Antibodies to ox40 and compositions thereof

Abstract

The present disclosure provides antibodies that bind to OX40, and methods of using them to modulate immunity in a patient in need thereof and to treat autoimmune disorders or immunoinflammatory disorders.

Description

ANTIBODIES TO 0X40 AND COMPOSITIONS THEREOFBACKGROUND OF THE INVENTION

[0001] 0X40 (also known as CD 134, TNFRSF4) is a member of the tumor necrosis factor (TNF) receptor superfamily and functions as a T cell costimulatory molecule. 0X40 is mainly expressed on activated immune cells, primarily CD4+T cells, CD8+T cells, and regulatory T cells (Tregs). 0X40’ s only known ligand is OX40L (CD252) and is mainly found on antigen-presenting cells activated by CD40, toll-like receptors, and inflammatory cytokines.

[0002] The OX40-OX40L complex is key to potentiating the expansion of effector T cells and the prolongation of their survival by suppressing apoptosis, enhancing T cell effector functions, such as cytokine production, and generating T helper memory cells. Even though resting memory T cells do not express 0X40, upon reactivation they become effector memory T cells and begin expressing 0X40. OX40-OX40L interaction promotes the expansion of these cells. 0X40 additionally suppresses the differentiation and activity of Tregs, further amplifying this process.

[0003] 0X40 expression is upregulated at sites of autoimmune inflammation and on peripheral circulating lymphocytes in autoimmunity. As a crucial T cell activator, blocking of OX40 / OX40L signaling has been shown to inhibit autoreactive T cells and reduce severity of disease in several mouse models for autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), systemic lupus erythematosus (SLE), and type 1 diabetes (T1D). Polymorphisms in the TNFSF4 gene are associated with SLE, systemic sclerosis, and Sjogren’s syndrome (SS). Thus, inhibition of the OX40-OX40L axis is an attractive approach to treating a broad range of T cell mediated autoimmune diseases.

[0004] In view of 0X40’ s role in immune responses and their repercussions in autoimmune and immunoinflammatory disorders, there is a need for new and improved therapies that target 0X40.SUMMARY OF THE INVENTION

[0005] The present disclosure provides anti-OX40 antibodies or antigen-binding portions thereof. In some embodiments, the antibody binds specifically to amino acids 80-104 and / or amino acids 110-122 of SEQ ID NO: 106. In some embodiments, the anti-OX40 antibody or antigen-binding portion further binds specifically to amino acids 70-72 and / or amino acidsportion thereof further binds specifically to one or more of amino acids 74, 82, 116, and 124 of SEQ ID NO: 106.

[0006] The present disclosure also provides anti-OX40 antibodies or antigen-binding portions thereof wherein the antibody binds to the same epitope as a reference antibody having a heavy chain variable domain (VH) and a light chain variable domain (VL) having SEQ ID NOs:5 and 6, respectively, SEQ ID NOs:l and 2, respectively, SEQ ID NOs:3 and 4, respectively, SEQ ID NOs:7 and 8, respectively, SEQ ID NOs:9 and 10, respectively, or SEQ ID NOs: l l and 12, respectively.

[0007] In some embodiments, the anti-OX40 antibody or antigen-binding portion thereof comprises a heavy chain that comprises heavy chain complementarity determining regions (H-CDR)-l-3 comprising SEQ ID NOs:37-39, respectively; a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to SEQ ID NO:5; a VH comprising SEQ ID NO:5; or SEQ ID NOs:5 and 97; and a light chain that comprises light chain complementarity determining regions (L-CDR)-l-3 comprising SEQ ID NOs:40-42, respectively; a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 6; a VL comprising SEQ ID NO: 6; or SEQ ID NOs: 6 and 98.

[0008] In some embodiments, the anti-OX40 antibody or antigen-binding portion thereof comprises a heavy chain that comprises H-CDR1-3 comprising SEQ ID NOs: 13-15, respectively; a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1; a VH comprising SEQ ID NO: 1; or SEQ ID NOs: 1 and 97; and a light chain that comprises L-CDR-1-3 comprising SEQ ID NOs: 16-18, respectively; a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2; a VL comprising SEQ ID NO:2; or SEQ ID NOs:2 and 98.

[0009] In some embodiments, the anti-OX40 antibody or antigen-binding portion thereof comprises a heavy chain that comprises H-CDR1-3 comprising SEQ ID NOs: 25-27, respectively; a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO:3; a VH comprising SEQ ID NO:3; or SEQ ID NOs:3 and 97; and a light chain that comprises L-CDR1-3 comprising SEQ ID NOs:28-30, respectively; a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NON; a VL comprising SEQ ID NON; or SEQ ID NOsN and 98.

[0010] In some embodiments, the anti-OX40 antibody or antigen-binding portion thereof comprises a heavy chain that comprises H-CDR1-3 comprising SEQ ID NOs:49-51,respectively; a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO:7; a VH comprising SEQ ID NO:7; or SEQ ID NOs:7 and 97; and a light chain that comprises L-CDR1-3 comprising SEQ ID NOs:52-54, respectively; a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:8; a VL comprising SEQ ID NO:8; or SEQ ID NOs:8 and 98.

[0011] In some embodiments, the anti-OX40 antibody or antigen-binding portion thereof comprises a heavy chain that comprises H-CDR1-3 comprising SEQ ID NOs:61-63, respectively; a VH comprising an amino acid sequence at least 90% identical to SEQ ID NOV; a VH comprising SEQ ID NOV; or SEQ ID NOsV and 97; and a light chain that comprises L-CDR1-3 comprising SEQ ID NOs:64-66, respectively; a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 10; a VL comprising SEQ ID NO: 10; or SEQ ID NOs: 10 and 98.

[0012] In some embodiments, the anti-OX40 antibody or antigen-binding portion thereof comprises a heavy chain that comprises H-CDR1-3 comprising SEQ ID NOs:73-75, respectively; a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 11; a VH comprising SEQ ID NO: 11; or an HC comprising SEQ ID NOs: l l and 97; and a light chain that comprises L-CDR1-3 comprising SEQ ID NOs:76-78, respectively; a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 12; a VL comprising SEQ ID NO: 12; or an LC comprising SEQ ID NOs: 12 and 98.

[0013] In some embodiments, the anti-OX40 antibody or antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs:37-42, respectively; SEQ ID NOs: 13-18, respectively; SEQ ID NOs:25-30, respectively; SEQ ID NOs:49-54, respectively; SEQ ID NOs:61-66, respectively; or SEQ ID NOs:73-78 respectively. In certain embodiments, the anti-OX40 antibody or antigen-binding portion thereof comprises a VH amino acid sequence and a VL amino acid sequence that are at least 90% identical to the amino acid sequences of SEQ ID NOs: 5 and 6, respectively; SEQ ID NOs: l and 2, respectively; SEQ ID NOs:3 and 4, respectively; SEQ ID NOs:7 and 8, respectively; SEQ ID NOsV and 10, respectively; or SEQ ID NOs: 11 and 12, respectively. In particular embodiments, the anti-OX40 antibody or antigen-binding portion thereof comprises a Vu and a VL comprising SEQ ID NOs:5 and 6, respectively; SEQ ID NOs: l and 2, respectively; SEQ ID NOs: 3 and 4, respectively; SEQ ID NOs: 7 and 8, respectively; SEQ ID NOs:9 and 10, respectively; or SEQ ID NOs: 11 and 12, respectively.

[0014] In some embodiments, the anti-OX40 antibody is of isotype subtype IgGi. In some embodiments, the anti-OX40 antibody comprises a mutant Fc domain with reducedbinding affinity for an Fc gamma receptor. In some embodiments, the anti-OX40 antibody comprises a heavy chain constant region comprising SEQ ID NO:97.In particular embodiments, the anti-OX40 or antigen-binding portion thereof comprises a heavy chain (HC) comprising SEQ ID NOs:5 and 97 and a light chain (LC) comprising SEQ ID NOs:6 and 98; an HC comprising SEQ ID NOs: l and 97 and an LC comprising SEQ ID NOs:2 and 98; an HC comprising SEQ ID NOs:3 and 97 and an LC comprising SEQ ID NOs:4 and 98; an HC comprising SEQ ID NOs:7 and 97 and an LC comprising SEQ ID NOs:8 and 98; an HC comprising SEQ ID NOs:9 and 97 and an LC comprising SEQ ID NOs: 10 and 98; or an HC comprising SEQ ID NOs: 11 and 97 and an LC comprising SEQ ID NOs: 12 and 98.

[0015] The present disclosure also provides a pharmaceutical composition comprising an anti-OX40 antibody or antigen-binding portion herein and a pharmaceutically acceptable excipient.

[0016] The present disclosure also provides isolated nucleic acid molecule(s) comprising a nucleotide sequence that encodes the heavy chain, or a nucleotide sequence that encodes the light chain, or both, of an anti-OX40 antibody or antigen-binding portion herein. In some embodiments, the isolated nucleic acid molecule(s) comprise any one of SEQ ID NOs: 85-96.

[0017] The present disclosure also provides vector(s) comprising isolated nucleic acid molecule(s) herein, wherein the vector(s) further comprise an expression control sequence.

[0018] The present disclosure also provides a host cell comprising a nucleotide sequence that encodes the heavy chain, and a nucleotide sequence that encodes the light chain, of an anti-OX40 antibody or antigen-binding portion herein.

