EA045547B1 - ANTIBODIES TO OX40 AND METHODS OF APPLICATION - Google Patents

Description

Disclosed herein are antibodies or antigen-binding fragments thereof that bind to human OX40, a composition containing said antibody, and methods of use for the treatment of cancer.

Prior Art

OX40 (also known as ACT35, CD134 or TNFRSF4) is an approximately 50 kDa type I transmembrane glycoprotein and 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 incomplete cysteine-rich domain (CRD - cysteine-rich domain). The intracellular domain of OX40 contains one conserved signaling-related QEE motif that mediates binding to several TNFR-associated factors (TRAFs), including TRAF2, TRAF3, and TRAF5, allowing OX40 to bind to intracellular kinases (Arch and Thompson, 1998; Willoughby et al. ., 2017).

OX40 was originally discovered on activated rat CD4 T cells, and mouse and human homologues were subsequently cloned from T cells (al-Shamkhani et al., 1996; Calderhead et al., 1993). In addition to expression on activated CD4 ⁺ T cells, including T helper (Th) 1 cells, Th2 cells, Th17 cells, and regulatory T (Treg) cells, OX40 expression has also been detected on the surface of activated CD8 + T cells, natural Killer T cells (NK - Natural Killer), neutrophils and NK cells (Croft, 2010). In contrast, low levels of OX40 expression are found on naive CD4 and CD8 T cells, as well as most resting memory T cells (Croft, 2010; Soroosh et al., 2007). Expression of OX40 on the surface of naïve T cells is transient. After TCR (T cell receptor) activation, the level of OX40 expression on T cells increases significantly within 24 hours and peaks after 2~3 days, lasting for 5-6 days (Gramaglia et al., 1998).

Ligand for OX40 (OX40L, also known as gp34, CD252 or TNFSF4) is one single ligand for OX40. Similar to 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). In nature, OX40L forms a homomeric trimeric complex on the cell surface. The ligand-trimer interacts with three copies of OX40 at the monomer-monomer interface of the ligand mainly through the CRD1, CRD2 and partially CRD3 regions of the receptor, but without the participation of CRD4 (Compaan and Hymowitz, 2006). OX40L is primarily 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 (pDC) (Ito et al., 2004), macrophages (Weinberg et al., 1999) and Langerhans cells (Sato et al., 2002). In addition, OX40L has been found to be expressed on other cell types such as NK, mast cells, a subset 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 through trimeric OX40L or dimerization by agonistic antibodies promotes the recruitment and docking of the adapter molecules TRAF2, TRAF3 and/or TRAF5 to their intracellular QEE motif. (Arch and Thompson, 1998; Willoughby et al., 2017) Recruitment and docking of TRAF2 and TRAF3 may further lead to activation of both canonical NF-kB1 and non-canonical NF-kB2 pathways, which play key roles in regulating survival, differentiation, proliferation , cytokine production and T cell effector functions (Croft, 2010; Gramaglia et al., 1998; Huddleston et al., 2006; Rogers et al., 2001; Ruby and Weinberg, 2009; Song et al., 2005a; Song et al., 2005a) al., 2005b; Song et al., 2008).

In normal tissues, OX40 expression is low and occurs primarily on lymphocytes in lymphoid organs (Durkop et al., 1995). However, up-regulation of OX40 expression on immune cells has been frequently 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 cancer (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 is inversely correlated with the occurrence of distant metastases and features of later stage tumors (Ladanyi et al., 2004; Petty et al., 2004). 2002; Sarff et al., 2008). It was also shown that treatment with antibodies to OX40 could induce antitumor efficacy in different mouse models (Aspeslagh et al, 2016), indicating the potential of OX40 as an immunotherapeutic target. In the first clinical trial in patients with cancer, Curti et al. observed evidence of antitumor efficacy and tumor-specific T cell activation with agonistic anti-OX40 monoclonal antibodies, indicating that anti-OX40 antibodies are useful in promoting 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 their inhibitory effect on the differentiation and function of Treg cells (Aspeslagh et al., 2016; Ito et al., 2006; St. Rose et al., 2013; Voo et al., 2013). Recent studies have shown that in both animal tumor models and cancer patients, tumor-infiltrating Tregs express OX40 at higher levels than effector T cells (both CD4+ and CD8+) and peripheral blood Tregs (Lai et al. al., 2016; Marabelle et al., 2013b; Montler et al., 2016; Soroosh et al., 2007; Timperi et al., 2016). Consequently, the secondary effects by which anti-OX40 antibodies trigger antitumor responses are due to their Fc-mediated effector functions in depleting intratumoral OX40 Treg cells through antibody-dependent cellular cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP). dependent cellular phagocytosis) (Aspeslagh et al., 2016; Bulliard et al., 2014; Marabelle et al., 2013a; Marabelle et al., 2013b; Smyth et al., 2014). This work demonstrates that agonistic anti-OX40 antibodies with Fc-mediated effector function could preferentially deplete intratumor Tregs and improve CD8 effector T cell to Treg ratios in the tumor microenvironment (TME), leading to improved antitumor immune responses, increased regression tumors and improved survival (Bulliard et al., 2014; Carboni et al., 2003; Jacquemin et al., 2015; Marabelle et al., 2013b). As follows from these data, there is an unmet medical need for the development of agonistic antibodies to OX40 with both agonist activities and Fc-mediated effector functions.

To date, agonistic antibodies to OX40 in the healthcare setting are primarily ligand-competitive antibodies that block the OX40-OX40L interaction (eg, WO2016196228A1). Because the OX40-OX40L interaction is essential for enhancing effective antitumor immunity, blockade of OX40-OX40L limits the effectiveness of these ligand-competitive antibodies. Thus, OX40 agonist antibodies that specifically bind to OX40 while not interfering with the interaction of OX40 with OX40L have utility in the treatment of cancer and autoimmune disorders.

Brief description of the invention

The present invention is directed to agonistic antibodies to OX40 and antigen-binding fragments thereof, which activate OX40 and induce signaling in immune cells, thereby stimulating antitumor immunity.

In one embodiment, the invention provides monoclonal antibodies that bind to human OX40, or antigen-binding fragments thereof. In one aspect, the antibody of the present invention does not compete with OX40L or interfere with the binding of OX40 to its ligand OX40L.

The present invention covers the following embodiments.

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

The antibody or antigen-binding fragment thereof according to claim 1 contains: (i) a heavy chain variable region (VH) containing an amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 26, and a light chain variable region (VL) containing an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 28 ; (ii) a heavy chain variable region (VH) containing an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 20, and a light chain variable region chain (VL) containing an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 22; (iii) a heavy chain variable region (VH) containing an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 14, and a variable region

- 2 045547 light chain (VL) containing an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 16; or (iv) a heavy chain variable region (VH) containing an amino acid sequence at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 9, and a light chain variable region (VL) containing an amino acid sequence at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 11.

An antibody or antigen binding fragment wherein SEQ ID NO: 26, 28, 20, 22, 14, 16, 9, and/or 11 contains one, two, three, four, five, six, seven, eight insertions, deletions or substitutions , nine or ten amino acids.

An antibody or antigen binding fragment that contains: (i) a heavy chain variable region (VH) that contains SEQ ID NO: 26, and a light chain variable region (VL) that 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, a light chain variable region (VL) that contains SEQ ID NO: 11.

An antibody or antigen binding fragment that is a monoclonal antibody, chimeric antibody, humanized antibody, human engineered antibody, single chain antibody (scFv), Fab fragment, Fab' fragment or F(ab')2 fragment.

An antibody or antigen-binding fragment that has OX40 agonist activity.

An antibody or antigen binding fragment that binds to human OX40 at an epitope containing one or more amino acid residues selected from the group consisting of human OX40 H153D170.

An antibody or antigen binding fragment that binds to human OX40 at the level of an epitope containing one or more amino acid residues selected from the group consisting of H153, T154, H65, E167 and D170 of human OX40.

An antibody or antigen binding fragment that binds to human OX40 at the level of an epitope containing one or more amino acid residues selected from the group consisting of H153, I165 and E167 of human OX40. An antibody or antigen-binding fragment thereof that binds to human OX40 at the level of an epitope containing one amino acid residue selected from the group consisting of H153, H65 and E167 of human OX40.

An antibody or antigen binding fragment that binds to human OX40 on or within SEQ ID NO. thirty.

An antibody or antigen-binding fragment that binds to human OX40 at an equilibrium dissociation constant (KD) equal to or greater than 7.28 nM, 9.47 nM, 13.5 nM, or 17.1 nM, as measured by surface plasmon resonance (SPR - surface plasmon resonance).

An antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

An antibody or antigen binding fragment, wherein the antibody or antigen binding fragment thereof has a reduced level of glycosylation, is not glycosylated, or is hypofucosylated.

An antibody or an antigen-binding fragment thereof, wherein the antibody or an antigen-binding fragment thereof has an increased content of structures having branching at GlcNac points.

An antibody or antigen-binding fragment where the Fc domain belongs to IgG1.

An antibody or antigen-binding fragment where the Fc domain belongs to IgG4.

An antibody or antigen binding fragment where IgG4 has the S228P substitution (according to the EU numbering system).

An antibody or antigen binding fragment where IgG4 has the substitutions S228P and R409K (according to the EU numbering system).

An antibody or antigen-binding fragment that has one or more of the following properties:

(i) capable of cross-reacting with cynoOX40;

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

(iii) capable of stimulating a T cell, particularly at an EC50 (half-maximal effective concentration) equal to or greater than 0.06 ng/ml;

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

(v) capable of mediating ADCC (antibody-dependent cellular cytotoxicity), particularly as measured in an ADCC assay based on lactate dehydrogenase (LDH) release;

- 3 045547 (vi) capable of depleting CD4 ⁺ Tregs;

(vii) capable of increasing the CD8 Teff (effector T cell)/Teg ratio; or (viii) is capable of mediating partial tumor regression in an animal tumor model.

A pharmaceutical composition containing an antibody or an antigen-binding fragment thereof, further containing a pharmaceutically acceptable carrier.

A method of treating cancer comprising administering to a patient in need thereof an effective amount of an antibody or antigen binding fragment.

The method, wherein the cancer includes 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, sarcoma, etc. ., but is not limited to them.

A method where the antibody or pharmaceutical composition is administered in combination with another therapeutic agent.

A method wherein the therapeutic agent is paclitaxel or a paclitaxel-based agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, 5azacytidine.

A method wherein the therapeutic agent is a paclitaxel-based agent, lenalidomide or 5-azacytidine.

An isolated nucleic acid that encodes an antibody or an antigen-binding fragment thereof.

A vector containing a nucleic acid.

A host cell containing a nucleic acid.

A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell and isolating the antibody or its antigen-binding fragment from the culture.

Antibody or antigen-binding fragment thereof for use in the treatment or reduction of: 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 , myelomas and sarcomas.

A diagnostic reagent containing an antibody or antigen-binding fragment that is labeled.

A diagnostic reagent, wherein the label is selected from the group consisting of a radiolabel, a fluorophore, a chromophore, an imaging agent, and a metal ion.

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

In another embodiment, the 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: 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, the 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, and HCDR3 having the amino acid sequence 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;

- 4 045547 and/or (b) a light chain variable region containing 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 LCDR3 having the amino acid sequence of SEQ ID NO: 8; or LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2 having the amino acid sequence of SEQ ID NO: 19, and LCDR3 having the amino acid sequence of SEQ ID NO: 8; or LCDR1 having the amino acid sequence of SEQ ID NO: 25, LCDR2 having the amino acid sequence of SEQ ID NO: 19, and LCDR3 having the amino acid sequence of SEQ ID NO: 8.

