time 0.

The response of the syngeneic MMTV-PyMT model to OX86 antibody treatment in combination with muTIGIT treatment is shown in FIG. 15 and in table. 10. At day 21, OX86 and muTIGIT, administered as monotherapy once weekly intraperitoneally, each inhibited tumor growth with TPO of 31% and 13%, respectively. In contrast, significantly improved antitumor activity was observed when OX86 was combined with muTIGIT with TPO equal to 56%, with a 25% increase observed compared to OX86 administered as a single agent (p less than 0.001, combination versus vehicle; p less than 0.05 , combination versus OX86 monotherapy; and p less than 0.001, combination versus muTIGIT monotherapy). These data demonstrate that anti-OX40 antibody in combination with anti-TIGIT antibody is effective in the syngeneic MMTV-PyM mouse model. Also, the combination of anti-OX40 antibody with anti-TIGIT antibody did not have a significant effect on the body weight of the animal throughout the study.

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Claims (9)

41. Stuber, E., Neurath, M., Calderhead, D., Fell, H. P., and Strober, W. (1995). Crosslinking of 0X40 ligand, a member of the TNF/NGF cytokine family, induces proliferation and differentiation in murine splenic B cells. Immunity 2, 507-521. 42. Szypowska, A., Stelmaszczyk-Emmel, A., Demkow, U., and Luczynski, W. (2014). High expression of 0X40 (CD134) and 4-1BB (CD137) molecules on CD4(+)CD25(high) cells in children with type 1 diabetes. Advances in medical sciences 59, 39-43. 43. Timperi, E., Pacella, I., Schinzari, V., Focaccetti, C., Sacco, L., Farelli, F., Caronna, R., Del Bene, G., Longo, F., Ciardi, A., et al. (2016). Regulatory T cells with multiple suppressive and potentially pro-tumor activities accumulate in human colorectal cancer. Oncoimmunology 5, el 175800. 44. Tourkova, I.L., Yurkovetsky, Z.R., Shurin, M.R., and Shurin, G.V. (2001). Mechanisms of dendritic cell-induced T cell proliferation in the primary MLR assay. Immunology letters 78, 75-82. 45. Vetto, J. T., Lum, S., Morris, A., Sicotte, M., Davis, J., Lemon, M., and Weinberg, A. (1997). Presence of the T-cell activation marker OX-40 on tumor infiltrating lymphocytes and draining lymph node cells from patients with melanoma and head and neck cancers. American journal of surgery 174, 258-265. 46. Voo, K.S., Bover, L., Harline, M.L., Vien, L.T., Facchinetti, V., Arima, K., Kwak, L.W., and Liu, Y.J. (2013). Antibodies targeting human 0X40 expand effector T cells and block inducible and natural regulatory T cell function. J Immunol 191, 3641-3650. 47. Weinberg, A. D., Rivera, M. M., Prell, R., Morris, A., Ramstad, T., Vetto, J. T., Urba, W. J., Alvord, G., Bunce, C., and Shields, J. (2000 ). Engagement of the OX-40 receptor in vivo enhances antitumor immunity. J Immunol 164, 2160-2169. 48. Weinberg, A.D., Wegmann, K.W., Funatake, C., and Whitham, R.H. (1999). Blocking OX-40/OX-40 ligand interaction in vitro and in vivo leads to decreased T cell function and amelioration of experimental allergic encephalomyelitis. J Immunol 162, 1818-1826. 49. Willoughby, J., Griffiths, J., Tews, I., and Cragg, M.S. (2017). 0X40: Structure and function - What questions remain? Molecular immunology 83, 13-22. 50. Zander, R.A., Obeng-Adjei, N., Guthmiller, J.J., Kulu, D.I., Li, J., Ongoiba, A., Traore, B., Crompton, P.D., and Butler, N.S. (2015). PD-1 Co-inhibitory and 0X40 Co-stimulatory Crosstalk Regulates Helper T Cell Differentiation and Anti-Plasmodium Humoral Immunity. Cell host & microbe 17, 628-641. 51. Zhang, T., Lemoi, B. A., and Sentman, C. L. (2005). Chimeric NK-receptor-bearing T cells mediate antitumor immunotherapy. Blood 106, 1544-1551. 52. Zhang, P., Tu H. G., Wei. J., Chaparro-Riggers J., Salek-Ardakani S., Yeung Y. A. (2019) Ligand-Blocking and Membrane-Proximal Domain Targeting Anti-0X40 Antibodies Mediate Potent T Cell-Stimulatory and Anti-Tumor Activity. Cell Reports 27, 3117-3123. CLAIM 1. A method of treating cancer, comprising administering to a subject an effective amount of a non-competitive anti-OX40 antibody or an antigen-binding fragment thereof in combination with an anti-TIGIT (Tissue immunoreceptor with Ig and ITIM domains) antibody or an antigen-binding fragment thereof, wherein OX40 is a member of the tumor necrosis factor receptor superfamily, wherein Anti-OX40 antibody specifically binds to human OX40 and contains: (i) a heavy chain variable region, which contains (a) an HCDR (complementary-determining region) - 36 046633 heavy chain region) 1 with SEQ ID NO: 3, (b) HCDR2 with SEQ ID NO: 24 and (c) HCDR3 with SEQ ID NO: 5, and a light chain variable region which contains (d) LCDR (light chain complementarity determining region) 1 with SEQ ID NO: 25, (e) LCDR2 with