[0019] The present disclosure also provides a method for producing an anti-OX40 antibody or an antigen-binding portion thereof, comprising providing a host cell herein, culturing said host cell under conditions suitable for expression of the antibody or antigenbinding portion, and isolating the resulting antibody or antigen-binding portion.

[0020] The present disclosure also provides a method of treating an autoimmune or inflammatory disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an anti-OX40 antibody or antigen-binding herein, or a pharmaceutical composition herein.

[0021] The present disclosure also provides an anti-OX40 antibody or antigen-binding portion herein, or a pharmaceutical composition herein, for use in treating an autoimmune or inflammatory disease in a patient in need thereof.

[0022] The present disclosure also provides use of an anti-OX40 antibody or antigenbinding portion herein, or a pharmaceutical composition herein, in the manufacture of a medicament for treating an autoimmune or inflammatory disease in a patient in need thereof.

[0023] In some embodiments, the autoimmune or inflammatory disease is selected from the group consisting of asthma, allergic asthma, chronic spontaneous urticaria (CSU), diabetes (e.g., type 1 diabetes or latent autoimmune diabetes), lupus (e.g., systemic lupus erythematosus or lupus nephritis), arthritis (e.g., rheumatoid arthritis), allergy, antibody- mediated rejection, organ graft rejection, graft-versus-host disease (GvHD), Addison’s disease, ankylosing spondylitis, anti-glomerular basement membrane disease, autoimmune hepatitis, celiac disease, autoimmune alopecia, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schonlein purpurajuvenile myositis, Kawasaki disease, inflammatory bowel diseases (such as Crohn’s disease and ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, pemphigus vulgaris, thrombocytopenic purpura, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis, vitiligo, and Vogt-Koyanagi-Harada Disease.

[0024] In some embodiments, the autoimmune or inflammatory disease is chronic spontaneous urticaria, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid, Sjogren’s syndrome, lupus (e.g., systemic lupus erythematosus or lupus nephritis), arthritis (e.g., rheumatoid arthritis), Graves’ disease, inflammatory bowel diseases (such as Crohn’s disease and ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis or vitiligo.

[0025] In some embodiments, the autoimmune or inflammatory disease is chronic spontaneous urticaria atopic dermatitis or pemphigus vulgaris.

[0026] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGs. 1A-B are line graphs showing the binding curves of anti-OX40 antibodies to human 0X40 receptors (FIG. 1A) and cynomolgus 0X40 receptors (FIG. IB) expressed on transiently transfected CHO-S cells. Data are shown as means ± SD of triplicates.

[0028] FIGs. 2A-B are line graphs showing the blocking curves of human 0X40 ligand (OX-40L) binding to human 0X40 (FIG. 2A) or cynomolgus 0X40 (FIG. 2B) by different anti-OX40 antibodies in CHO-S cells. Data are presented as duplicate means ± SEM.

[0029] FIG. 3 is a pair of structures showing the common epitope of antibodies 16683, 16706 and 16713, mapped on the structure of the OX40L and 0X40 complex (PDB entry: 2HEV). The three 0X40 receptors are colored light grey and the three OX40Ls are represented as ribbon diagram. Linear epitope is shown as dark gray and contact residues are shown as black. Panel A shows the structure seen from the N-terminal top of OX40:OX40L and Panel B is the structure viewed from the side.

[0030] FIGs. 4A-F are line graphs showing antibody-dependent cellular cytotoxicity (ADCC) FcyR (FcGR) reporter assays using the OX40-expressing target cell line MYLA treated with anti-OX40 antibodies 16683, 16706 and 16713, or with Glenmark analogue, Kirin analogue or Inmagene analogue. Bioluminescence reporter signal (NF AT reporter) shows raw luminescence units (RLUs). Untreated single concentration reference at 167 nM and a negative control wildtype IgGl antibody reference are shown for clarity. FIGs. 4A-E show positive signal FcGRs (FcGRIa (FIG. 4A), FcGRIIa-H (FIG. 4B) / FcGRIIa-R (FIG. 4C), FcGRIIIa-V (FIG. 4D) / FcGRIIIa-F (FIG. 4E), while FIG. 4F shows negative signal receptor FcGR2b. Horizontal dotted lines represent untreated signal level. Error bars show SEM of duplicate measurements.

[0031] FIG. 5 is a line graph showing the specific lysis of OX40-expressing MYLA target cells as measured by calcein release upon treatment with anti-OX40 antibodies, no antibodies (untreated) or a negative control wildtype IgGl antibody, in the presence of healthy donor peripheral blood mononuclear cells (PBMCs). Spontaneous lysis (no PBMCs present) was deducted, and values normalized to maximal lysis. Horizontal dotted line represents untreated signal level. Error bars represent SEM of triplicate measurements.

[0032] FIG. 6 is a line graph showing 0X40 signaling reporter bioluminescence activity (counts per second) from 0X40 reporter cells upon treatment with several anti-OX40 antibodies. The 0X40 reporter cells were cultured in the presence of FcGR-expressing Raji cells. Error bars represent mean ± SEM of measurements.

[0033] FIG. 7 is a line graph showing the effect of anti-OX40 antagonist antibodies (16706 antibody and Kirin analogue) on IL-2 production induced by OX40 / OX40L interaction in a PBMC assay. Data represent the mean with standard deviation of IL-2 concentration measured in the cell culture supernatant.

[0034] FIG. 8 is a line graph showing the effect of anti-OX40 antagonist antibodies on the differentiation of induced Treg (iTreg) in the presence of OX40L. Data represent the mean with standard deviation of Foxp3+ / CD25+percentage.

[0035] FIGs. 9A-B are line graphs showing the effect of anti-OX40 antagonist antibodies on CD4+T cell polarization induced by OX40 / OX40L interaction. Data represent the mean with standard deviation of IL-2 (FIG. 9A) or IL-21 (FIG. 9B) concentration measured in the cell culture supernatant.

[0036] FIGs. 10A-C are line graphs showing development of GvHD symptoms displayed as scoring of clinical observation (FIG. 10A), body weight reduction (FIG. 10B), and overall survival (FIG. 10C). Mice were treated two times weekly for a total of ten treatments by intraperitoneal injection of vehicle or 16706 dosed at 10 mg / kg, 1 mg / kg or 0.1 mg / kg. One group of mice not engrafted with PBMCs and treated with vehicle was included as control. Data are presented as means ± SEM.DETAILED DESCRIPTION OF THE INVENTION

[0037] The present disclosure provides new antibodies that specifically bind to 0X40 (“anti-OX40 antibodies”), or antigen-binding portions thereof, that can be used to block the interaction of 0X40 with its ligand OX40L. The present antibodies can modulate the immune system in a patient and may be used for treatment of autoimmune disorders or inflammatory disorders.

[0038] The present anti-OX40 antibodies antagonize and do not agonize 0X40 activity. The present anti-OX40 antibodies promote differentiation of regulatory T cells and reverse polarization of naive CD4+T cells, and the antibodies display high potency in reversing production of pro-inflammatory cytokines such as IL-2, IL-21, and interferon-gamma by effector T cells.

[0039] Unless otherwise stated, “0X40” refers to human 0X40. A human 0X40 polypeptide sequence is available under UniProt Accession No. P43489, as shown below:1 MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN 51GMVSRCSRSQ NTVCRPCGPG FYNDWSSKP CKPCTWCNLR SGSERKQLCT101 ATQDTVCRCR AGTQPLDSYK PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA151 GKHTLQPASN SSDAICEDRD PPATQPQETQ GPPARPITVQ PTEAWPRTSQ201 GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL RRDQRLPPDA251 HKPPGGGSFR TPIQEEQADA HSTLAKI ( SEQ ID NO : 106 )In this sequence, the signal peptide spans amino acids 1-28; the extracellular domain (ECD) spans amino acids 29-214 (underlined); the transmembrane domain spans amino acids 215- 235; and the cytoplasmic domain spans amino acids 236-277.

[0040] A cynomolgus monkey 0X40 polypeptide sequence is available under UniProt Accession No. A0A2K5TJH8, as shown below:1 MCVGARRLGR GPCAALLLLG LGLSTTAKLH CVGDTYPSND RCCQDAAGNG51 MVSRCNRSQN TVCRPCGPGF YNDWSAKPC KACTWCNLRS GSERKQPCTA 101 TQDTVCRCRA GTQPLDSYKP GVDCAPCPPG HFSPGDNQAC KPWTNCTLAG 151 KHTLQPASNS SDAICEDRDP PPTQPQETQG PPARPTTVQP TEAWPRTSQR 201 PSTRPVEVPR GPTVAAILGL GLALGLLGPL AMLLALLLLG GTRGCPDAPK 251 PLGEAVSGPP SKRSRLTRTP PWPRSDPGPP RWTLGPARLE PEVCWASRAA 301 GRPPAAPGPL CAVLGAAGCL RLSAYVCHAY LLPRRATIKP WQTGVSNWHC 351 GLRTCGREAW AVPASGWRRV LGQGGGSR ( SEQ ID NO : 107 )In this sequence, the signal peptide spans amino acids 1-28; the ECD spans amino acids 29- 213 (underlined); the transmembrane domain spans amino acids 214-240; and the cytoplasmic domain spans amino acids 241-378. The ECDs of human 0X40 and cynomolgus 0X40 are 94% homologous.