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: 4, and HCDR3 having the amino acid sequence of SEQ ID NO: 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 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 an 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 the 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 an 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 the 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 an amino acid 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

- 5 045547 (c) a heavy chain variable region having the amino acid sequence 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.

In one embodiment, the antibody of the present invention belongs to 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 called human IgG1wt or hulgG1) or IgG2. In another embodiment, the antibody of the present invention contains a human IgG4 Fc domain with substitutions S228P and/or R409K (according to the EU numbering system).

In one embodiment, an antibody of the present invention binds to OX40 with a binding affinity ( _KD ) of 1x10 ^-6 M to 1x10 ^-10 M. In another embodiment, an antibody of the present invention binds to OX40 with a binding affinity (KD) of about 1x10 ^-6 M, approximately 1x10 ^-7 M, approximately 1x10 ^-8 M, approximately 1x10 ^-9 M or approximately 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 an epitope of human OX40 outside the OX40-OX40L interaction zone. 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 of OX40 with its ligand OX40L.

The antibodies of the present invention are agonistic and significantly enhance the immune response. According to the invention, a method is proposed for analyzing the agonistic ability of antibodies to OX40. In one embodiment, an antibody of the present invention can significantly stimulate primary T cell production of IL-2 (interleukin-2) in a mixed lymphocyte response (MLR) assay.

In one embodiment, the antibodies of the present invention have potent Fc-mediated effector functions. Antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) to OX40 ^I1 target cells, such as regulatory T cells (Treg cells), under the influence of NK cells. In one aspect, the invention provides a method for assessing anti-OX40 antibody-mediated in vitro depletion of specific T cell subsets as a function of different levels of OX40 expression.

Antibodies or antigen binding fragments of the present invention do not block the OX40-OX40L interaction. In addition, anti-OX40 antibodies demonstrate dose-dependent antitumor activity in vivo, as demonstrated in animal models. The dose-dependent activity differs from the activity profile of anti-OX40 antibodies, 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 comprises a VH nucleotide sequence of SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 21, or SEQ ID NO: 27, or a nucleotide sequence of at least 95, 96, 97, 98, or 99 % identical to any of 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 nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 23 or SEQ ID NO: 29, or a nucleotide sequence of at least 95, 96, 97, 98 or 99% identical to SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 23 or SEQ ID NO: 29, and encodes the VL region of an antibody or antigen binding fragment of the present invention.

In another aspect, the present invention provides a pharmaceutical composition comprising an anti-OX40 antibody or antigen-binding fragment thereof and optionally a pharmaceutically acceptable excipient.

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

The present invention relates to the use of an antibody or an antigen binding fragment thereof or a pharmaceutical composition based on an anti-OX40 antibody for the treatment of a disease such as cancer or autoimmune diseases.

Brief description of graphic materials

Fig. 1 is a schematic representation of the OX40-mIgG2a, OX40-huIgG1 and

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OX40-His. ECD OX40: 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 by surface plasmon resonance (SPR).

In fig. 3 demonstrates the determination of OX40 binding by flow cytometry. OX40-positive HuT78/OX40 cells were incubated with different antibodies to OX40 (ch445, 445-1, 445-2, 445-3 and 445-3 IgG4 antibodies) and subjected to FACS (fluorescence-activated cell sorting) analysis ). The result is shown by mean fluorescence intensity (MFI - mean fluorescence intensity, 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 mean fluorescence intensity (MFI, shown on the Y axis) was determined by flow cytometry.

In fig. 5 depicts affinity determination of Fab 445-3 for wild-type OX40 and point mutations by surface plasmon resonance (SPR).

In fig. 6 details the interactions between antibody 445-3 and its epitopes on OX40. Antibody 445-3 and OX40 are shown in light gray and black, respectively. Hydrogen bonding or salt bridge, π-π stacking and Van der Waals (VDW) interactions are shown as dotted, double dotted and solid lines, respectively.

In fig. 7 shows that antibody 445-3 does not interfere with OX40L binding. 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 lA7.grl (+lA7.grl, see US 2015/0307617) in a 1:1 molar ratio. The binding of OX40L to the OX40-mIgG2a/anti-OX40 complex was determined by co-incubation of HEK293/OX40L cells with the OX40mIgG2a/anti-OX40 complex followed by reaction with a secondary anti-mouse IgG Ab and by flow cytometry. The results are shown as the mean plus/minus SD (Standard Deviation) of two replicates. Statistical significance: *: P less than 0.05; **: P less than 0.01.

In fig. 8 shows the structural alignment of OX40/Fab 445-3 with the described 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 conjunction with TCR stimulation. OX40-positive HuT78/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 a recording value for stimulation of T cells (Fig. 9B). IL-2 in the culture supernatant was detected by ELISA. The results are shown as follows: mean plus/minus SD of three replicates.

In fig. 10 shows that antibodies to OX40 enhance MLR responses. In vitro differentiated dendritic cells (DC) were co-cultured 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 by ELISA. All analyzes were performed in quadruplicate, and the results are shown as follows: mean plus/minus SD. Statistical significance: *: P less than 0.05; **: P less than 0.01.

In fig. 11 shows that anti-OX40 antibody 445-3 causes 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 controls. Equal numbers of effector cells and target cells were cocultured for 5 hours before detection of lactate dehydrogenase (LDH) release. Percent cytotoxicity (Y-axis) was calculated based on the manufacturer's protocol as described in Example 12. Results are shown as follows: mean plus/minus SD of triplicates.

In fig. 12A-12C show that anti-OX40 antibody 445-3 in combination with NK cells increases CD8 effector T cell to Treg ratios in activated PBMC (human peripheral blood mononuclear cell) 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 by flow cytometry. Additionally, the ratios of CD8+ effector T cells to Tregs were calculated. In fig. 12A shows the CD8/total T cell ratio. Fig. 12B represents the Treg/Total T cell ratio. In fig. 12C shows the CD8/Treg ratio. Data are shown as the mean plus/minus SD of two replicates. Statistical significance is shown between 445-3 and lA7.gr1 at the indicated concentrations. *: P less than 0.05; **: P less than 0.01.

In fig. 13A, 13B show that anti-OX40 antibody 445-3, but not 1 A7.gr1, exhibits dose-dependent antitumor activity in the syngeneic MC38 model of colon and rectal cancer in OX40-7045547 humanized mice. MC38 murine colon carcinoma cells (2x10 ⁷ ) were subcutaneously implanted into female mice transgenic for human OX40. After randomization according to tumor volume, animals were intraperitoneally injected with either anti-OX40 antibodies or isotype control once a week 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 that particular dose. Data are presented as mean tumor volume plus/minus standard error of the mean (SEM) with 6 mice per group. Statistical significance: *: P less than 0.05, compared with isotype control.

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

Definitions

Unless specifically defined elsewhere herein, all other technical and scientific terms used herein have meanings commonly understood by one of ordinary skill in the art.

As used herein, including the appended claims, words in the singular include corresponding references therein in the plural unless the context clearly dictates otherwise.

The term or is used to refer to and is used interchangeably with the term and/or unless the context clearly dictates otherwise.

The term anticancer agent, as used herein, refers to any agent that can be used to treat a cell proliferation disorder such as cancer, including cytotoxic agents, chemotherapeutic agents, radiation therapy and radiotherapy agents, targeted anticancer agents actions and immunotherapies, but not limited to them.

The term OX40 refers to a type I transmembrane glycoprotein 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 with accession number NP_003318, and the nucleotide sequence encoding the OX40 protein is accession number: X75962.1. The term OX40 ligand or OX40L refers to one single OX40 ligand and is interchangeable with gp34, CD252 or TNFSF4.

The terms administration, administration, treatment and treatment, as used herein, when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, mean bringing an exogenous pharmaceutical, therapeutic, diagnostic agent or composition into contact with the animal, human , subject, cell, tissue, organ or biological fluid. Treatment of the cell includes bringing the reagent into contact with the cell, as well as bringing the reagent into contact with a liquid, where the liquid is in contact with the cell. The term administration and treatment also means in vitro or ex vivo treatments, eg, with 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. Treating any disease or disorder refers in one aspect to improving the condition of the disease or disorder (namely, 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 the alleviation or improvement of at least one physical parameter, including parameters that may not be noticeable to the patient. In yet another aspect, the term treat, treatment, or treatment refers to modulating a disease or disorder, either physically (eg, stabilizing an expressed symptom) or physiologically (eg, stabilizing a physical parameter), or both. In yet another aspect, the term 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 of having a disorder described herein).

The term affinity, as used herein, refers to the strength of interaction between an antibody and an antigen. Within an antigen, the variable arm of an antibody interacts through non-covalent forces with the antigen at multiple sites; the more interactions, the stronger the affinity.

The term antibody, as used herein, refers to a polypeptide of the immunoglobulin family that can bind a corresponding antigen in a non-covalent, reversible and specific manner. For example, a naturally occurring IgG antibody is a tetramer containing at least two heavy (H) chains and two (L) light chains, interrelated

- 8 045547 containing disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a single CL domain. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with regions that are more conserved called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from N-terminus to C-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 (Clq) of the classical complement system.

The term antibody includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and anti-idiotic antibodies (anti-Ids). Antibodies can belong to 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 region 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, that is, the antibody molecules contained in a given 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 include many different antibodies having different amino acid sequences in their variable domains, especially in their complementarity determining regions (CDRs), which are often specific for different epitopes. The definition of monoclonal indicates the nature of the antibody obtained from an essentially homogeneous population of antibodies, and should not be construed as requiring the antibody to be produced by 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 256:495497; Pat. US 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. Antibodies disclosed herein may belong to the class of immunoglobulins including IgG, IgM, IgD, IgE, IgA, and any subclass thereof, such as IgG1, IgG2, IgG3, IgG4. The monoclonal antibody-producing hybridoma can be cultured in vitro or in vivo. High titers of monoclonal antibodies can be produced by in vivo production, in which cells from individual hybridomas are injected intraperitoneally into mice, such as the pristine Balb/c premium mice, to produce free peritoneal fluid containing high concentrations of the desired antibodies. Monoclonal antibodies of the IgM or IgG isotype can be purified from such free peritoneal fluids or 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 comprises two identical pairs of polypeptide chains, each pair having one light chain (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa). The N-terminal portion of each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The C-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. In addition, human heavy chains are generally referred to as α, δ, ε, γ, or μ, and define antibody isotypes as IgA, IgD, IgE, IgG, and IgM, respectively. Within the light and heavy chains, the variable and constant regions are connected by a J region of about 12 or more amino acids, with the heavy chain also including a D region of about 10 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, these two binding sites are usually 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 to framework regions to allow binding to a specific epitope. In general, from N-terminus to C

- 9 045547 end variable domains of both 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 well-known definitions in the art, for example, Kabat, Chothia 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); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)). Definitions of antigen binding sites are also described in the following references: 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 the combined Kabat and Chotia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of the Kabat CDR, the 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.

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 (namely, VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable domain and VHCDR1, VH-CDR2 and VH-CDR3 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. (determination of antibody CDR regions by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (identification of antibody CDR regions by structure). The term FR residues or framework region residues means variable domain residues other than hypervariable region residues, defined herein as CDR residues.