SEQ ID NO: 19 and (f) LCDR3 with SEQ ID NO: 8; (ii) a heavy chain variable region which contains (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 18 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region which contains (d) LCDR1 having SEQ ID NO: 6, (e) LCDR2 having SEQ ID NO: 19 and (f) LCDR3 having SEQ ID NO: 8; (iii) a heavy chain variable region which contains (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 13 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region which contains (d) LCDR1 having SEQ ID NO: 6, (e) LCDR2 having SEQ ID NO: 7 and (f) LCDR3 having SEQ ID NO: 8; or (iv) a heavy chain variable region which comprises (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 4 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region, which comprises (d) LCDR1 of SEQ ID NO: 6, (e) LCDR2 of SEQ ID NO: 7, and (f) LCDR3 of SEQ ID NO: 8, and wherein the anti-TIGIT antibody or antigen binding fragment thereof comprises an antibody antigen binding domain that specifically binds human TIGIT, and contains a heavy chain variable region containing HCDR1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33 and HCDR3 of SEQ ID NO: 34; and a light chain variable region comprising LCDR1 of SEQ ID NO: 35, LCDR2 of SEQ ID NO: 36 and LCDR3 of SEQ ID NO: 37. 2. The method according to claim 1, in which the antibody to OX40 or its antigen-binding fragment contains: (i) a heavy chain variable region (VH), which contains SEQ ID NO: 26, and a light chain variable region (VL), which contains SEQ ID NO: 28; (ii) a heavy chain variable region (VH), which contains SEQ ID NO: 20, and a light chain variable region (VL), which contains SEQ ID NO: 22; (iii) a heavy chain variable region (VH), which contains SEQ ID NO: 14, and a light chain variable region (VL), which contains SEQ ID NO: 16; or (iv) a heavy chain variable region (VH) that contains SEQ ID NO: 9, and a light chain variable region (VL) that contains SEQ ID NO: 11. 3. The method of claim 1, wherein the anti-TIGIT antibody comprises an antigen-binding domain of an antibody that specifically binds human TIGIT, and comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 39, and a light chain variable region (VL ), containing the amino acid sequence SEQ ID NO: 41. 4. The method of claim 1, wherein the anti-OX40 antibody or antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv and (Fab') ₂ fragments. 5. The method of claim 1, wherein the anti-TIGIT antibody or antigen binding fragment is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv and (Fab') ₂ fragments. 6. The method of claim 1, wherein the cancer is breast cancer, colon cancer, head and neck cancer, stomach cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma , lymphoma, leukemia, myeloma or sarcoma. 7. The method of claim 6, wherein the breast cancer is metastatic breast cancer. 8. The method of any one of claims 1 to 7, wherein the treatment results in a sustained anti-cancer response in the subject after cessation of treatment. 9. A method of enhancing, enhancing or stimulating an immune response or function, which includes administering to a subject an effective amount of a non-competitive anti-OX40 antibody or antigen-binding fragment thereof in combination with an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the anti-OX40 antibody or antigen-binding fragment thereof specifically binds to OX40 human and contains: (i) a heavy chain variable region that contains (a) HCDR (heavy chain complementarity 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 which comprises (d) LCDR (light chain complementarity 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 which contains (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 18 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region which contains (d) LCDR1 having SEQ ID NO: 6, (e) LCDR2 having SEQ ID NO: 19 and (f) LCDR3 having SEQ ID NO: 8; (iii) a heavy chain variable region which contains (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 13 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region which contains (d) LCDR1 having SEQ ID NO: 6, (e) LCDR2 having SEQ ID NO: 7 and (f) LCDR3 having SEQ ID NO: 8; or (iv) a heavy chain variable region which comprises (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 4 and (c) HCDR3 of SEQ ID NO: 5, and a light chain variable region, which contains (d) LCDR1 having SEQ ID NO: 6, (e) LCDR2 having SEQ ID NO: 7 and (f) LCDR3 having SEQ ID NO: 8; and the anti-TIGIT antibody or antigen-binding fragment thereof comprises an antibody antigen-binding domain that specifically binds human TIGIT, and contains a heavy chain variable region comprising HCDR1 of SEQ ID NO: 32, HCDR2 of SEQ ID NO: 33, and HCDR3 of SEQ ID NO: 34 , and va -