[0041] The term “antibody” (Ab) or “immunoglobulin” (Ig), as used herein, refers to a tetramer comprising two heavy chains (HCs) (about 50-70 kDa) and two light chains (LCs) (about 25 kDa) inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be subdivided further into regions of hypervariability, termed “complementarity determining regions” (CDRs), interspersed with regions that are more conserved, termed “framework regions” (FRs). Each VH and VL is composed of three CDRs (H-CDR herein designates a CDR from the heavy chain; and L-CDR herein designates a CDR from the light chain) and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acid numbers, and of FR and CDR regions, in the heavy or light chain may be in accordance with IMGT® definitions (Lefranc et al., Dev Comp Immunol. (2003) 27(l):55-77); or the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J Mol Biol (1987)196:901-17; Chothia et al., Nature (1989) 342:878-83; MacCallum et al., J Mol Biol. (1996) 262:732-45; or Honegger and Pluckthun, J Mol Biol. (2001) 309(3):657-70.

[0042] The term “recombinant antibody” refers to a non-naturally occurring antibody that is expressed from a cell or cell line comprising the nucleotide sequence(s) that encode the antibody, wherein said nucleotide sequence(s) are not naturally associated with the cell.

[0043] The term “isolated protein,” “isolated polypeptide” or “isolated antibody” refers to a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, and / or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. Such naturally associated components may include, e.g., nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, cellular debris, growth medium, etc.

[0044] The term “affinity” refers to a measure of the attraction between an antigen and an antibody. The intrinsic attractiveness of the antibody for the antigen is typically expressed as the binding affinity equilibrium constant (KD) of a particular antibody-antigen interaction.An antibody is said to specifically bind to an antigen when the KD for the binding is < 1 pM, e.g., < 100 nM or < 10 nM. A KD binding affinity constant can be measured, e.g., by surface plasmon resonance (Biacore™) using the Biacore™ T200 system, the IBIS MX96 SPR system from IBIS Technologies, or the Carterra LSA SPR platform, or by bio-layer interferometry, for example using the Octet™ system from ForteBio.

[0045] The term “epitope” as used herein refers to a portion (determinant) of an antigen that is bound by an antigen-binding protein (e.g., an antibody or an antigen-binding portion thereof). For example, an epitope may refer to the portion of an antigen that specifically binds to an antigen-binding site of an antigen-binding protein (e.g., an antibody or antigenbinding portion thereof), wherein the antigen-binding site is known as a paratope. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three- dimensional structural characteristics, as well as specific charge characteristics. An antigen may have more than one epitope.

[0046] An epitope may be “linear” or “conformational.” In a linear epitope, all of thepoints of interaction between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another in the primary amino acid sequence. A “functional” epitope refers to residues that directly contribute to the affinity of the antigen / antigen-binding protein interaction. Epitopes may be determined using methods such as, e.g., alanine scanning mutational analysis, peptide blot analysis, peptide cleavage analysis, crystallographic studies, NMR analysis, and hydrogen / deuterium exchange detected by mass spectrometry.

[0047] Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope using techniques well known in the art. For example, an antibody to a linear epitope may be generated, e.g., by immunizing an animal with a peptide having the amino acid residues of the linear epitope. An antibody to a conformational epitope may be generated, e.g., by immunizing an animal with a mini-domain containing the relevant amino acid residues of the conformational epitope. An antibody to a particular epitope can also be generated, e.g., by immunizing an animal with the target molecule of interest (e.g., 0X40) or a relevant portion thereof, then screening for binding to the epitope. An antibody to a particular epitope also may be generated using phage display methods.

[0048] One can determine whether an antibody binds to the same epitope as or competes for binding to 0X40 with an anti-OX40 antibody of the present disclosure by using methods known in the art, including, without limitation, competition assays, epitope binning, and alanine scanning. In some embodiments, one allows the antibody of the present disclosure to bind to the antigen under saturating conditions, and then measures the ability of the test antibody to bind to that antigen. If the test antibody is able to bind to the antigen at the same time as the reference antibody, then the test antibody binds to a different epitope than the reference antibody. However, if the test antibody is not able to bind to the antigen at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the antibody of the present disclosure. To test whether an antibody cross-competes with another antibody, one may use the competition method described above in two directions, i.e., determining if the known antibody blocks the test antibody and vice-versa. Competition or cross-competition experiments can be performed using, e.g., ELISA, RIA, Biacore™, SPR, Bio-Layer Interferometry or flow cytometry. For example, the experiments may be performed, e.g., using a Biacore™ T200, IBIS MX96, or Carterra LSA SPR instrument or the Octet™ system.

[0049] The term “human antibody” refers to an antibody in which the variable domains and constant region sequences are derived from human sequences. The term encompasses antibodies with sequences that are derived from human genes but have been modified, e.g., to decrease immunogenicity, increase affinity, and / or increase stability. Further, the term encompasses antibodies produced recombinantly in nonhuman cells, which may impart glycosylation not typical of human cells. The term also encompasses antibodies produced in transgenic nonhuman organisms with human antibody genes (e.g., OmniRat® rats).

[0050] The term “antigen-binding portion” or “antigen-binding fragment” of an antibody, or simply “antibody portion,” as used herein, refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human 0X40, or portion(s) thereof). It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” include (i) a Fab fragment: a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment: a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) capable of specifically binding to an antigen (e.g., a CDR3 peptide or a FR3- CDR3-FR4 peptide). Further, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form a monovalent molecule (known as single chain Fv (scFv)). Also within the present disclosure are antigen-binding molecules comprising a VH and / or a VL. In the case of a VH, the molecule may also comprise one or more of a CHI, hinge, CH2, or CH3 region. Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigenbinding sites. Also contemplated are tribodies, tetrabodies, nanobodies (e.g., monovalent or bivalent nanobodies), minibodies, domain-specific antibodies, single domain antibodies, and domain-deleted antibodies.

[0051] Antigen-binding portions, such as Fab and F(ab’)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies. Moreover, antibodies, antigen-binding portions and immunoadhesin molecules can be obtained using standard recombinant DNA techniques, e.g., as described herein.

[0052] The class (isotype) and subclass of anti-OX40 antibodies described herein may be determined by any method known in the art. In general, the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are available commercially. The class and subclass can be determined by ELISA or Western blot as well as other techniques. Alternatively, the class and subclass may be determined by sequencing all or a portion of the constant regions of the heavy and / or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.

[0053] Unless otherwise indicated, the numbering of all antibody amino acid residues in this disclosure is according to the IMGT® numbering scheme.I. Anti-QX40 Antibodies and Binding Proteins

[0054] The present disclosure provides anti-OX40 antibodies and antigen-binding portions thereof, as well as binding proteins comprising said antibodies or antigen-binding portions. In a certain aspect, the antibodies disclosed herein are human antibodies generated from transgenic animals (e.g., rats) that are able to produce antibodies encoded by rearranged human antibody genes. In particular embodiments, the human antibodies may contain certain mutations, e.g., to change primer-derived mutations back to the germline sequence.

[0055] In some embodiments, the anti-OX40 antibodies of the present disclosure have the “LALA” mutations (L234A / L235A) in the Fc region. These mutations hinder binding of the antibodies to human FcyR (Fc gamma receptors). Additionally or alternatively, the anti- 0X40 antibodies may have a G237A mutation in the Fc region. “IgGl-3f ’ refers to the presence of the L234A, L235E, and G237A mutations, which are known to reduce effector function of the Fc region of IgGl antibodies (W088 / 07089). All Fc region positions are defined according to Eu numbering unless otherwise stated.

[0056] In some embodiments, the anti-OX40 antibody or antigen-binding portion competes or cross-competes for binding to human 0X40 with, or binds to the same epitope of human 0X40 as, an antibody comprising HC and LC sequences that comprise:a) SEQ ID NOs: 1 and 2, respectively; b) SEQ ID NOs: 3 and 4, respectively; c) SEQ ID NOs: 5 and 6, respectively; d) SEQ ID NOs: 7 and 8, respectively; e) SEQ ID NOs: 9 and 10, respectively; or f) SEQ ID NOs: 11 and 12, respectively.

[0057] In some embodiments, the anti-OX40 antibody or antigen-binding portion has H- CDR1-3 comprising SEQ ID NOs: 13-15, 25-27, 37-39, 49-51, 61-63, or 73-75, respectively.

[0058] In some embodiments, the anti-OX40 antibody or antigen-binding portion has a VH amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, or 11. In certain embodiments, any variations from the selected sequence are in the framework regions (FRs).

[0059] In some embodiments, the anti-OX40 antibody or antigen-binding portion has a VH comprising SEQ ID NO: 1, 3, 5, 7, 9, or 11.

[0060] In some embodiments, the anti-OX40 antibody has a VH amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 3, 5, 7, 9, or 11 (optionally wherein any variations from the selected sequence are in the FRs); and a heavy chain constant region amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 97 or 99.

[0061] In some embodiments, the anti-OX40 antibody comprises a VH amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11, and a heavy chain constant region amino acid sequence of SEQ ID NO: 97 or 99.

[0062] In some embodiments, the anti-OX40 antibody or antigen-binding portion has L- CDR1-3 comprising SEQ ID NOs: 16-18, 28-30, 40-42, 52-54, 64-66, or 76-78, respectively.

[0063] In some embodiments, the anti-OX40 antibody or antigen-binding portion has a VL amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, 4, 6, 8, 10, or 12. In certain embodiments, any variations from the selected sequence are in the FRs.

[0064] In some embodiments, the anti-OX40 antibody or antigen-binding portion has a VL comprising SEQ ID NO: 2, 4, 6, 8, 10, or 12.