Unless specifically stated otherwise, the term antigen binding fragment means antigen binding fragments of antibodies, that is, fragments of an antibody that retain the ability to specifically bind an antigen that is bound by a full-length antibody, such as 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; diatels; linear antibodies; single chain antibody molecules, for example single chain Fv (ScFv); nanobodies and multispecific antibodies formed from antibody fragments.

An antibody specifically binds to a target protein, meaning 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 detects 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 useful in the present invention 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. An antibody herein is said to specifically bind to a polypeptide containing a given amino acid sequence, for example, the amino acid sequence of a human OX40 molecule, if it binds to polypeptides containing that sequence, but does not bind to proteins not containing 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 when produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Likewise, the terms mouse antibody or rat antibody refer to an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively.

The term humanized antibody refers to forms of antibodies that contain sequences from non-human (eg, mouse) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, a humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the FR regions represent is the FR region of the human immunoglobulin sequence. The humanized antibody will also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. The prefix hum, hu, Hu, or h is added to antibody clone designations when it is necessary to distinguish between humanized antibodies and the original rodent antibodies. Humanized forms of rodent antibodies will generally contain the same CDR sequences of the parent 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.

The term corresponding human germline sequence refers to a nucleic acid sequence encoding a human variable region amino acid sequence, or a subsequence, that shares the highest determined amino acid sequence identity with a reference amino acid sequence or variable region subsequence, compared to all other known variable region amino acid sequences encoded by human germline immunoglobulin variable region sequences. 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 a framework region only, a complementarity-determining region only, a framework region and a complementarity-determining region, a variable segment (as defined above), or other combinations of sequences or subsequences that comprise a variable region. Sequence identities can be determined using methods described herein, for example, by aligning two sequences using BLAST, ALIGN, or other alignment algorithm known in the art. The corresponding human germline nucleotide or amino acid sequence may be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identical to the reference variable region nucleotide or amino acid sequence.

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 will typically 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 intended 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 multi-part 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 covers the administration of each type of therapeutic agent sequentially, or at approximately the same time, or at different points in time. In any case, the treatment regimen will provide a beneficial effect of the drug combination in treating the conditions or disorders described herein.

In the context of the present invention, when referring to an amino acid sequence, the term conservative substitution means replacing the original amino acid with a new amino acid that does not substantially change the chemical, physical and/or functional properties of the antibody or fragment, for example, its binding affinity for OX40. Specifically, commonly occurring conservative amino acid substitutions are shown in the following table and are well known in the art.

Illustrative conservative amino acid substitutions.

Original amino acid residue Single letter and three letter codes Conservative substitution Alanin A or Ala Gly; Ser

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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 Glutamine Q or Gin Asn Glutamic acid E or Glu Asp; Gin Glycine G or Gly Ala G istidin 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, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. BLAST analysis software is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSP) by identifying short words of length W in the search sequence that match or meet some threshold similarity score with a positive T value when aligned with a word of the same length in the database sequence. T is called the threshold for matching neighboring words. These initial matches of adjacent words act as values to start a search to find longer HSPs containing them. Word matches continue in both directions along each sequence as long as the cumulative alignment score can be increased. Aggregate scores are calculated using, in the case of nucleotide sequences, the parameters M (reward score for pairs of matching residues; always greater than 0) and N (penalty score for mismatched residues; always less than 0). For amino acid sequences, a weighting matrix is used to calculate the aggregate score. Expansion of word matches in each direction stops when: the cumulative alignment score decreases by a quantitative amount X from its maximum achieved value; the cumulative score approaches zero or lower due to the accumulation of one or more balance adjustments with a negative score; or the end of one of the sequences 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 default word length (W) of 11, expectation (E) of 10, M equals 5, N equals minus 4, and compares both strands. For amino acid sequences, BLAST uses a default word length of 3 and an expectation (E) of 10, and a BLOSUM62 weight matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915), number of alignments ( B) 50, expect(E) 10, M is 5, N is -4, and compare both threads.

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 similarity criterion proposed by the BLAST algorithm is the smallest cumulative probability (P(N)), which provides a measure of the probability with which a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the lowest overall probability when comparing the nucleic acid being analyzed 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 of two amino acid sequences can be determined using an algorithm by E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11-17, (1988), which is included in the ALIGN program (version 2.0) using a table of substitution weights for RAM120 residues, a gap extension penalty of 12 and a gap penalty of 4. In addition, the percentage identity of two amino acid sequences can be determined using Needleman and Wunsch, J. Mol. Biol. 48:444-453, (1970), an algorithm that is 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 extension 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 polymers thereof, either in single-stranded or double-stranded form. The term covers nucleic acids, including known nucleotide analogues or modified backbone residues or linkages, that are synthetic, naturally occurring or non-naturally occurring, 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, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNA).

The term operably linked in the context of nucleic acids refers to a functional link between two or more segments of polynucleotides (eg, DNA). Generally, it refers to the functional relationship of a transcriptional regulatory sequence to 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, transcriptional promoter sequences that are operably linked to the transcribed sequence are physically adjacent to the transcribed sequence, that is, they are cis-acting. However, some regulatory sequences that regulate transcription, such as enhancers, do not necessarily need to be physically adjacent or located 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 or all solvents, dispersion media, isotonic and absorption retarding agents, and the like 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 a variety of forms. These forms include, for example, liquid, semi-liquid and solid dosage forms such as liquid solutions (eg, injectables 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 injectable or infusion solutions. One suitable route of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered via intravenous infusion or injection. In some embodiments, the antibody is administered via intramuscular or subcutaneous injection.

The term therapeutically effective amount, as used herein, 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 given example may be apparent to those skilled in the art or can be determined by routine experimentation. In the case of combination therapy, a therapeutically effective amount refers to the total amount of the combination entities to effectively treat a disease, disorder or condition.

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Detailed description

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 or treat cancer. In addition, the present invention provides pharmaceutical compositions containing antibodies and methods for preparing and using such pharmaceutical compositions for the prevention and treatment of cancer and associated disorders.

Antibodies to OX40.

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 formed 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) comprise 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, wherein said antibodies or antigen binding fragments comprise a VH CDR comprising 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 SEQ ID NO: 16, 22 or 28 (Table 3). The present invention also provides antibodies or antigen binding fragments that specifically bind to OX40, wherein said antibodies or antigen binding fragments comprise 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 comprise (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 still have a percentage identity of at least 60, 70, 80, 90, 95 or 99% in the CDR regions with the CDR regions depicted in the sequences described in table 3. In some aspects, mutant amino acid sequences are included where 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 antibodies in which amino acids or nucleic acids encoding amino acids are mutated; still have a percentage identity of at least 60, 70, 80, 90, 95 or 99% with the sequences described in table. 3. In some aspects, mutant amino acid sequences are included where no more than 1, 2, 3, 4 or 5 amino acids are mutated in the variable regions when compared to the variable regions depicted in the sequence described in table. 3, maintaining essentially the same therapeutic activity.

The present invention also provides nucleic acid sequences that encode VH, VL, the full length heavy chain and the full length light chain of antibodies that specifically bind to OX40. Such nucleic acid 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 an epitope of human OX40. In some aspects, antibodies and antigen binding fragments thereof may bind to the same OX40 epitope.

The present invention also provides antibodies or antigen-binding fragments thereof that bind to the same epitope as the anti-OX40 antibodies described in Table 1. 3. Additional antibodies and antigen binding fragments thereof can thus 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 that antibody or antigen binding fragment thereof for binding to OX40. Such an antibody can

- 14 045547 without being bound by any theory, bind to the same or related (eg, structurally similar or spatially close) epitope on OX40 as the antibody or antigen-binding fragments thereof with which it competes. In a particular 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.

Additional modification to the Fc region framework

In yet other aspects, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has altered affinity for the effector ligand but retains the antigen-binding ability of the original antibody. The effector ligand for which the affinity is changed may be, for example, an Fc receptor or a complement component C1. This approach is described, for example, in US Pat. US Nos. 5624821 and 5648260, both to Winter et al.

In another aspect, one or more amino acid residues can be replaced with one or more different amino acid residues such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described, for example, in US Pat. US No. 6194551 Idusogie et al.

In yet another aspect, one or more amino acid residues are changed, 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, in the case of the IgG1 subclass and the kappa isotype. Allotypic amino acid residues also include the heavy chain constant region of the IgG1, IgG2 and IgG3 subclasses, as well as the light chain constant region of the kappa isotype, as described by Jefferis et al., MAbs. 1:332-338 (2009), but not limited to them.

In another aspect, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or 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 on human IgG1 for FcyRI, FcyRII, FcyRIII and FcRn have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276:6591-6604, 2001).

In yet another aspect, the glycosylation of the antibody is modified. For example, an aglycosylated antibody may be produced (ie, the antibody has no or reduced levels of glycosylation). Glycosylation can be altered, for example by increasing the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished, for example, by changing one or more glycosylation sites within 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 backbone, 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 Pat. US No. 5714350 and No. 6350861 Co et al.

Additionally or alternatively, an antibody may be produced that has an altered glycosylation pattern, such as a hypofucosylated antibody having reduced amounts of fucosyl residues, or an antibody having an increased content of structures having branching at GlcNac points. Such altered glycosylation profiles have been demonstrated to enhance the ADCC capacity 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 are described in the art and can be used as host cells in which recombinant antibodies are expressed to thereby produce altered glycosylation antibodies. For example, in EP 1176195 Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which 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 that host cell (see also Shields et al, (2002) J. Biol. Chem. 277:26733-26740). In PCT publication WO 99/54342 Umana et al. describes 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

- 15 045547 showed an increased content of structures having branching at GlcNac points, which leads to increased ADCC activity of antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

In another aspect, if reduction in ADCC is desired, the human IgG4 antibody subclass has been shown in many previous reports to have only moderate ADCC and little to no CDC effector function (Moore G L, et al. 2010 MAbs, 2:181-189 ). On the other hand, natural IgG4 has been found to be less stable under stressful conditions, such as in acidic buffer or increasing 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 reduce or eliminate FcyR or Clq 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 intrinsic properties of IgG4 is the dynamic separation of its two heavy chains in solution to form a half-antibody, resulting in bispecific antibodies formed in vivo through a process called Fab exchange ( Van der Neut Kolfschoten M, et al. 2007 Science, 317:1554-157). Mutation of serine to proline at position 228 (EU numbering system) has been shown to be inhibitory for 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 the yFc region have been reported to influence the interaction of the antibody 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; Arnold J. N., 2007 Annu Rev immunol, 25:21-50). In addition, some rare IgG4 isoforms in the human population may also cause different physicochemical properties (Brusco, A. et al. 1998 Eur J Immunogenet, 25:349-55; Aalberse et al. 2002 Immunol, 105:9-19 ). To produce OX40 antibodies with low ADCC, CDC and instability, it is possible to modify the hinge region and Fc region of human IgG4 and introduce a variety of changes. These modified IgG4 Fc molecules can be found in SEQ ID NO: 83-88, US Patent No. 8735553 Li et al.

Obtaining antibodies to OX40.