[0065] In some embodiments, the anti-OX40 antibody has a VL amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, 4, 6, 8, 10, or 12 (optionally wherein any variations from the selectedsequence are in the FRs); and a light chain constant region amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 98.

[0066] In some embodiments, the anti-OX40 antibody comprises a VL amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 12, and a light chain constant region amino acid sequence of SEQ ID NO: 98.

[0067] In certain embodiments, the anti-OX40 antibody or antigen-binding portion comprises any one of the above-described heavy chain sequences and any one of the abovedescribed light chain sequences.

[0068] In some embodiments, the anti-OX40 antibody or antigen-binding portion of the present disclosure comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of: a) SEQ ID NOs: 13-18, respectively; b) SEQ ID NOs: 25-30, respectively; c) SEQ ID NOs: 37-42, respectively; d) SEQ ID NOs: 49-54, respectively; e) SEQ ID NOs: 61-66, respectively; or f) SEQ ID NOs: 73-78, respectively.

[0069] In some embodiments, the anti-OX40 antibody or antigen-binding portion of the present disclosure comprises a VH and a VL that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., at least 90% identical) to: a) SEQ ID NOs: 1 and 2, respectively; b) SEQ ID NOs: 3 and 4, respectively; c) SEQ ID NOs: 5 and 6, respectively; d) SEQ ID NOs: 7 and 8, respectively; e) SEQ ID NOs: 9 and 10, respectively; or f) SEQ ID NOs: 11 and 12, respectively.In certain embodiments, any variations from the selected sequences are in the FRs.

[0070] In some embodiments, the anti-OX40 antibody or antigen-binding portion of the present disclosure comprises a VH and a VL that comprise: a) SEQ ID NOs: 1 and 2, respectively; b) SEQ ID NOs: 3 and 4, respectively; c) SEQ ID NOs: 5 and 6, respectively; d) SEQ ID NOs: 7 and 8, respectively; e) SEQ ID NOs: 9 and 10, respectively; orf) SEQ ID NOs: 11 and 12, respectively.

[0071] In some embodiments, the anti-OX40 antibody of the present disclosure comprises: a) an HC comprising SEQ ID NOs: 1 and 97 and an LC comprising SEQ ID NOs: 2 and 98; b) an HC comprising SEQ ID NOs: 3 and 97 and an LC comprising SEQ ID NOs: 4 and 98; c) an HC comprising SEQ ID NOs: 5 and 97 and an LC comprising SEQ ID NOs: 6 and 98; d) an HC comprising SEQ ID NOs: 7 and 97 and an LC comprising SEQ ID NOs: 8 and 98; e) an HC comprising SEQ ID NOs: 9 and 97 and an LC comprising SEQ ID NOs: 10 and 98; or f) an HC comprising SEQ ID NOs: 11 and 97 and an LC comprising SEQ ID NOs: 12 and 98.

[0072] In some embodiments, the anti-OX40 antibody of the present disclosure comprises: a) an HC comprising SEQ ID NOs: 1 and 99 and an LC comprising SEQ ID NOs: 2 and 98; b) an HC comprising SEQ ID NOs: 3 and 99 and an LC comprising SEQ ID NOs: 4 and 98; c) an HC comprising SEQ ID NOs: 5 and 99 and an LC comprising SEQ ID NOs: 6 and 98; d) an HC comprising SEQ ID NOs: 7 and 99 and an LC comprising SEQ ID NOs: 8 and 98; e) an HC comprising SEQ ID NOs: 9 and 99 and an LC comprising SEQ ID NOs: 10 and 98; or f) an HC comprising SEQ ID NOs: 11 and 99 and an LC comprising SEQ ID NOs: 12 and 98.

[0073] The present disclosure also provides an anti-OX40 antibody or an antigen-binding portion thereof that competes or cross-competes for binding to 0X40 with, or binds to the same epitope of 0X40 as, antibody 16598, 16683, 16706, 16713, 16718, or 16790.

[0074] In some embodiments, the anti-OX40 antibody or antigen-binding portion of the present disclosure comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences ofantibody 16598, 16683, 16706, 16713, 16718, or 16790.

[0075] In some embodiments, the anti-OX40 antibody or antigen-binding portion of the present disclosure comprises a VH and a VL that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the VH and VL, respectively, of antibody 16598, 16683, 16706, 16713, 16718, or 16790. In certain embodiments, any variations from the VH and VL sequences of the selected antibody are in the FRs.

[0076] In some embodiments, the anti-OX40 antibody or antigen-binding portion of the present disclosure comprises a VH and a VL that are the VH and VL, respectively, of antibody 16598, 16683, 16706, 16713, 16718, or 16790.

[0077] In some embodiments, the anti-OX40 antibody of the present disclosure is antibody 167598, 16683, 16706, 16713, 16718, or 16790, or an antibody with the same amino acid sequences as said antibody.

[0078] Also contemplated by the present disclosure is an anti-OX40 antibody or an antigen-binding portion thereof, wherein said anti-OX40 antibody- competes or cross-competes for binding to human 0X40 with, or binds to the same epitope of human 0X40 as, an antibody comprising an HC comprising the amino acid sequences of SEQ ID NOs: 5 and 97 or SEQ ID NOs: 5 and 99 and an LC comprising the amino acid sequences of SEQ ID NOs: 6 and 98;- comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of SEQ ID NOs: 37-42, respectively;- comprises a VH and a VL that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (e.g., at least 90% identical) in amino acid sequence to SEQ ID NOs: 5 and 6, respectively (optionally wherein any variations from SEQ ID NOs: 5 and 6 are in the FRs);- comprises a VH and a VL that comprise the amino acid sequences of SEQ ID NOs: 5 and 6, respectively;- comprises an HC comprising the amino acid sequences of SEQ ID NOs: 5 and 97 and an LC comprising the amino acid sequences of SEQ ID NOs: 6 and 98; or- comprises an HC comprising the amino acid sequences of SEQ ID NOs: 5 and 99 and an LC comprising the amino acid sequences of SEQ ID NOs: 6 and 98.

[0079] In some embodiments, the anti-OX40 antibody- competes or cross-competes for binding to 0X40 with, or binds to the same epitope of 0X40 as, antibody 16706;- comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 16706;- comprises a VH and a VL that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the VH and VL, respectively, of antibody 16706 (optionally wherein any variations from the VH and VL of antibody 16706 are in the FRs);- comprises a VH and a VL that are the VH and VL, respectively, of antibody 16706; or- is antibody 16706, or an antibody with the same amino acid sequences as said antibody.

[0080] In some embodiments, a “variant” antibody or antigen-binding portion, having amino acid substitutions (which may be conservative or non-conservative) from an antibody or antigen-binding portion exemplified herein, does not have substantially altered biologic activity from the exemplified antibody or antigen-binding portion. For example, the variant antibody or antigen-binding portion may retain at least 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the binding affinity of the parent antibody or antigen-binding portion, or may exceed the binding affinity of the parent antibody or antigen-binding portion. In some embodiments, a variant antibody or an antigen-binding portion thereof may have mutations, e.g., that increase its half-life, alter its immunogenicity, provide a site for covalent or non- covalent binding to another molecule, etc. In certain embodiments, the variant antibody or antigen-binding portion thereof may have mutations in its FRs (e.g., in one, two, three, four, five, six, seven, or eight of its FRs). In certain embodiments, the variant antibody or antigenbinding portion thereof may have mutations in its CDRs (e.g., in one, two, three, four, five, or six of its CDRs). In certain embodiments, the variant antibody or antigen-binding potion thereof may have mutations in its constant regions.

[0081] The class of an anti-OX40 antibody described herein may be changed or switched with another class or subclass. In some embodiments of the present disclosure, a nucleic acid molecule encoding the VL or VH of the antibody is isolated using methods well known in the art such that it does not include nucleic acid sequences encoding CL or CH, respectively. The nucleic acid molecules encoding VL or VH then are operatively linked to a nucleic acid sequence encoding a CL or CH, respectively, from a different class or subclass of immunoglobulin molecule. This may be achieved using a vector or nucleic acid molecule that comprises a CL or CH sequence, as described above. For example, an anti-OX40 antibody that was originally IgM may be class switched to IgG. Further, the class switching may be used to convert one IgG subclass to another, e.g., from IgGi to IgG?. A K light chain constant region can be changed, e.g., to a light chain constant region, or vice-versa.

[0082] The anti-OX40 antibody of the present disclosure can be an IgG, an IgM, an IgE,an IgA, or an IgD molecule, but is typically of the IgG isotype, e.g., of IgG subclass IgGi, IgG2aor IgG2b, IgG? or IgG . In some embodiments, the antibody is of the isotype subclass IgGi.

[0083] In some embodiments, the anti-OX40 antibody may comprise at least one mutation in the Fc region. A number of different Fc mutations are known, where these mutations alter, e.g., the antibody’s effector functions or half-life. For example, in some embodiments, the anti-OX40 antibody comprises at least one mutation in the Fc region that reduces effector function.

[0084] In some embodiments, the anti-OX40 antibody or antigen-binding portion of the present disclosure is antagonistic.

[0085] In some embodiments, the anti-OX40 antibody binds to human 0X40 with a KD of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nM or less (e.g., 12 nM or less or 2 nM or less) as measured by surface plasmon resonance (e.g., as described in Example 4).

[0086] In some embodiments, the anti-OX40 antibody binds to cynomolgus 0X40 with a KD of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nM or less (e.g., 8 nM or less or 2 nM or less) as measured by surface plasmon resonance (e.g., as described in Example 4).

[0087] In some embodiments, the anti-OX40 antibody or antigen-binding portion binds to cell surface-expressed human and / or cynomolgus 0X40 (e.g., expressed on CHO-S cells). In certain embodiments, the anti-OX40 antibody or antigen-binding portion binds to human and / or cynomolgus 0X40 with an efficacy (MFI) of at least 50000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, 500000, 550000, 600000, or 650000 (e.g., at least 300000 or at least 400000). In certain embodiments, additionally or alternatively, the anti- 0X40 antibody or antigen-binding portion binds to human and / or cynomolgus 0X40 with an EC5O of 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 nM or less (e.g., with an ECso of 1 nM or less for human 0X40 or with an ECso of 0.5 nM or less for cynomolgus 0X40). The efficacy and ECso may be determined, for example, using assays as described in Example 2.

[0088] In some embodiments, the anti-OX40 antibody or antigen-binding portion blocks binding of human OX40L to human or cynomolgus 0X40 receptor (e.g., expressed on the surface of cells such as CHO-S cells), for example with an IC50 of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM or less (e.g., with an IC50 of 1.5 nM or less or 0.5 nM or less for human 0X40, and / or an IC50 of 15 nM or less or 4 nM or less for cynomolgus 0X40). The IC50 may be determined, for example, using assays as described in Example 3.

[0089] In some embodiments, the anti-OX40 antibody or antigen-binding portion blocks binding of human OX40L to human and / or cynomolgus 0X40 receptor (e.g., expressed onthe surface of cells such as CHO-S cells), e.g., with an efficacy (MFI) of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. The efficacy may be determined, for example, using assays as described in Example 3.

[0090] In some embodiments, the anti-OX40 antibody or antigen-binding portion, e.g., in IgG1.3f format as described herein, does not enhance killing of OX40+cells in a PBMC- ADCC assay (e.g., as described in Example 7).

[0091] In some embodiments, the anti-OX40 antibody or antigen-binding portion, e.g., in IgG1.3f format, does not bind to FcGRs (e.g., on the surface of cells such as Raji cells). In certain embodiments, the anti-OX40 antibody or antigen-binding portion does not have agonistic activity (i.e., activate downstream signaling of 0X40).

[0092] In some embodiments, the anti-OX40 antibody or antigen-binding portion inhibits OX40L-induced 0X40 signaling, e.g., as measured by an 0X40 reporter cell line, for example with an IC50 of 3, 2, 1, 0.9, 0.8, 0.7, 0.6, or 0.5 nM or less (e.g., 1 nM or less). The IC50 may be determined, for example, using an assay as described in Example 9.

[0093] In some embodiments, the anti-OX40 antibody or antigen-binding portion reverses IL-2 production on peripheral blood mononuclear cells following OX40 / OX40L engagement, for example with an IC50 of 5xl0'10, 4xlO'10, 3xl0'10, 2xlO'10, IxlO'10nM or less (e.g., with an IC50 of 3xl0'10or less). The IC50 may be determined, for example, using an assay as described in Example 10.

[0094] In some embodiments, the anti-OX40 antibody or antigen-binding portion inhibits activation of T cells. In certain embodiments, the anti-OX40 antibody or antigen-binding portion inhibits OX40L-induced IFNy secretion in primary CD4+T cells, for example with an IC50 of 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 nM or less (e.g., 0.2 nM or less). The IC50 may be determined, for example, using an assay as described in Example 11.

[0095] In some embodiments, the anti-OX40 antibody or antigen-binding portion restores iTreg differentiation inhibited by OX40 / OX40L engagement, for example with an IC50 of 10xl0'9, 9xl0'9, 8xl0'9, 7xl0'9, 6xl0'9, or 5xlO'9M or less (e.g., 8xlO'9M or less). The IC50 may be determined, for example, using an assay as described in Example 12.

[0096] In some embodiments, the anti-OX40 antibody or antigen-binding portion reverses the polarization of naive CD4+T cells induced by OX40 / OX40L engagement. In some embodiments, the anti-OX40 antibody or antigen-binding portion reverses production of IL-2 producing cells, for example with an IC50 of 5xl0'9, 4xl0'9, 3xl0'9, 2xl0'9, or IxlO'9M or less. In some embodiments, the anti-OX40 antibody or antigen-binding portion reverses production of IL-21 producing cells, for example with an IC50 of 5xl0'10, 4xlO'10, or3xlO'10M or less. The IC50S may be determined, for example, using assays as described in Example 13.

[0097] In some embodiments, the anti-OX40 antibody or antigen-binding portion suppresses development of Graft-versus-Host Disease (GvHD) in vivo. Suppression may comprise reduction in symptoms such as, e.g., anemia, weight loss, and hair loss, and / or may comprise prolonged survival. The suppression may be determined, for example, using assays as described in Example 14.

[0098] In some embodiments, the anti-OX40 antibody or antigen-binding portion binds to an epitope in the cysteine-rich domains (CRDs) of human 0X40. In certain embodiments, the epitope comprises CRD2, CRD3, or both. In some embodiments, the epitope comprises or overlaps with the contact surface of the receptor-ligand interaction. In certain embodiments, the epitope comprises human 0X40 residues P80-D104, R110-G122, or both. In certain embodiments, the epitope comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or all 14 of human 0X40 residues G70-Y72, D74, K82, W86-G92, LI 16, and D124 (in any combination).

[0099] In certain embodiments, the epitopes are determined using the methods described in Examples 5 and 6 below. In certain embodiments, the anti-OX40 antibody or antigenbinding portion binds to a different epitope of human 0X40 than the anti-OX40 antibody analogues from Inmagene, Glenmark, and / or Kirin as described in Example 2, or the anti- 0X40 antibody 11D4. In particular embodiments, the anti-OX40 antibody or antigenbinding portion binds to a different epitope than all of said analogues and / or 11D4.

[0100] In some embodiments, an anti-OX40 antibody or antigen-binding portion described herein has at least one (e.g., any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19) of the following properties: a) binds to human 0X40 with a KD of 12 nM or less as measured by surface plasmon resonance (SPR); b) binds to cynomolgus 0X40 with a KD of 8 nM or less as measured by surface plasmon resonance (SPR); c) binds to cell surface-expressed human 0X40 with an EC50 of 1 nM or less as measured by flow cytometry; d) binds to cell surface-expressed cynomolgus 0X40 with an EC50 of 0.5 nM or less as measured by flow cytometry; e) blocks binding of human OX40L to human and / or cynomolgus 0X40, e.g., with an IC50 of 1.5 nM or less or 15 nM or less, respectively;f) does not enhance killing of OX40+cells in a PBMC-ADCC assay (e.g., in IgG1.3f format); g) does not bind to FcGRs (e.g., in IgGl ,3f format); h) does not have agonistic activity (i.e., activate downstream signaling of 0X40); i) inhibits OX40L-induced 0X40 signaling, e.g., with an IC50 of 1 nM or less; j) reverses IL-2 production on peripheral blood mononuclear cells following OX40 / OX40L engagement, e.g., with an IC50 of 5xlO'10or less; k) inhibits OX40L-induced IFNy secretion in primary CD4+T cells, e.g., with an IC50 of 1 nM or less; l) restores iTreg differentiation inhibited by OX40 / OX40L engagement, e.g., with an IC50 of 10x1 O'9or less; m) reverses polarization of naive CD4 T cells induced by OX40 / OX40L engagement; n) reverses production of IL-2 producing cells, e.g., with an IC50 of 5xl0'9nM or less; o) reverses production of IL-21 producing cells, e.g., with an IC50 of 5xlO'10nM or less; p) suppresses development of Graft-versus-Host Disease (GvHD) in vivo, q) binds to an epitope in the cysteine-rich domains (CRDs) of human 0X40 (e.g., in CRD2 and partially in CRD3); r) binds to a human 0X40 epitope comprising residues P80-D104 and / or R110-G122; and s) binds to an epitope comprising any combination of human 0X40 residues G70-Y72, D74, K82, W86-G92, LI 16, and D124.

[0101] In some embodiments, the anti-OX40 antibody or antigen-binding portion may comprise, for instance, all of properties a)-s) (e.g., antibody 16706), properties a-g, i, k, 1, and q (e.g., antibody 16713), properties a-g, i, k, and q (e.g., antibody 16683), properties c-e (e.g., antibody 16598 or 16790), or properties a and b (e.g., antibody 16718).

[0102] In some embodiments, the present disclosure provides an anti-OX40 binding protein that comprises, or has the binding specificity of, the anti-OX40 antibody or antigenbinding portion described herein. In some embodiments, the binding protein is a multispecific (e.g., bispecific) binding molecule. The multispecific binding molecule has the binding specificity of an anti-OX40 antibody described herein (e.g., may comprise the six CDRs or the VH and VL of said anti-OX40 antibody). In some embodiments, the multispecific binding molecule additionally has the binding specificity of a) another, distinct anti-OX40 antibody, e.g., an antibody that targets different epitope(s) on the same protein(s), or b) another, distinct antibody that targets a different protein, such as another cell surfacemolecule whose activity mediates the immune response or a disease condition such as an inflammatory or autoimmune disease. Multispecific binding molecules are known in the art, and examples of different types of multispecific binding molecules (e.g., bispecific binding molecules) are given elsewhere herein.