Antibodies to OX40 and antigen-binding fragments thereof can be produced by any means known in the art, including, but not limited to, recombinant expression, chemical synthesis and enzymatic cleavage of antibody tetramers, while full-length monoclonal antibodies can be produced by, for example, hybridoma or recombinant products. 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 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, a polynucleotide encoding heavy chain variable regions is at least 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 identical in nucleotide sequence to a polynucleotide selected from the group consisting of from SEQ ID NO: 15, 21, or 27. In some aspects, a polynucleotide encoding light chain variable regions of at least 85, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % identical in nucleotide sequence 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 may also encode 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 exemplary anti-OX40 antibodies. Certain 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 an antigen-binding fragment thereof. In some aspects, an inducible promoter is used to prevent expression of inserted sequences other than those under the control of inducing conditions. Inducible promoters include, for example, the arabinose, lacZ, metallothionein, or heat shock promoter. Cultures of transformed organisms can be propagated under non-inducing conditions without affecting the population with respect to coding sequences whose expression products are better tolerated by host cells. In addition to promoters, other regulatory elements may also be required or desirable for efficient expression of an anti-OX40 antibody or antigen binding fragment. These elements typically include an ATG start codon and an adjacent ribosome binding site or other sequences. In addition, the efficiency of expression can be enhanced by the inclusion of enhancers native to the cell system 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 increase the level of expression in mammalian host cells.

The host cells for transfer and expression of anti-OX40 antibody chains can be either prokaryotic or eukaryotic. E. coli is one prokaryotic host useful 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 generated in these prokaryotic hosts, which typically contain expression control sequences compatible with the host cell (eg, origin of replication). In addition, any number of a variety of well known promoters will be present, such as the lactose promoter system, the tryptophan promoter (trp) 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 and the like. to initiate and complete 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 carrying an exogenous expression vector. These cells include any normal immortalized or normal or abnormal immortalized cell 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 cell lines (SV-40 transformed monkey kidney cells), HEK 293 cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell culture for the expression of polypeptides is generally discussed 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 an origin of replication, a promoter and enhancer (see, for example, Queen et al, Immunol. Rev. 89:49-68, 1986), and essential messenger RNA processing sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription termination sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type specific, developmental stage specific, and/or modulated or regulated. Useful promoters include, but are not limited to, metallothionein promoter, constitutive adenoviral major late promoter, dexamethasone-inducible MMTV (mouse mammary tumor virus) promoter, SV40 promoter, MRP polIII promoter, constitutive MPSV (myeloproliferative sarcoma virus) promoter sarcoma virus), a tetracycline-inducible CMV promoter (such as the human CMV immediate-early response gene promoter), a constitutive CMV promoter, and promoter-enhancer combinations known in the art.

Methods of detection and diagnosis.

The antibodies or antigen binding fragments thereof of the present invention are useful in a variety of 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. The term identification, as used herein, includes quantitative or qualitative identification. In some 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 relative to other tissues.

In one aspect, the present invention provides a method for detecting the presence of OX40 in a biological sample. In some aspects, the method includes contacting a biological sample with an anti-OX40 antibody under conditions that are permissive for antibody-antigen binding and determining whether a complex is formed 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 some aspects, the method includes contacting a cell under analysis with an anti-OX40 antibody;

- 17 045547 determination of the level of expression (either quantitatively or qualitatively) of OX40 in the analyzed cell by detecting the binding of an antibody to OX40 to the OX40 polypeptide; and comparing the expression level in the test cell with the level of OX40 expression in a control cell (eg, a normal cell of the same tissue origin as the test cell or a cell not expressing OX40), wherein the test cell has a higher level of OX40 expression than with a 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 useful in a variety of applications, including, but not limited to, methods of treating an OX40-associated disorder or disease. In one aspect, the OX40-associated disorder or disease is cancer.

In one aspect, the present invention provides a method for treating cancer. In some aspects, the method includes 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 and sarcoma.

The antibody or antigen binding fragment of the invention can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and, if necessary for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be carried out 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 continuous. Various dosing regimens are discussed herein, including, but not limited to, single or multiple administrations over different time points, bolus administration, and pulse infusion.

Antibodies or antigen binding fragments of the invention could be prepared, dosed and administered in a manner consistent with good medical practice. Factors 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 the practitioner. The antibody need not be, but may be prepared by, one or more agents currently used to prevent or treat the disorder in question. The effective amount of other such agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These agents are generally used at the same dosages and routes of administration as described herein, or from about 1 to 99% of the dosages described herein, or at any dosage and route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of a disease, the appropriate dosage of an antibody or antigen binding fragment of the invention will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the clinical history of the patient and antibody response and the discretion of the treating physician. The antibody is suitably administered to the patient once or over a number of courses of treatment. Depending on the type and severity of the disease, from about 1 to 100 mg/kg of antibody may be a possible initial dosage to administer to a patient, whether, for example, through one or more separate administrations or through a continuous infusion. One typical daily dosage could range from about 1 mcg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment will usually be continued until the desired suppression of disease symptoms occurs. Such doses may be administered intermittently, for example, every week or every three weeks (for example, such that the patient receives from about two to about twenty or, for example, about six doses of the antibody). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be useful. The effectiveness of this therapy was easily monitored using traditional techniques and assays.

Combination therapy.

In one aspect, the anti-OX40 antibodies of the present invention can be used in combination with other therapeutic agents. Other therapeutic agents that can be used with the anti-OX40 antibodies of the present invention include: a chemotherapeutic agent (e.g., paclitaxel or a paclitaxel-based agent; (e.g., 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, pemetrexed di

- 18 045547 sodium), tyrosine kinase inhibitor (e.g. EGFR (epidermal growth factor receptor) inhibitor (e.g. erlotinib), multikinase inhibitor (e.g. MGCD265, RGB-286638), CD-20 targeting agent ( e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52-targeting agent (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bc1-2 inhibitor (e.g., oblimersen sodium), aurora kinase inhibitor (e.g., 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 inhibitor (eg, temsirolimus, everolimus), BCR/ABL inhibitor (eg, imatinib), ET-A receptor antagonist (eg, ZD4054), TRAIL 2 receptor (TR2) agonist (eg, CS-1008), HGF/ SF (eg, AmG 102), EGEN-001, Polo-like kinase 1 inhibitor (eg, BI 672), but not limited to.

Pharmaceutical compositions and preparations.

Also provided are compositions, including pharmaceutical preparations, containing an anti-OX40 antibody or antigen-binding fragment, or polynucleotides containing sequences encoding an anti-OX40 antibody or antigen-binding fragment. In some 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 preparations of anti-OX40 antibody or antigen binding fragments as described herein are prepared by admixing such antibody or antigen binding fragment having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized preparations 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 such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; pyrocatechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as 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). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral hyaluronidase glycoproteins (sHASEGPs), e.g., PH-20 human soluble hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc. .). Some exemplary sHASEGPs and methods of use involving rHuPH20 are described in US Patent No. 7871607 and No. 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

Exemplary lyophilized antibody preparations are described in US Pat. No. 6,267,958. Aqueous antibody preparations include those described in US Pat. No. 6,171,586 and WO2006/044908, the latter formulations including 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 of which are in the form of shaped particles, such as films or microcapsules.

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

Examples

Example 1. Preparation of monoclonal antibody to OX40.

Monoclonal antibodies to OX40 were generated using conventional fusion hybridoma 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) assays were selected for further characterization.

- 19 045547 tions.

Recombinant OX40 proteins for immunization and binding assays cDNA encoding full-length human OX40 (SEQ ID NO: 1) was synthesized through Sino Biological (Beijing, China) based on the GenBank sequence (Accession No.: X75962.1). The coding region of the signal peptide and the extracellular domain (ECD - extracellular domain), consisting of amino acids (aa) 1-216 OX-40 (SEQ ID NO: 2), were amplified by PCR (polymerase chain reaction) and cloned into independently developed expression vectors with the C terminus fused to the Fc domain of mouse IgG2a, the heavy chain Fc domain of wild-type human IgG1, or a His tag, resulting in three recombinant fusion protein expression plasmids, OX40-mIgG2a, OX40huIgG1, and OX40-His, respectively. A schematic representation of the OX40 fusion proteins is shown in FIG. 1. To obtain recombinant fusion proteins OX40-mIgG2a, OX40-huIgG1 and OX40-His, 293G cells were transiently transfected with expression plasmids and cultured for 7 days in a CO ₂ incubator equipped with a rotating shaker. The supernatant containing the recombinant protein was collected and clarified by centrifugation. OX40-mIgG2a and OX40-huIgG1 were purified using a Protein A column (Cat. No: 17-5438-02, GE Life Sciences). OX40-His was purified using a Ni-Sepharose column (Cat. No: 17-5318-02, GE Life Science). Proteins OX40-mIgG2a, OX40-huIgG and OX40-His were dialyzed against phosphate buffered saline (PBS) and stored in a freezer at minus 80°C in small aliquots.

Cell lines with stable expression.

To obtain stable cell lines that express full-length human OX40 (OX40) or cynoOX40, these genes were cloned into the retroviral vector pFB-Neo (Cat. No: 217561, Agilent, USA). Retroviral transduction was performed based on a previously described protocol (Zhang et al, 2005). Retroviral transduction of HuT78 and HEK293 cells was carried out with a virus containing human OX40 or cynoOX40, respectively, to obtain the cell lines HuT78/OX40, HEK293/OX40 and HuT78/cynoOX40.

Immunization, Hybridoma Fusion and Cloning Eight to twelve week old Balb/c mice (from FIFK BIOSCIENCE CO., LTD, Beijing, China) were immunized intraperitoneally with 200 μl of antigen in a mixture containing 10 μg of OX40-mIgG2a and Quick-Antibody Immuno-Adjuvant (Cat. No: 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 titers of antibodies to OX40 in serum were re-immunized by i.p. (intraperitoneal) injection of 10 μg OX40-mIgG2a. Three days after booster immunization, splenocytes were isolated and fused with a mouse myeloma cell line, SP2/0 cells (ATCC, Manassas VA) using standard techniques (Somat Cell Genet, 1977 3:231).

Evaluation of OX40 binding activity by antibodies by ELISA and FACS.

Initial screening of hybridoma clone supernatants was performed by ELISA as described in (Methods in Molecular Biology (2007) 378:33-52) with some modifications. Briefly, 96-well plates were coated with OX40-His protein 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 hours 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. HRP (horse-radish peroxidase)-linked anti-mouse IgG antibody (cat. no.: 115035-008, Jackson ImmunoResearch Inc, peroxidase-labeled goat anti-mouse IgG antibody AffmiPure, specific Fcy fragment) and substrate (cat. no.: 00-4201-56, eBioscience, USA) was used to develop the absorbance signal at a wavelength of 450 nm, which was measured using a plate reader (SpectraMax Paradigm, Molecular Devices/PHERAstar, BMG LABTECH). Positive parent clones were selected from the fusion by screening by indirect ELISA. ELISA-positive clones were further verified by FACS using HuT78/OX40 and HuT78/cynoOX40 cells described above. OX40-expressing cells (10 cells/well) were incubated with ELISA-positive hybridoma cell supernatants, followed by coupling with eFluor® 660 anti-mouse IgG (Cat. No: 50-4010-82, eBioscience, USA). Cell fluorescence was quantified using a flow cytometer (Guava easyCyte 8HT, Merck-Millipore, USA).

Conditioned media from hybridomas that showed positive signals in both ELISA and FACS screening were subjected to functional assays to identify antibodies with good functional activity in human immune cell-based assays (see following sections). Antibodies with desired functional activities 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

- 20 045547 above, positive hybridoma clones were subcloned by limiting dilution to ensure clonality. The best antibody subclones were verified by functional assays and adapted for growth in CDM4MAb medium (Cat. No: SH30801.02, Hyclone, USA) with 3% FBS (Fetal Bovine

Serum - fetal calf serum).