[0103] In some embodiments, the anti-OX40 binding protein is a fusion antibody or an immunoadhesin.

[0104] In some embodiments, the anti-OX40 binding protein is an immunoconjugate, wherein the anti-OX40 antibody or antigen-binding portion thereof is conjugated to another molecule, e.g., a therapeutic moiety. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins.

[0105] In some embodiments, the anti-OX40 binding protein is a small modular immunopharmaceutical (SMIP).

[0106] In some embodiments, the anti-OX40 binding protein is a chimeric antigen receptor (CAR). Such a CAR may be used in CAR-T therapy, in which T cells are engineered to express the CAR targeting 0X40.II. Nucleic Acid Molecules and Vectors

[0107] The present disclosure also provides nucleic acid molecules and sequences encoding anti-OX40 antibodies or antigen-binding portions thereof described herein, or binding proteins described herein. In some embodiments, different nucleic acid molecules encode the heavy chain and light chain amino acid sequences of the anti-OX40 antibody or antigen-binding portion. In other embodiments, the same nucleic acid molecule encodes the heavy chain and light chain amino acid sequences of the anti-OX40 antibody or antigenbinding portion.

[0108] A reference to a nucleotide sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The term “polynucleotide” as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single- and double-stranded forms.

[0109] In some embodiments, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence that encodes the heavy chain sequence, or a nucleotide sequence that encodes the light chain sequence, or both, of an anti-OX40 antibody or antigenbinding portion thereof described herein. In some embodiments, the present disclosureprovides a set (e.g., a pair) of nucleic acid molecules comprising a nucleotide sequence that encodes the heavy chain sequence, and a nucleotide sequence that encodes the light chain sequence, of an anti-OX40 antibody or antigen-binding portion thereof described herein.

[0110] The present disclosure also provides nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to one or more nucleotide sequences recited herein, e.g., to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 85-96. The term “percent sequence identity” in the context of nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48, or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (see, e.g., Pearson, Methods Enzymol. (1990) 183:63-98; Pearson, Methods Mol. Biol. (2000) 132: 185-219; Pearson, Methods Enzymol. (1996) 266:227-58 (1996); and Pearson, J. Mol. Biol. (1998) 276:71-84; incorporated herein by reference).[OHl] In some embodiments, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 85-96.

[0112] In some embodiments, the present disclosure provides a set (e.g., a pair) of nucleic acid molecules comprising the nucleotide sequences of a) SEQ ID NOs: 85 and 86; b) SEQ ID NOs: 87 and 88; c) SEQ ID NOs: 89 and 90; d) SEQ ID NOs: 91 and 92; e) SEQ ID NOs: 93 and 94; or f) SEQ ID NOs: 95 and 96.

[0113] In any of the above embodiments, the nucleic acid molecules may be isolated. Nucleic acid molecules referred to herein as “isolated” or “purified” are nucleic acids which (1) have been separated away from the nucleic acids of the genomic DNA or cellular RNA oftheir source of origin; and / or (2) do not occur in nature.

[0114] In a further aspect, the present disclosure provides a vector suitable for expressing one or both of the chains of an antibody or antigen-binding portion thereof as described herein. The term “vector”, as used herein, means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded piece of DNA into which additional DNA segments may be ligated. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).

[0115] The present disclosure provides vectors comprising nucleic acid molecules that encode the heavy chain sequence, the light chain sequence, or both the heavy and light chain sequences, of an anti-OX40 antibody or antigen-binding portion thereof as described herein. In certain embodiments, a vector of the present disclosure comprises a nucleic acid molecule described herein, or a pair or set of nucleic acid molecules described herein. In certain embodiments, the present disclosure provides a set (e.g., a pair) of vectors comprising a set or pair of nucleic acid molecules described herein. The present disclosure further provides vectors comprising nucleic acid molecules encoding binding proteins, fusion proteins, modified antibodies, etc. as described herein. The vectors may further comprise an expression control sequence.

[0116] The term “expression control sequence” as used herein means polynucleotide sequences that are necessary to effect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.III. Making of Antibodies and Binding Proteins

[0117] The anti-OX40 antibodies and antigen-binding portions thereof of the present disclosure, or related binding proteins as described herein, may be produced recombinantlyusing isolated nucleic acid molecules such as expression constructs. The encoding sequences for each polypeptide chain may be cloned into a single vector or cloned into separate vectors (e.g., a pair or set of vectors).

[0118] The antibodies or antigen-binding portions thereof or binding proteins may be produced in host cells, e.g., mammalian host cells, using appropriate expression constructs. Mammalian cell lines available as hosts for expression include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells, and yeast cell lines. In certain embodiments, the cell lines are not derived from a human embryo. Cell lines may be selected based on their expression levels. The antibodies, antigen-binding portions, or binding proteins may be isolated and purified from the host cell culture using well known methods, such as centrifugation, ultracentrifugation, protein A, protein G, protein A / G, or protein L purification, and / or ion exchange chromatography.

[0119] In some embodiments, a host cell described herein comprises nucleic acid molecule(s) comprising a nucleotide sequence that encodes the heavy chain sequence, or a nucleotide sequence that encodes the light chain sequence, or both, of an anti-OX40 antibody or antigen-binding portion thereof described herein. In certain embodiments, the host cell comprises nucleic acid molecule(s) encoding both the heavy and light chain sequences. In certain embodiments, the host cell comprises nucleic acid molecule(s) encoding only the heavy or light chain sequence, such chains being useful, e.g., as intermediates for the expression of an antibody or antigen-binding fragment that includes said heavy or light chain.

[0120] Host cells used to produce the antibodies, antigen-binding portions, or binding proteins are also termed “recombinant host cells.” A “recombinant host cell” (or simply “host cell”), as used herein, means a cell into which a recombinant expression construct has been introduced. By definition, a recombinant host cell does not occur in nature. A protein produced from a recombinant host cell is a recombinant protein.IV. Pharmaceutical Compositions and Uses

[0121] Another aspect of the present disclosure is a pharmaceutical composition comprising as an active ingredient (or as the sole active ingredient) an anti-OX40 antibody orantigen-binding portion thereof or binding protein of the present disclosure. The pharmaceutical composition may additionally comprise a pharmaceutically acceptable excipient. A “pharmaceutically acceptable excipient” may include appropriate solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, and the like. Examples of pharmaceutically acceptable excipients are water and saline (e.g., phosphate-buffered saline).

[0122] The pharmaceutical compositions herein may be used to treat an inflammatory or autoimmune disease. In some embodiments, a method of treating a disease (such as an inflammatory or autoimmune disease) in a subject comprises administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion disclosed herein. In some embodiments, the inflammatory or autoimmune disease is selected from the group consisting of asthma, allergic asthma, chronic spontaneous urticaria (CSU), diabetes (e.g., type 1 diabetes or latent autoimmune diabetes), lupus (e.g., systemic lupus erythematosus or lupus nephritis), arthritis (e.g., rheumatoid arthritis), allergy, antibody-mediated rejection, organ graft rejection, graft-versus-host disease (GvHD), Addison’s disease, ankylosing spondylitis, anti-glomerular basement membrane disease, autoimmune hepatitis, celiac disease, autoimmune alopecia, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schonlein purpurajuvenile myositis, Kawasaki disease, inflammatory bowel diseases (such as Crohn’s disease and ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, thrombocytopenic purpura, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis, vitiligo, and Vogt- Koyanagi-Harada Disease. In some embodiments, the antibodies or antigen-binding portions thereof, compositions, or binding proteins of the present disclosure may be used to treat CSU, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid, Sjogren’s syndrome, lupus (e.g., systemic lupus erythematosus or lupus nephritis), arthritis (e.g., rheumatoid arthritis), Graves’ disease, inflammatory bowel diseases (such as Crohn’s disease and ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis or vitiligo. In some embodiments, the antibodies or antigenbinding portions thereof, compositions, or binding proteins of the present disclosure may be used to treat CSU, atopic dermatitis or pemphigus vulgaris.

[0123] Treat,” “treating,” and “treatment” refer to a method of alleviating or abrogating a biological disorder and / or at least one of its attendant symptoms. As used herein, to “alleviate” a disease, disorder or condition means reducing the severity and / or occurrence frequency of the symptoms of the disease, disorder, or condition.

[0124] “Therapeutically effective amount” refers to the amount of the therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated.

[0125] The anti-OX40 antibodies or antigen-binding portions thereof, antibody compositions, or binding proteins of the present disclosure may be administered without additional therapeutic treatments, i.e., as a stand-alone therapy (monotherapy). Alternatively, treatment with the anti-OX40 antibodies or antigen-binding portions thereof, antibody compositions, or binding proteins of the present disclosure may include at least one additional therapeutic treatment (combination therapy), e.g., another immunomodulatory agent.

[0126] The pharmaceutical compositions herein may be delivered to the patient through parenteral administration. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, or intravenous injection (e.g., intravenous infusion). Particular embodiments include the intravenous route (e.g., intravenous infusion) and the subcutaneous route (e.g., subcutaneous injection).V. Diagnostic Uses

[0127] The antibodies and antigen-binding portions or binding proteins of the present disclosure also are useful in diagnostic processes (e.g., in vitro or ex vivo). For example, the antibodies, antigen-binding portions, or binding proteins can be used to detect and / or measure the level of 0X40 in a biological sample from a patient (e.g., a tissue sample, or a blood sample). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays, radioimmunoassays, and immunohistochemistry. The present disclosure further encompasses kits (e.g., diagnostic kits) comprising the antibodies, antigen-binding portions, or binding proteins described herein.