Expression and purification of monoclonal antibodies.

Hybridoma cells expressing the best antibody clones were cultured in CDM4MAb medium (Cat. No.: 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 through a 0.22 μm membrane before purification. Mouse antibodies in the supernatants were applied to and coupled to a Protein A column (Cat. No: 17-5438-02, GE Life Sciences) according to the manufacturer's instructions. This procedure typically resulted in antibodies with a purity greater than 90%. Antibodies purified by a Protein A affinity column were either dialyzed against PBS or, if necessary, further purified using a HiLoad 16/60 Superdex 200 column (Cat. No: 28-9893-35, GE Life Sciences) to removal of 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 RNA using the Ultrapure RNA kit (Cat. No: 74104, QIAGEN, Germany) based on the manufacturer's protocol. Single-stranded cDNAs were synthesized using a cDNA synthesis kit from Invitrogen (Cat. No.: 18080-051), and PCR amplification of VH and VL hybridoma antibodies was performed using a PLR kit (Cat. No.: CW0686, CWBio, Beijing, China). Oligoprimers used for cloning variable heavy chain (VH) and variable light chain (VL) antibody cDNAs were synthesized at Invitrogen (Beijing, China) based on the sequences reported previously (Brocks et al. 2001 Mol Med 7:461). PCR products were used directly for sequencing or subcloned into the pEASY-Blunt cloning vector (Cat. No: CB101 TransGen, China), then sequenced using Genewiz (Beijing, China). The amino acid sequences of the VH and VL regions were determined based on DNA sequencing results.

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

Table 1

Amino acid sequences of the VH and VL regions of Mu445

VH Mi445 SEQ ID NO: 9 EVQLQQSGPELVKPGASVKMSCKASGYKFT SYIIHWVKQKPGQGLEWIGYINPYNDGTRY NEKFKGKATLTSDKSSSTAYMEYSSLTSEDS AVYYC ARGY YGS S YAMD YWGQGT S VT VS S VL Mu445 SEQ ID NO: 11 DIQMTQTTSSLSASLGDRVTISCSASQGISNY LNWYQQKPDGTIKLLIYDTSTLYSGVPSRFS GSGSGTDYFLTISNLEPEDIATYYCQQYSKL PYTFGGGTKLEKK

table 2

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

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

Example 3: Humanization of Mouse Anti-Human Antibody OX40 445.

Humanization and antibody construction.

To humanize Mu445, human germline IgG genes were screened for sequences that share high degrees of homology with Mu445 variable region cDNA sequences by sequence comparison to the human immunoglobulin gene database at IMGT. The human IGHV and IGKV genes, which are found in the human antibody repertoire at high frequencies (Glanville et al, 2009 PNAS 106:20216-20221) and are highly homologous to Mu445, were selected as templates for humanization.

Humanization was performed by CDR grafting (Methods in Molecular Biology, Antibody Engineering, Methods and Protocols, Vol 248: Humana Press), and humanized antibodies were constructed in wild-type human IgG1 format using a self-developed expression vector. In the initial round of humanization, mutations of amino acid residues in the framework regions from murine to human were manipulated through analysis of the modeled 3D structure, and the murine framework residues with structure importance for maintaining canonical CDR structures were retained in the first version of the humanized antibody 445 (see 445-1, Table 3). The six CDRs 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 an 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 an 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 onto the human germline variable region gene framework IGVK1-39, retaining two residues of the mouse framework region (I44 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 onto the human germline variable region gene backbone IGHV1-69, retaining two residues of the mouse backbone region (L ₇₀ and S ₇₂ ) (SEQ ID NO: 14). In humanization variants, 445 (445-1) was grafted with only the N-terminal half of HCDR2 Kabat because only the N-terminal half was predicted to be important for antigen binding according to the modeled 3D structure.

445-1 was constructed as a humanized full-length antibody using in-house developed expression vectors that contain human IgG1 wild-type (IgG1wt) and kappa chain constant regions, respectively, with easy adaptation of subcloning sites. Antibody 445-1 was expressed by co-transfecting 293G cells with the above two constructs and purified using a Protein A column (Cat. No: 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 substitutions were made, converting the remaining mouse residues in the framework region of VH and VL into the corresponding human germline residues, such as I44P and Y71F in VL and L70I and S72A in VH. In addition, several single amino acid substitutions were made in the CDR to reduce the risk of possible isomerization and to increase the level of humanization. For example, in LCDR2 changes were made to T51A and D50E and in HCDR2 changes were made to D56E, G57A and N61A. All humanization changes were made using primers containing mutations at specific positions and a site-directed mutagenesis kit (Cat. No: AP231-11, TransGen, Beijing, China). Desirable substitutions were verified by sequencing.

Amino acid substitutions in antibody 445-1 were evaluated for their OX40 binding and thermostability. Antibody 445-2 containing HCDR1 with SEQ ID NO: 3, HCDR2 with SEQ ID NO: 18, HCDR3 with SEQ ID NO: 5, LCDR1 with SEQ ID NO: 6, LCDR2 with SEQ ID NO: 19 and LCDR3 with SEQ ID NO:8 (see Table 3) was designed based on the combination of specific substitutions described above. When comparing the two antibodies, the results showed that both antibodies 445-2 and 445-1 showed comparable binding affinities (see Tables 4 and 5 below).

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

Humanized antibodies 445 were further engineered by introducing specific amino acid substitutions into CDRs and framework regions to improve molecular and biophysical properties for therapeutic use in humans. Criteria included removal of deleterious post-translational modifications, improved thermal stability (T _m ), surface hydrophobicity and isoelectric points (pI) while maintaining binding activities.

Humanized monoclonal antibody, 445-3, containing HCDR1 with SEQ ID NO: 3, HCDR2 with SEQ ID NO: 24, HCDR 3 with SEQ ID NO: 5, LCDR1 with SEQ ID NO: 25, LCDR2 with SEQ ID NO: 19 and LCDR3 with SEQ ID NO: 8 (see Table 3) was constructed based on the maturation method described above and characterized in detail. Antibody 445-3 was also converted to an Fc domain-containing version of IgG2 (IgG2 445-3)

- 22 045547 wild-type human IgG2 heavy chain, and a version of IgG4 containing the human Fc domain

IgG4 with mutations S228P and R409K (IgG4 445-3). The results showed that 445-3 and 445-2 exhibited comparable binding affinities (see Tables 4 and 5).

Table 3

Antibody sequences 445

Antibody SEQ ID NO Subsequence 445-1 SEQ ID NO: 3 HCDR1 (Kabat) SYIIH SEQ ID NO: 13 HCDR2 (Kabat) YINPYNDGTRYNQKFQG SEQ ID NO: 5 HCDR3 (Kabat) GYYGSSYAMDY SEQ ID NO: 6 LCDR1 (Kabat) SASQGISNYLN SEQ ID NO: 7 LCDR2 (Kabat) DTSTLYS SEQ ID NO: 8 LCDR3 (Kabat) QQYSKLPYT SEQ ID NO: 14 VH QVQLVQSGAEVKKPGSSVKVSCKASGY KFTSYIIHWVRQAPGQGLEWMGYINPYN DGTRYNQKFQGRVTLTSDKSTSTAYMEL S SLRSEDT AVYYC ARGYYGS S Y AMD YW GQGTTVTVSS SEQ ID NO: 16 VL DIQMTQSPSSLSASVGDRVTITCSASQGIS NYLNWYQQKPGKAIKLLIYDTSTLYSGV PSRFSGSGSGTDYTLTISSLQPEDFATYYC QQYSKLPYTFGGGTKVEIK 445-2 SEQ ID NO: 3 HCDR1 (Kabat) SYIIH SEQ ID NO: 18 HCDR2 (Kabat) YINPYNEGTRYAQKFQG SEQ ID NO: 5 HCDR3 (Kabat) GYYGSSYAMDY SEQ ID NO: 6 LCDR1 (Kabat) SASQGISNYLN SEQ ID NO: 19 LCDR2 (Kabat) DASTLYS SEQ ID LCDR3 QQYSKLPYT

- 23 045547

NO: 8 (Kabat) SEQ ID NO: 20 VH QVQLVQSGAEVKKPGSSVKVSCKASGY KFTSYIIHWVRQAPGQGLEWMGYINPYN EGTRYAQKFQGRVTLTADKSTSTAYMEL S SLRSEDT AVYYC ARGYYGS S YAMD YW GQGTTVTVSS SEQ ID NO: 22 VL DIQMTQSPSSLSASVGDRVTITCSASQGIS NYLNWYQQKPGKAIKLLIYDASTLYSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYSKLPYTFGGGTKVEIK 445-3 SEQ ID NO: 3 HCDR1 (Kabat) SYIIH SEQ ID NO: 24 HCDR2 (Kabat) YINPYNEGTRYNQKFQG SEQ ID NO: 5 HCDR3 (Kabat) GYYGSSYAMDY SEQ ID NO: 25 LCDR1 (Kabat) RASQGISNYLN SEQ ID NO: 19 LCDR2 (Kabat) DASTLYS SEQ ID NO: 8 LCDR3 (Kabat) QQYSKLPYT SEQ ID NO: 26 VH QVQLVQSGAEVKKPGSSVKVSCKASGY KFTSYIIHWVRQAPGQGLEWMGYINPYN EGTRYNQKFQGRVTLTADKSTSTAYMEL S SLRSEDT AVYYC ARGYYGS S YAMD YW GQGTTVTVSS SEQ ID NO: 28 VL DIQMTQSPSSLSASVGDRVTITCRASQGIS NYLNWYQQKPDGAIKLLIYDASTLYSGV PSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYSKLPYTFGGGTKVEIK

Example 4 Determination of OX40 antibody binding kinetics and affinity by SPR.

Antibodies to OX40 were characterized for their kinetics and binding affinity by SPR assays using BIAcore™ T-200 (GE Life Sciences). Briefly, anti-human IgG was immobilized onto an activated CM5 biosensor chip (Cat. No: BR100530, GE Life Sciences). An antibody with the Fc region of human IgG was passed through the surface of the chip and captured with an anti-human IgG antibody. Then, a serial dilution of His-tagged recombinant OX40 protein (Cat. No.: 10481-H08H, Sino Biological) was passed through the chip surface, and changes in surface plasmon resonance signals were analyzed to calculate association rates (ka) and dissociation rates (KD) by using the 1:1 Langmuir binding model (BIA evaluation software, GE Life Sciences). The equilibrium dissociation constant (KD) was calculated as the ratio kd/ka. The results of the SPR-determined anti-OX40 antibody binding profiles are summarized in FIG. 2 and in table. 4. The binding profile to the average KD of antibody 445-3 (9.47 nM) is slightly better than that of antibodies 445-2 (13.5 nM) and 445-1 (17.1 nM), and is similar to the binding profile of ch445. The binding profile of IgG4 445-3 was similar to 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.