[0128] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in thepractice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0129] According to the present disclosure, back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Any compound disclosed herein can be used in any of the treatment method here, wherein the individual to be treated is as defined anywhere herein. Further, headers herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any manner.

[0130] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.EXAMPLESExample 1: Cloning of Anti-OX40 Antibodies from Rat B Cells

[0131] Antibodies against human 0X40 were isolated from an antibody repertoire derived from OmniRat® rats (Osborn et al., J Immunol. (2013) 190(4): 1481-90), a transgenic rat strain from Ligand Pharmaceuticals Inc. that produces antibodies with fully human idiotypes. Cloning of rat-derived antibody genes from single-cell sorted antibody-secreting B cells (ASC) was performed by means of Symplex™ antibody discovery technology (Meijer et al., J Mol Biol. (2006) 358(3):764-72).

[0132] Antibody repertoire constructs encoding fully human immunoglobulins in IgGl-3f format (see below) were transfected into HEK293 cells. Cell culture supernatants were screened for binding to 0X40 expressed on the surface of CHO cells using flow cytometry in a high-throughput format. 0X40 reactive clones were analyzed by DNA sequencing and antibody-encoding DNA sequences were extracted. Selected antibody clones were expressed and tested functionally as described below.

[0133] Missense mutations in the amino termini of heavy and light chains that were introduced by the use of degenerate primers in the Symplex™ cloning of the antibodyencoding cDNA fragments were corrected back to germline sequence (“germlined”). Table 1 shows the heavy and light chain variable domain nucleotide sequences of the germlined antibodies designated 16598, 16683, 16706, 16713, 16718 and 16790. The correction process involved amino terminal sequence correction to germline as well as codon usage optimization. The targets for matching to human germline sequences were identified by BLAST® homology searches for the heavy chain and light chain variable regions.Table 1: Variable domain nucleotide sequences of antibodies 16598, 16683, 16706, 16713, 16718, and 16790

[0134] Amino acid sequences of the variable domains of antibodies 16598, 16683, 16706, 16713, 16718 and 16790 are shown in Table 2, where the CDRs annotated according to IMGT® are underlined.Table 2: Variable domain amino acid sequences of antibodies 16598, 16683, 16706, 16713, 16718 and 16790

[0135] Amino acid sequences of the complementarity-determining regions (CDRs) of antibodies 16598, 16683, 16706, 16713, 16718 and 16790 as defined by the IMGT® and Chothia systems are shown in Table 3.Table 3: CDR amino acid sequences of antibodies 16598, 16683, 16706, 16713, 16718 and 16790

[0136] Amino acid sequences of the constant regions of antibodies 16598, 16683, 16706, 16713, 16718 and 16790 are shown in Table 4. “IgGl-3f’ refers to the presence of L234A, L235E, G237A mutations in the heavy chain (numbered according to the Eu numbering scheme), which are known to reduce effector function of the Fc region of IgGl antibodies (W088 / 07089).Table 4: Constant region amino acid sequences of antibodies 16598, 16683, 16706, 16713, 16718 and 16790

[0137] Table 5 shows SEQ ID NO information for antibodies 16598, 16683, 16706, 16713, 16718 and 16790, including the SEQ ID NOs of the CDRs according to the IMGT® system (nt: nucleotide; aa: amino acid).Table 5: SEQ ID NOs for antibodies 16598, 16683, 16706, 16713, 16718 and 16790Example 2: Direct Binding of Anti-OX40 Antibodies to CHO-S Cells Transfected with Human or Cynomolgus 0X40

[0138] This example describes binding of anti-OX40 antibodies to CHO-S cells transiently expressing human or cynomolgus 0X40 receptor. Efficacy and binding values of anti-OX40 antibodies 16598, 16683, 16706, 16713, 16718 and 16790 and of Inmagene, Glenmark, and Kirin analogues are reported herein. The sequences of the Inmagene, Glenmark, and Kirin analogues are described in Table 6.Table 6: Variable domain amino acid sequences of Inmagene, Glenmark, and Kirin anti-OX40 analogues

[0139] The anti-OX40 antibodies were incubated for 30 minutes at 4°C with hamster CHO-S cell line transiently expressing human or cynomolgus 0X40 receptors. The cells were washed twice and subsequently incubated for an additional 20 minutes with AF647- conjugated secondary anti-human IgG (H+L) antibody. After washing, antibody binding to the cells was detected using the high-throughput flow cytometer iQue® Screener PLUS (Sartorius) measuring the GeoMean of AF647 signal in each well. A 12-point curve was generated for each antibody and every concentration was assayed in triplicate.

[0140] All anti-OX40 antibodies bound to human 0X40 with different potency and efficacy (FIG. 1A). In particular, anti-OX40 antibody 16706 bound to human 0X40 receptor with the highest potency (ECso in nM) and efficacy compared to the other antibodies.

[0141] Anti-OX40 antibodies and Kirin antibody analogue bound to cynomolgus 0X40 with comparable potency and efficacy, while Glenmark and Inmagene antibody analogues bound to cynomolgus 0X40 receptor with significantly lower potency and efficacy compared to the other antibodies tested (FIG. IB). IgGl control showed no binding to CHO-S cells expressing either human or cynomolgus OX-40.

[0142] Efficacy and ECso values for binding of anti-OX40 antibodies to CHO-S cells transfected with human or cynomolgus OX-40 were calculated using GraphPad Prism 10 software. They are shown in Table 7.Table 7: Efficacy and ECso values for binding of anti-OX40 antibodiesExample 3: Blocking of Human 0X40 Ligand (OX40L) Binding to Human or Cynomolgus 0X40 Receptor-Transfected CHO-S Cells by Anti-OX40 Antibodies

[0143] This example describes blocking of human OX40L binding to human or cynomolgus 0X40 receptor transiently expressed on CHO-S cells by anti-OX40 antibodies.

[0144] Hamster CHO-S cells expressing human or cynomolgus 0X40 receptor were encoded with different intensities of violet encoder dye (Sartorius) to allow detection of each population seeded in the same well. After seeding the cells, the anti-OX40 antibodies were serially diluted from 200 nM to 3 pM in a 3-fold serial dilution and subsequently added to the cells in each well and incubated for 30 minutes at 4°C. After washing, 100 ng / mL recombinant human His-tagged OX40L was added to the wells and incubated for a further 30 minutes at 4°C. After washing, AF647-labelled anti-His tag antibody was added to the wells and incubated for further 30 minutes at 4°C. After a final washing step, binding of OX40L to 0X40 receptor expressed on CHO-S cells was detected using the high-throughput flowcytometer iQue® Screener PLUS (Sartorius) measuring the GeoMean of AF647 signal in each well. A 12-points curve was generated for each antibody and every concentration was analyzed in duplicates.

[0145] All anti-OX40 antibodies tested, apart from Glenmark analogue, fully blocked the binding of human OX40L to human 0X40 receptor expressed on CHO-S (FIG. 2A, Table 8), while Glenmark antibody analogue only partially blocked binding of OX40L to human 0X40 receptor. Notably, the blocking capacity of 16706 was superior to all the reference antibody analogues tested (Kirin, Glenmark and Inmagene antibody analogues).

[0146] Further, anti-OX40 antibodies 16598, 16683, 16706 and 16716 and Kirin antibody analogue fully blocked the binding of human OX40L to cynomolgus 0X40 receptor expressed on CHO-S with similar potency (FIG. 2B, Table 8), while Inmagene analogue and 16790 antibody partially blocked binding of human OX40L to cynomolgus 0X40 receptor. Glenmark antibody analogue did not block binding of OX40L to cynomolgus 0X40 receptor.

[0147] As expected, IgGl control antibody did not block binding of OX40L to human 0X40 receptor in CHO-S cells, while in cynomolgus OX40-transfected cells a partial blocking effect was observed. Despite this mild effect that was not observed in previous experiments (data not shown), it is possible to discriminate between blocking and nonblocking antibodies for cynomolgus 0X40 receptor.Table 8: Efficacy and IC50 values for blocking of OX40L binding to human or cynomolgus 0X40 receptor-transiently transfected CHO-S cells by anti-OX40 antibodiesExample 4: Measurement of Antibody Binding Kinetics Towards Human and Cynomolgus 0X40 Extracellular Domain

[0148] This example demonstrates the binding of anti-OX40 antibodies to recombinant human and cynomolgus 0X40 extracellular domains (ECDs) as measured by surface plasmon resonance (SPR).

[0149] The anti-OX40 antibodies were tested for binding to recombinant human and cynomolgus 0X40 (ACRO Biosystems) by Surface Plasmon Resonance (SPR) using Carterra® LSA™. An HC30M (Carterra®) chip was functionalized by goat anti -human Ig Fc (Southern Biotech) using amine-coupling. The chip was activated by freshly prepared 0.4 M EDC, 0.1 M sulfo-NHS, and 0.1 M MES, pH 5.5 (1 : 1 :1 v / v / v) for 5 min, coupled with 75 pg / mL anti-human Ig Fc in 10 mM sodium acetate, pH 4.5, for 10 min, and excess reactive esters were quenched for 3 min by injection of 1 M ethanolamine, pH 8.5. The instrument was primed in running buffer (PBS pH 7.4, 0.01% Tween-20, 0.5 mg / mL BSA). After priming and washing, antibodies were captured onto individual spots of the chip for 12 minutes as replicates (n=8). Kinetic analysis was performed by applying kinetic titration series of each antigen as increasing concentrations. Antigen association was performed for 10 minutes, and antigen dissociation was recorded for 15 minutes. After each cycle of antigen injections, the surface was regenerated by 0.45% H3PO4 for 2x20 s and washed for 5 min in running buffer. Binding responses were processed and analyzed using Carterra®’ s KIT software tool. Processed data were fitted to a simple Langmuir 1 : 1 binding model for calculation of the on-rate (konor ka), off-rate (koir or kd) and affinity (KD) constants.