- 24 045547

Table 4

Binding affinities of antibodies to OX40 according to SPR

Analyzed parameters ch445* 445-1 445-2 445-3 IgG4 445-3 Test 1 ka (M'V ¹ ) 1.74^10 ⁵ 1.56χ10 ⁵ 2.76χ10 ⁵ 1.82χ10 ⁵ Ι,όΙχΙΟ ⁵ kd (c' ¹ ) 1.43 χ ΙΟ' ⁵ 2.77χ10' ⁵ 3.90χ10' ⁵ 1.67χ10' ⁵ 1.61 X1O ^J KD (nM) 8.26 17.8 14.2 9.16 10.0 KA (M ^-1 ) ^{1.22x10s 0.56x10s 0.71x10s 1.09x10s 1.00x10s} Test 2 ka (M'A ¹ ) 2.65χ10 ⁵ 2.37χ10 ⁵ 2.06χ10 ⁵ 1.63χ10 ⁵ - kd (c' ¹ ) 1.67χ10' ⁵ 3.89χ10' ⁵ 2.64χ10' ⁵ 1.59χ10' ⁵ - KD(hM) 6.3 16.4 12.8 9.77 - KA (M ^-1 ) ^{1.59x10s 0.61x10s 0.78x10s 1.03x10s} - Average KD(hM) 7.28 17.1 13.5 9.47 10.0 KA (M ^-1 ) ^{1.41x10s 0.59x10s 0.75x10s 1.06x10s 1.00x10s}

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

Example 5 Determination of the binding affinity of antibodies to OX40 to 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-expressing line. Live HuT78/OX40 cells were seeded in a 96-well plate and incubated with serial dilutions of different OX40 antibodies. Goat anti-human IgG-FITC antibody (Cat: A0556, Beyotime) was used as a secondary antibody to detect cell surface binding antibody. EC ₅₀ 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 table. 5, antibodies to OX40 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 of Table 5), indicating a slower dissociation of the antibody from OX40, which is a more desirable binding profile.

Table 5

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

Antibody ECso (µg/ml) Upper level (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 IgG4 445-3 0.263 n/a 0.263 892

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

To assess the cross-reactivity of antibody 445-3 against human OX40 and cyno OX40, cells expressing human OX40 (HuT78/OX40) and cyno OX40 (HuT78/cynoOX40) were seeded in 96-well plates and incubated with a series of dilutions of antibodies to OX40. Goat anti-human IgG-FITC antibody (Cat. No: A0556, Beyotime) was used as a secondary detection antibody. EC ₅₀ 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 in the table below. 6. Antibody 445-3 cross-reacts with both human OX40 and cynomolgus OX40, with similar EC ₅₀ values as shown below.

- 25 045547

Table 6

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

Cell line EC50 (µg/ml) 445-3 Upper level (MFI) NiT78/OX40 0.174 575 HuT78/cynoOX40 0.171 594

Example 7. Determination of co-crystallization and structure of OX40 with Fab 445-3.

To understand the binding mechanism of OX40 to the antibodies of the present invention, the co-crystal structure of OX40 and Fab 445-3 was solved. Mutations at positions T148 and N160 were introduced to block OX40 glycosylation and to improve protein homogeneity. DNA encoding mutant human OX40 (residues M1-D170 with two mutant sites, T148A and N160A) was cloned into an expression vector incorporating a hexa-His tag, and 293G cells were transiently transfected with this construct to express the protein at 37°C for 7 days. Cells were harvested, and the supernatant was collected and incubated with His tag 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 using 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 293G cells were transiently co-transfected with this vector to express the protein at 37°C for 7 days. The purification steps for Fab 445-3 were the same as those used above for the OX40 mutant protein.

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

Cocrystal screening was performed by mixing the protein complex with the reservoir solution in a 1:1 volume ratio. Cocrystals were prepared from a hanging drop cultured at 20°C by vapor diffusion using a reservoir solution containing 0.1 M HEPES, pH 7.0, 1% PEG 2000 MME, and 0.95 M sodium succinate.

Nylon loops were used to collect cocrystals, and these crystals were immersed in a reservoir solution supplemented with 20% glycerol for 10 seconds. Diffraction data were collected at BL17U1, a synchrotron radiation facility, Shanghai, and processed through XDS software. The phase was solved by the PHASER program using the Fab IgG structure (chain C and D PDB: 5CZX) and the structure of OX40 (chain R PDB: 2HEV) as molecular replacement study models. The Phenix.refine GUI was used to perform rigid body, TLS, and limited refinement based on the radiographic data, followed by correction using the COOT program and additional refinement in the Phenix.refine program. The totality of radiographic examination data and refinement statistics are briefly summarized in Table. 7.

- 26 045547

Table 7

Data set and refinement statistics

Data set Beam channel BL17U1,SSRF Space group R 31 2 1 Cell size (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 reflections 988771 (81305) Number of characteristic reflections 50306 (4625) Completeness (%) 99.9 (99.9) Average redundancy 19.7 (17.6) Yasliyaniea 0.059 (0.962) Ι/sigma (I) 29.4 (3.5) Wilson factor B (A) 73.9 Clarification Resolution (A) 60.22-2.55 Number of reflections 50008 rmsd (standard deviation) of bond lengths (A) 0.010 rmsd connection angles (°) 0.856 Kraboch (%) 19.27 Kfree (%) 21.60 Average protein B factors 97.10 Ramachandran Maps (%) preferred 96.34 acceptable 3.48 Emissions 0.17

Values in parentheses refer to the highest resolution shell.

^a RcnHMHHe=£Sil/(A)i - №)>Ι/ΣΣΦ(Α)ίΙ, ^g de </(L.)> represents the average intensity of the equivalent.

%a ₆ <^|Fo-Fc|^|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.

calculated using the analysis data set, 5% of the total data randomly selected from the observed reflections.

Example 8 Antibody 445-3 Epitope Identification by SPR.

Based on the co-crystal structure of OX40 and the Fab 445-3 antibody, we selected and generated a variety 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 created in the human OX40/IgG1 fusion construct using a site-directed kit

- 27 045547 mutagenesis (cat. no.: AP231-11, TransGen). Desirable mutations were verified by sequencing.

Expression and production of OX40 mutants was achieved through transfection of 293G cells and purified using a Protein A column (Cat. No: 17-5438-02, GE Life Sciences).

The binding affinity of OX40 point mutations for Fab 445-3 was characterized by SPR assays using BIAcore 8K (GE Life Sciences). Briefly, immobilization of wild-type OX40 and OX40 mutants was carried out on a CM5 biosensor chip (Cat. No: BR100530, GE Life Sciences) using EDC and NHS. A serial dilution of Fab 445-3 in HBS-EP+buffer (Cat. No: BR-1008-26, GE Life Sciences) was then passed through the chip surface using 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 the 1:1 Langmuir binding model (BIA trial release, GE Life Sciences). The equilibrium dissociation constant (KD) was calculated as the ratio kd/ka. The KD fold shift of the mutant was calculated as the ratio of KD mutant/KDWT. The epitope identification profiles determined by SPR 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 resulted in a moderate decrease in binding of antibody 445-3 to OX40.

Detailed relationships between antibody 445-3 and residues H153, T154, 1165, E167, and D170 of 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 _strand S31 and _strand G102 and π-π stacking with _strand Y101. The side chain of E167 formed hydrogen bonds with _strand U50 and _strand N52, while D170 formed a hydrogen bond and salt bridge with _strand S31 and _strand K28, respectively, which may further stabilize the complex. Van der Waals (VDW) interactions of T154 with _strand Y105, I165 and _strand R59 contributed to the high affinity of antibody 445-3 for OX40.

In conclusion, residues H153, I165 and E167 of OX40 were identified as important residues for interaction with antibody 445-3. In addition, amino acids T154 and D170 of OX40 are also important residues for contact with antibody 445-3. These data showed that the epitopes of antibody 445-3 are residues H153, T154, I165, E167 and D170 of OX40. These epitopes are located in the sequence HTLQPASNSSDAICEDRD (SEQ ID NO: 30), with residues important for contact being highlighted in bold and underlined.

Table 8 Identification of Antibody 445-3 Epitope Determined by SPR

Mutants Kd mutant/KDWT H153A No binding detected T154A 8 Q156A 1.9 S161A 1.1 S162A 0.6 I165A 28 E167A 135 D170A 8

Significant effect: no binding was detected, or the mutant/KD WT value was greater than 10. Moderate effect: the mutant KD value/WT KD was between 5 and 10. Non-significant effect: the mutant KD value/WT KD was less than 5.

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

To determine whether antibody 445-3 interferes with the OX40-OX40L interaction, flow cytometry analysis was performed on cells. In this assay, antibody 445-3, reference antibody lA7.grl, control huIgG, or medium alone were preincubated with human OX40-mouse IgG2a Fc fusion protein (OX40-mIgG2a). The antibody and fusion protein complex were then added to OX40L-expressing HEK293 cells. If the 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 using a secondary anti-mouse Fc antibody.

As shown in FIG. 7, antibody 445-3, even at high concentration, did 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 does not bind tightly enough to sterically hinder OX40L binding. In contrast, the positive control antibody, 1A7.gr1, completely blocked OX40 binding to OX40L, as shown in FIG. 7.

- 28 045547

In addition, the cocrystal structure of OX40 complexed 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. In summary, antibody 445-3 and the OX40L trimer bind at the level of different corresponding 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 above Examples. CRD4 OX40 is located at amino acids 127-167, and the epitope of antibody 445-3 partially overlaps with this region. The sequence of CRD4 OX40 (amino acids 127-167) is shown below, and the partial overlap of the 445-3 epitope is shown in bold and underlined: PCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICE (SEQ ID NO: 31).

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

To examine the agonistic functions of the 445-3 antibody, an OX40-positive T cell line, HuT78/OX40, was co-cultured with an artificial antigen presenting cell (APC) line (HEK293/OS8 ^low -FcyRI) in the presence or absence of 445-3 or lA7.gr1 in overnight, and IL2 production was used as a recording value for T cell stimulation. In HEK293/OS8 ^Low -FcyRI genes encoding the 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 immune activation induced by anti-OX40 antibody is dependent on antibody cross-linking (Voo et al, 2013), FcyRJ on HEK293/OS8 ^Low -FcyRI provides the basis for anti-OX40 antibody-mediated cross-linking of OX40 in a dual interaction of anti-OX40 antibody with OX40 and FcyRI. As shown in FIG. 9, the anti-OX40 antibody 445-3 was highly active in enhancing TCR signaling in a dose-dependent manner with an EC ₅₀ of 0.06 ng/ml. Slightly weaker activities of the reference Ab lA7.gr1 were also observed. In contrast, control human IgG (10 μg/ml) or blank showed no effect on IL-2 production.

Example 11 Anti-OX40 antibody 445-3 stimulated immune responses in a mixed lymphocyte reaction (MLR) assay.

To determine whether antibody 445-3 could stimulate T cell activation, a mixed lymphocyte response (MLR) assay was performed as previously described (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 (lipopolyssacharide) stimulation. Next, mitomycin C-treated DCs were co-cultured 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 the coculture was detected by ELISA as a recorded indicator of the MLR 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 activities in the MLR assay.

Example 12 Antibody OX40 445-3 demonstrated ADCC activity.

An ADCC lactate dehydrogenase (LDH) release assay was performed to examine whether antibody 445-3 could kill OX40 ^Hi -expressing target cells. The NK92MI/CD16V cell line was generated as effector cells by co-transduction of the CD16v158 (V158 allele) and FcRy genes into the NK cell line, NK92MI (ATCC, Manassas VA). An OX40-expressing T cell line, HuT78/OX40, was used as target cells. Equal numbers (3x10 ⁴ ) of target and effector cells were co-cultured for 5 hours in the presence of OX40 antibody (0.004-3 μg/ml) or control Ab. Cytotoxicity was assessed by LDH release using the CytoTox 96 Nonradioactive Cytotoxicity Assay Kit (Promega, Madison, WI). Specific lysis was calculated using the formula shown below.

Experimental. - Effector, spontaneous. - Target, spontaneous.

%, Specific lysis =-------------------------------------------- x 100

Target, maximum - Target, spontaneous.

As shown in FIG. 11, antibody 445-3 showed high activity in killing OX40 ^Hi targets via ADCC in a dose-dependent manner ( _EC50 : 0.027 μg/ml). The ADCC effect of antibody 445-3 was similar to that of control antibody 1 A7.grl. In contrast, 445-3 Fc format IgG4 with mutations S228P and R409K (445-3-IgG4) did not show any significant ADCC effects compared to control human IgG or blank. The results are consistent with previous results that suggest that IgG4 Fc is weak or silent against ADCC (An Z, et al. mAbs 2009).

Example 13 Anti-OX40 antibody 445-3 preferentially depletes CD4 ⁺ Tregs and increases CD8 ⁺ Teff/Treg ratios in vitro.

Several animal tumor models showed that anti-OX40 antibodies could deplete tumor-infiltrating OX40 ^Hi Tregs and increase CD8+ T cell to Treg ratios (Bulliard et al., 2014; Carboni et al., 2003; Jacquemin et al., 2015; Marabelle et al., 2013b). Consequently, the immune response is enhanced

- 29 045547 was achieved, leading to tumor regression and improved survival.

Given the fact that in vitro activated or intratumoral CD4 ⁺ Foxp3 ⁺ Tregs express OX40 preferentially over other T cell subsets (Lai et al., 2016; Marabelle et al., 2013b; Montler et al., 2016; Soroosh et al., 2007; Timperi et al., 2016), an assay based on human PBMCs was performed to investigate the ability of antibody 445-3 to destroy OX40 ^Hi cells, in particular Tregs. 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, 5x104) 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 by flow cytometry. As shown in FIG. 12A and 12B, treatment with antibody 445-3 caused an increase in the percentage of CD8 T cells and a decrease in the percentage of CD4 Foxp3 Tregs in a dose-dependent manner. As a result, CD8 T cell to Treg ratios were significantly improved (Figure 12C). Less improvement results were obtained with 1A7.gr1 treatment. This result demonstrates the therapeutic applications of 445-3 in inducing antitumor immunity by stimulating CD8 ⁺ T cell functions while limiting Treg-mediated immune tolerance.

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

The effectiveness of the antibody to OX40 445-3 was demonstrated in a mouse tumor model. MC38 murine colon tumor cells were subcutaneously implanted into human OX40 transgenic C57 mice (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 (axb ² ), where a and b were the tumor dimensions along the long and short axes, respectively. When the tumors reached an average volume of approximately 190 mm ³ in size, mice were randomly assigned to 7 groups and were injected intraperitoneally with either antibody 445-3 or antibody 1A7.gr1 once a week for three weeks. Human IgG was administered as an isotype control. Partial regression (PR) was defined as tumor volume being less than 50% of initial tumor volume on the first day of dosing in three consecutive measurements. Tumor growth inhibition (TGI) was calculated using the following formula:

. Λ /(process t) - (process t ₀ ) \\

Growth inhibition,% = JO x I 1 - I ------------ _r . I ^g \ ((placebo t) - (placebo QJ) treatment t means the volume of the treated tumor at time t; treatment t ₀ means the volume of the treated tumor at time 0; placebo t means the volume of the tumor in the presence of placebo at time t ; placebo t ₀ means tumor volume in the presence of placebo at time 0. The results demonstrated that 445-3 had dose-dependent antitumor efficacy when given by intraperitoneal injection at doses of 0.4, 2, and 10 mg/kg. Administration of 445-3 resulted in 53 % (0.4 mg/kg), 69% (2 mg/kg) and 94% (10 mg/kg) inhibition of tumor growth and led to 0% (0.4 mg/kg ), 17% (2 mg/kg) and 33% (10 mg/kg) partial regression from baseline. In contrast, no partial regression was observed with antibody 1A7.gr1. 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 (Fig. 13A and 13B, Table 9).

Table 9

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

Treatment QW (weekly) Dose (mg/kg) N Degree of partial regression Average tumor volume per day 21 (mm ³ ) TGI in day 21 (%) 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 1. Changes in the amino acid composition of antibodies to OX40.

Several amino acids were selected for modification in order to improve OX40 antibodies. Amino acid substitutions were made to improve affinity or to increase the level of humanization. Sets of PCR primers were designed for appropriate changes in amino acid composition, synthesized and used to modify antibodies to OX40. For example, replacing K28T in the heavy chain and

- 30 045547 substitution of S24R in the light chain resulted in a 1.7-fold increase in EC50 determined by

FACS compared to parent antibody 445-2. The Y27G substitution in the heavy chain and the S24R substitution in the light chain resulted in a 1.7-fold increase in the KD determined by Biacore compared to the parent antibody 445-2. These changes are summarized in FIG. 14A, 14B.

Links.

al-Shamkhani, A., Birkeland, M. L., Puklavec, M., Brown, M. H., James, W., and Barclay,

A.N. (1996). 0X40 is differentially expressed on activated rat and mouse T cells and is the sole receptor for the 0X40 ligand. European journal of immunology 26, 1695-1699.

An Z, Forrest G, Moore R, Cukan M, Haytko P, Huang L, Vitelli S, Zhao JZ, Lu P, Hua

J, Gibson CR, Harvey BR, Montgomery D, Zaller D, Wang F, Strohl W. (2009). IgG2m4, an engineered antibody isotype with reduced Fc function. MAbs. 1.572-579.

Arch, R.H., and Thompson, C.B. (1998). 4-1BB and 0x40 are members of a tumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bind TNF receptor-associated factors and activate nuclear factor kappaB. Molecular and cellular biology 18, 558-565.

Aspeslagh, S., Postel-Vinay, S., Rusakiewicz, S., Soria, J.C., Zitvogel, L., and Marabelle,

A. (2016). Rationale for anti-OX40 cancer immunotherapy. Eur J Cancer 52, 50-66.

Bulliard, Y., Jolicoeur, R., Zhang, J., Dranoff, G., Wilson, N.S., and Brogdon, J.L.

(2014). OX40 engagement depletes intratumoral Tregs via activating FcgammaRs, leading to antitumor efficacy. Immunology and cell biology 92, 475-480.

Calderhead, D.M., Buhlmann, J.E., van den Eertwegh, A.J., Claassen, E., Noelle, R.J., and Fell, H.P. (1993). Cloning of mouse 0x40: a T cell activation marker that may mediate T-B cell interactions. J Immunol 151, 5261-5271.

Carboni, S., Aboul-Enein, F., Waitzinger, C., Killeen, N., Lassmann, H., and Pena-Rossi,

C. (2003). CD134 plays a crucial role in the pathogenesis of EAE and is upregulated in the CNS of patients with multiple sclerosis. Journal of neuroimmunology 145, 1-11.

Compaan, D.M., and Hymowitz, S.G. (2006). The crystal structure of the costimulatory

OX40-OX40L complex. Structure 14, 1321-1330.

- 31 045547

Croft, M. (2010). Control of immunity by the TNFR-related molecule 0X40 (CD 134). Annual review of immunology 28, 57-78.

Croft, M., So, T., Duan, W., and Soroosh, P. (2009). The significance of OX40 and OX40L to T-cell biology and immune disease. Immunological reviews 229, 173-191.

Curti, B.D., Kovacsovics-Bankowski, M., Morris, N., Walker, E., Chisholm, L., Floyd, K., Walker, J., Gonzalez, I., Meeuwsen, T., Fox, B.A., et al. (2013). OX40 is a potent immune stimulating target in late-stage cancer patients. Cancer research 73, 7189-7198.

Durkop, H., Latza, U., Himmelreich, P., and Stein, H. (1995). Expression of the human OX40 (hOX40) antigen in normal and neoplastic tissues. British journal of hematology 91, 927931.

Gough, M.J., and Weinberg, A.D. (2009). OX40 (CD134) and OX40L. Advances in experimental medicine and biology 647, 94-107.

Gramaglia, I., Weinberg, A. D., Lemon, M., and Croft, M. (1998). Ox-40 ligand: a potent costimulatory molecule for sustaining primary CD4 T cell responses. J Immunol 161, 65106517.

Guo, Z., Cheng, D., Xia, Z., Luan, M., Wu, L., Wang, G., and Zhang, S. (2013). Combined TIM-3 blockade and CD 137 activation affords the long-term protection in a murine model of ovarian cancer. Journal of translational medicine 11,215.

Hori, S., Nomura, T., and Sakaguchi, S. (2003). Control of regulatory T cell development by the transcription factor Foxp3. Science 299, 1057-1061.

Huddleston, C.A., Weinberg, A.D., and Parker, D.C. (2006). OX40 (CD134) engagement drives differentiation of CD4+ T cells to effector cells. European journal of immunology 36, 1093-1103.

Ito, T., Amakawa, R., Inaba, M., Hori, T., Ota, M., Nakamura, K., Takebayashi, M., Miyaji, M., Yoshimura, T., Inaba, K., and Fukuhara, S. (2004). Plasmacytoid dendritic cells regulate Th cell responses through OX40 ligand and type I IFNs. J Immunol 172, 4253-4259.

Ito, T., Wang, Y.H., Duramad, 0., Hanabuchi, S., Perng, O.A., Gilliet, M., Qin, F.X., and Liu, Y.J. (2006). OX40 ligand shuts down IL-10-producing regulatory T cells. Proceedings of the National Academy of Sciences of the United States of America 103, 13138-13143.

Jacquemin, C., Schmitt, N., Contin-Bordes, C., Liu, Y., Narayanan, P., Seneschal, J., Maurouard, T., Dougall, D., Davizon, E.S., Dumortier, H. , et al. (2015). OX40 Ligand Contributes to Human Lupus Pathogenesis by Promoting T Follicular Helper Response. Immunity 42, 1159-1170.

- 32 045547

Kjaergaard, J., Tanaka, J., Kim, J. A., Rothchild, K., Weinberg, A., and Shu, S. (2000). Therapeutic efficacy of OX-40 receptor antibody depends on tumor immunogenicity and anatomic site of tumor growth. Cancer research 60, 5514-5521.

Ladanyi, A., Somlai, B., Gilde, K., Fejos, Z., Gaudi, I., and Timar, J. (2004). T-cell activation marker expression on tumor-infiltrating lymphocytes as prognostic factor in cutaneous malignant melanoma. Clinical cancer research: an official journal of the American Association for Cancer Research 10, 521-530.

Lai, C., August, S., Albibas, A., Behar, R., Cho, S.Y., Polak, M.E., Theaker, J., MacLeod, A.S., French, R.R., Glennie, M.J., et al. (2016). OX40+ Regulatory T Cells in Cutaneous Squamous Cell Carcinoma Suppress Effector T-Cell Responses and Associate with Metastatic Potential. Clinical cancer research: an official journal of the American Association for Cancer Research 22, 4236-4248.

Marabelle, A., Kohrt, H., and Levy, R. (2013a). Intratumoral anti-CTLA-4 therapy: enhancing efficacy while avoiding toxicity. Clinical cancer research: an official journal of the American Association for Cancer Research 19, 5261-5263.

Marabelle, A., Kohrt, H., Sagiv-Barfi, 1., Ajami, B., Axtell, R.C., Zhou, G., Rajapaksa, R., Green, M.R., Torchia, J., Brody, J., et al. (2013b). Depleting tumor-specific Tregs at a single site eradicates disseminated tumors. The Journal of clinical investigation 123, 2447-2463.

Montier, R., Bell, R.B., Thalhofer, C., Leidner, R., Feng, Z., Fox, B.A., Cheng, A.C., Bui, T.G., Tucker, C., Hoen, H., and Weinberg, A. (2016). OX40, PD-1 and CTLA-4 are selectively expressed on tumor-infiltrating T cells in head and neck cancer. Clinical & translational immunology 5, e70.

Morris, N.P., Peters, C., Montier, R., Hu, H.M., Curti, B.D., Urba, W.J., and Weinberg, A.D. (2007). Development and characterization of recombinant human Fc:OX40L fusion protein linked via a coiled-coil trimerization domain. Molecular immunology 44, 3112-3121.

Ohshima, Y., Tanaka, Y., Tozawa, H., Takahashi, Y., Maliszewski, C., and Delespesse, G. (1997). Expression and function of OX40 ligand on human dendritic cells. J Immunol 159, 3838-3848.

Petty, J.K., He, K., Corless, C.L., Vetto, J.T., and Weinberg, A.D. (2002). Survival in human colorectal cancer correlates with expression of the T-cell costimulatory molecule OX-40 (CD134). American journal of surgery 183, 512-518.

Redmond, W.L., and Weinberg, A.D. (2007). Targeting OX40 and OX40L for the treatment of autoimmunity and cancer. Critical reviews in immunology 27, 415-436.

- 33 045547

Rogers, P. R., Song, J., Gramaglia, I., Killeen, N., and Croft, M. (2001). 0X40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells. Immunity 15, 445-455.

Ruby, C.E., and Weinberg, A.D. (2009). OX40-enhanced tumor rejection and effector T cell differentiation decreases with age. J Immunol 182, 1481-1489.

Sarff, M., Edwards, D., Dhungel, B., Wegmann, K.W., Corless, C., Weinberg, A.D., and Vetto, J.T. (2008). OX40 (CD 134) expression in sentinel lymph nodes correlates with prognostic features of primary melanomas. American journal of surgery 195, 621-625; discussion 625.

Sato, T., Ishii, N., Murata, K., Kikuchi, K., Nakagawa, S., Ndhlovu, L. C., and Sugamura, K. (2002). Consequences of 0X40-0X40 ligand interactions in langerhans cell function: enhanced contact hypersensitivity responses in OX40L-transgenic mice. European journal of immunology 32, 3326-3335.

Smyth, M.J., Ngiow, S.F., and Teng, M.W. (2014). Targeting regulatory T cells in tumor immunotherapy. Immunology and cell biology 92, 473-474.

Song, A., Tang, X., Harms, K. M., and Croft, M. (2005a). OX40 and Bcl-xL promote the persistence of CD8 T cells to recall tumor-associated antigen. J Immunol 175, 3534-3541.

Song, J., So, T., Cheng, M., Tang, X., and Croft, M. (2005b). Sustained survivin expression from OX40 costimulatory signals drives T cell clonal expansion. Immunity 22, 621631.

Song, J., So, T., and Croft, M. (2008). Activation of NF-kappaBl by OX40 contributes to antigen-driven T cell expansion and survival. J Immunol 180, 7240-7248.

Soroosh, P., Ine, S., Sugamura, K., and Ishii, N. (2007). Differential requirements for OX40 signals on generation of effector and central memory CD4+ T cells. J Immunol 179, 50145023.

St Rose, M.C., Taylor, R.A., Bandyopadhyay, S., Qui, H.Z., Hagymasi, A.T., Vella, A.T., and Adler, A.J. (2013). CD134/CD137 dual costimulation-elicited IFN-gamma maximizes effector T-cell function but limits Treg expansion. Immunology and cell biology 91, 173-183.

Stuber, E., Neurath, M., Calderhead, D., Fell, H. P., and Strober, W. (1995). Cross-linking of OX40 ligand, a member of the TNF/NGF cytokine family, induces proliferation and differentiation in murine splenic B cells. Immunity 2, 507-521.

Szypowska, A., Stelmaszczyk-Emmel, A., Demkow, U., and Luczynski, W. (2014). High expression of OX40 (CD 134) and 4-IBB (CD 137) molecules on CD4(+)CD25(high) cells in children with type 1 diabetes. Advances in medical sciences 59, 39-43.

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

- 34 045547 and potentially pro-tumor activities accumulate in human colorectal cancer. Oncoimmunology 5, el 175800.

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.

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.

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 OX40 expand effector T cells and block inducible and natural regulatory T cell function. J Immunol 191, 3641-3650.

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.

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.

Willoughby, J., Griffiths, J., Tews, I., and Cragg, M.S. (2017). OX40: Structure and function - What questions remain? Molecular immunology 83, 13-22.

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 OX40 Co-stimulatory Crosstalk Regulates Helper T Cell Differentiation and Anti-Plasmodium Humoral Immunity. Cell host & microbe 17, 628-641.

Zhang, T., Lemoi, B.A., and Sentman, C.L. (2005). Chimeric NK-receptor-bearing T cells mediate antitumor immunotherapy. Blood 106, 1544-1551.

Claims (31)

1. An antibody or antigen-binding fragment thereof that specifically binds to human OX40 and contains: (i) a heavy chain variable region which contains (a) HCDR (heavy chain complementarity determining region) 1 with SEQ ID NO: 3, (b) HCDR2 with SEQ ID NO: 24, (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, (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: 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, (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; or (iv) a heavy chain variable region that contains (a) HCDR1 of SEQ ID NO: 3, (b) HCDR2 of SEQ ID NO: 4, (c) HCDR3 of SEQ ID NO: 5; and a light chain variable region which contains: (d) LCDR1 of SEQ ID NO: 6, (e) LCDR2 of SEQ ID NO: 7, and (f) LCDR3 of SEQ ID NO: 8. 2. Antibody or antigen-binding fragment according to claim 1, containing: (i) a heavy chain variable region (VH) containing an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 26, and a light chain variable region chain (VL) containing an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 28; (ii) a heavy chain variable region (VH) containing an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 20, and a light chain variable region chain (VL) containing an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 22; - 35 045547 (iii) a heavy chain variable region (VH) containing an amino acid sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 14, and a light chain variable region (VL) containing an amino acid sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 16; or (iv) a heavy chain variable region (VH) containing an amino acid sequence at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 9, and a light chain variable region (VL) containing an amino acid sequence at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 11. 3. The antibody or antigen binding fragment of claim 2, wherein (i) SEQ ID NO: 26 and/or 28 contains one, two, three, four, five, six, seven, eight, nine or ten insertions, deletions or substitutions amino acids; (ii) SEQ ID NO: 20 and/or 22 contains insertions, deletions or substitutions of one, two, three, four, five, six, seven, eight, nine or ten amino acids; (iii) SEQ ID NO: 14 and/or 16 contains insertions, deletions or substitutions of one, two, three, four, five, six, seven, eight, nine or ten amino acids; or (iv) SEQ ID NO: 9 and/or 11 contains insertions, deletions or substitutions of one, two, three, four, five, six, seven, eight, nine or ten amino acids. 4. Antibody or antigen-binding fragment according to claim 1, containing: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 26 and a light chain variable region (VL) comprising SEQ ID NO: 28; (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 20 and a light chain variable region (VL) comprising SEQ ID NO: 22; (iii) a heavy chain variable region (VH) comprising SEQ ID NO: 14 and a light chain variable region (VL) comprising SEQ ID NO: 16; or (iv) a heavy chain variable region (VH) comprising SEQ ID NO: 9 and a light chain variable region (VH) comprising SEQ ID NO: 11. 5. An antibody or antigen binding fragment according to any one of claims 1 to 4, being a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a single chain antibody (scFv), a Fab fragment, a Fab' fragment or an F(ab')2 fragment . 6. Antibody or antigen-binding fragment according to any one of claims 1-5, having agonistic activity against OX40. 7. An antibody or antigen binding fragment according to any one of claims 1 to 6, binding to human OX40 at an epitope containing one or more amino acid residues selected from the group consisting of H153-D170 of human OX40. 8. An antibody or antigen binding fragment according to any one of claims 1 to 6, binding to human OX40 at an epitope containing one or more amino acid residues selected from the group consisting of H153, T154, I165, E167 and D170 of human OX40. 9. An antibody or antigen binding fragment according to any one of claims 1 to 6, binding to human OX40 at an epitope containing one or more amino acid residues selected from the group consisting of H153, I165 and E167 of human OX40. 10. An antibody or antigen binding fragment according to any one of claims 1 to 6, binding to human OX40 on or within SEQ ID NO. thirty. 11. An antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). 12. An antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof has a reduced level of glycosylation, or does not have glycosylation, or is hypofucosylated. 13. An antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof has an increased content of structures having branching at GlcNac points. 14. An antibody or antigen binding fragment according to any one of claims 1 to 6, wherein the Fc domain belongs to IgG1. 15. An antibody or antigen binding fragment according to any one of claims 1 to 6, wherein the Fc domain belongs to IgG4. 16. The antibody or antigen binding fragment according to claim 15, wherein IgG4 has the substitution S228P (according to the EU numbering system). 17. The antibody or antigen binding fragment according to claim 16, wherein IgG4 has the substitutions S228P and R409K (according to the EU numbering system). 18. An antibody or antigen-binding fragment according to any one of claims 1 to 17, which has one or more of the following properties: (i) capable of cross-reacting with cynoOX40; (ii) does not interfere with the OX40-OX40L interaction; - 36 045547 (iii) capable of co-stimulating T cells to produce IL-2; (iv) capable of co-activating CD4+ T cells, particularly as measured in a mixed lymphocyte response (MLR) assay; (v) capable of mediating ADCC, particularly as measured in an ADCC assay based on lactate dehydrogenase (LDH) release; (vi) capable of depleting CD4 ⁺ Treg (regulatory T cell); (vii) capable of increasing the CD8 ⁺ Teff (effector T cell)/Teg ratio; or (viii) is capable of mediating partial regression of tumors in an animal tumor model. 19. A pharmaceutical composition containing an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, further containing a pharmaceutically acceptable carrier. 20. A method of treating cancer, comprising administering to a patient in need thereof an effective amount of an antibody or antigen binding fragment according to any one of claims 1 to 18. 21. The method of claim 20, wherein the cancer is 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 and sarcoma. 22. The method of claim 21, wherein the antibody or antigen binding fragment is administered in combination with another therapeutic agent. 23. The method of claim 22, wherein the therapeutic agent is paclitaxel or a paclitaxel-based agent, docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide or 5-azacytidine. 24. The method of claim 23, wherein the therapeutic agent is a paclitaxel-based agent, lenalidomide or 5-azacytidine. 25. An isolated nucleic acid encoding an antibody or antigen-binding fragment according to any one of claims 1 to 18. 26. A vector for encoding an antibody or antigen-binding fragment according to any one of claims 1 to 18, containing a nucleic acid according to claim 25. 27. A host cell containing the nucleic acid of claim 25 or the vector of claim 26. 28. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell according to claim 27 and isolating the antibody or antigen-binding fragment from the culture. 29. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 for treating or reducing the likelihood of breast cancer, head and neck cancer, gastric cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma and sarcoma. 30. A diagnostic reagent comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof is labeled. 31. The diagnostic reagent according to claim 30, wherein the label is selected from the group consisting of a radiolabel, a fluorophore, a chromophore, an imaging agent and a metal ion.