[0150] The binding kinetics of anti-OX40 antibodies are shown in Table 9. The antibodies exhibited high affinities in the single digit nanomolar range for binding to the extracellular domain of recombinant human 0X40 and demonstrated similar affinities for cynomolgus 0X40. The antibodies 16683, 16706, 16713, and 16718 exhibited binding kinetics to human 0X40 that were comparable to those measured for the 11D4 analogue, Inmagene analogue, Glenmark analogue and Kirin analogue.Table 9: Binding kinetics, kon, koff, and KD, of anti-OX40 antibodies binding to human and cynomolgus 0X40 ECD as measured by SPR* Standard deviation (SD) for each is presented in the table.Example 5: Epitope Binning of Anti-OX40 Antibodies

[0151] This example measures paired competition of anti-OX40 monoclonal antibodies measured by Biolayer Interferometry (BLI). Antibodies belonging to different epitope groups or bins recognize different epitope binding regions on the 0X40 ECD.

[0152] Paired competition of anti-OX40 monoclonal antibodies binding to recombinant human 0X40 ECD His-tagged receptor (Sino Biological) were measured by BLI on an Octet® RH 96 biosensor instrument (ForteBio). In-tandem assays were performed by capturing antigen (0.5 pg / mL) on a pre-equilibrated Anti-Penta-HIS (HIS IK) Biosensor (ForteBio) for 300 s, followed by 180 s association of first antibody and hereafter second antibody at saturating conditions (250 nM). Sensors were regenerated in 10 mM glycine, pH 1.5, 3 x 5 s. All experiments were carried out at 25°C. Data were analyzed in Octet® Analysis Studio (13.0.1.35) program. Data were analyzed by the epitope binning application.

[0153] Table 10 shows the in-tandem epitope binning data as the responses of the second anti-OX40 antibodies in the in-tandem epitope binning assay. A threshold above 0.2 nm in response units is set to differentiate antibodies binding different epitopes (white) from those competing for the same epitope and blocking each other in the assay (underlined text and bold text). Bold text indicates self-blocking of antibodies, underlined text indicates crossblocking activity of antibodies, and white cells indicate non-competing antibodies. Epitope bins are shown in the table.Table 10: Competition of anti-OX40 antibodies

[0154] The four anti-OX40 antibodies, designated as 16683, 16706, 16713, 16718, exhibited competition with each other and were grouped in bin 1.

[0155] The Glenmark and Inmagene analogues and the panel of anti-OX40 antibodies engaged in competition. However, unlike antibodies in epitope bin 1, the Glenmark and Inmagene analogues did not compete with the Kirin analogue; therefore, they are classified into bin 2.

[0156] The Kirin analogue competed with the four anti-OX40 antibodies but not with the Inmagene and Kirin analogues, thus forming its distinct epitope group, bin 3.

[0157] None of the antibodies exhibited competition with 11D4, suggesting this antibody binds to an epitope distinct from the other antibodies; thus it is assigned to bin 4.

[0158] In summary, antibodies 16683, 16706, 16713 and 16718 are grouped in bin 1, indicate that the antibodies bind to a shared epitope, overlapping but yet distinct from the epitopes recognized by the Glenmark and Inmagene analogues in bin 2, and from the epitope recognized by the Kirin analogue in bin 3. Antibody 11D4 binds to a unique epitope.Example 6: Epitope Mapping of Anti-OX40 Antibodies by Mutagenesis and Surface Plasmon Resonance

[0159] Linear and conformational epitopes were characterized using a mutagenesis approach and surface plasmon resonance (SPR). This example demonstrates that monoclonal anti-OX40 antibodies 16683, 16706 and 16713 recognize epitopes located within domain 2 and partially in domain 3 of the extracellular domains (ECD) of 0X40.

[0160] The protein sequences of human and rat (Rattus norvegicus) 0X40 were downloaded from UniProt (P43489 and Pl 5725 respectively) and aligned. To map linearepitopes, human 0X40 ECD Fc fusion proteins were generated having 10 amino acids sequentially exchanged with corresponding rat 0X40 sequence in segments overlapping by five amino acids. Conformational epitopes were characterized by alanine scanning mutagenesis of 0X40 ECD.

[0161] The cDNA coding for human 0X40 ECD was synthesized and cloned into a vector containing CMV promoter and human Ig Fc sequence (residues P101-K330), resulting in fusion of Ig Fc to the 0X40 ECD C-terminus. Fc fusion constructs with wildtype and mutated human 0X40 ECD were generated by standard gene synthesis techniques and proteins were expressed transiently in an ExpiCHO™ expression system. After harvesting, supernatants were tested for binding to anti-OX40 Fabs by surface plasmon resonance (SPR) using the Carterra® LSA™ Platform (Carterra, USA). An HC200M (Carterra, USA) chip was functionalized with goat anti -human Ig Fc (Southern Biotech) by amine-coupling. The chip was activated with freshly prepared 0.4 M EDC, 0.1 M sulfo-NHS, and 0.1 M MES, pH 5.5 (1 : 1 :1 v / v / v) for 5 minutes, coupled with 75 pg / mL anti-human Ig Fc in 10 mM sodium acetate, pH 4.5, for 10 minutes, and excess reactive esters were quenched for 3 minutes by injection of 1 M ethanolamine, pH 8.5. The instrument was primed in running buffer (PBS pH 7.4, 0.01% Tween 20, 0.5 mg / mL BSA).

[0162] After priming and washing, 0X40 fusion proteins in culture supernatants were captured onto individual spots of the chip for 12 minutes as duplicates. Fab analytes were each prepared in running buffer. Kinetic analysis was performed by applying a kinetic titration series of monomeric Fabs as increasing concentrations from 0.8 nM and up to 300 nM. Fab association was performed for 15 minutes, and dissociation was recorded for 15 minutes. After each cycle of Fab injections, the surface was regenerated by 0.45% H3PO4 for 2x20 s and washed for 5 min in running buffer.

[0163] Binding responses were analyzed in Carterra’s KIT software tool by referencing to spots of wildtype rat 0X40, buffer blanked and aligned to y-axis. Processed data was fitted to a simple Langmuir 1 : 1 binding model for calculation of the on-rate (konor ka), off- rate (koff or ka) and affinity (KD) constants. Mutations generating inactive proteins common for all antibodies were deselected and a 5-fold affinity reduction compared to the KD of antibodies binding to wildtype 0X40 was used to define the epitopes.

[0164] The linear and conformational epitopes of anti-OX40 antibodies 16683, 16706, 16713 were identified by measuring binding of Fab fragments to mutated receptor constructs by SPR. A criterion of a minimum 5-fold decrease in Fab binding affinity compared towildtype human 0X40 was applied to identify amino acids causing a substantial loss of binding, thus defining the epitopes (Table 11).Table 11: Summary of epitopes of 16683, 16706 and 16713

[0165] The extracellular part of 0X40 comprises four cysteine-rich domains (CRDs), designated as CRD 1-4. Each domain exhibits an elongated configuration stabilized by 1-2 disulfide bridges. Upon forming an active complex with OX40L, 0X40 assembles into a functional trimer, with three receptor molecules binding as monomer-monomer configuration to the trimeric ligand (Compaan and Hymowitz, Structure. (2006) 14(8): 1321-30).

[0166] The linear epitopes and contact residues determined for antibodies 16683, 16706 and 16713 exhibit complete similarity, suggesting their binding to a shared epitope as also shown by the epitope binning analysis performed in Example 5. The epitope is located primarily within CRD2, with partial involvement of CRD3, as shown in FIG. 3, overlapping with the contact surface of the receptor-ligand interaction. An antibody targeting this epitope can disrupt the interaction between the 0X40 receptor and its ligand, thereby inhibiting downstream signaling events.

[0167] In conclusion, antibodies 16683, 16706 and 16713 share the same epitope. The location of the conformational epitope at the receptor-ligand interaction interface elucidates the mechanism behind the antagonistic activity of the antibodies.Example 7: ADCC (PBMC Cell-Mediated Killing) and FcGR Signaling Activity of Anti- 0X40 Antibodies Using the OX40-Positive MYLA Cell Line

[0168] This example describes in vitro functional evaluation of several anti-OX40 antibodies (16683, 16706, 16713) in the Fc-effector function attenuated format IgG1.3f with the purpose of examining ADCC.

[0169] The antibodies were firstly evaluated for their ability to induce Fc-mediated reporter signaling of “ADCC” assays using Jurkat cell lines stably expressing either of a series of Fc-Gamma receptors (FcGRs). This included the high affinity receptor FcGRIa, the two main variants of FcGRIIa (-R and -H with arginine and histidine in the 131 amino acidposition, respectively, with the -H variant generally exhibiting higher Fc affinity), as well as the two variants of FcGRIIIa (-F and -V with valine and phenylalanine in the 158 amino acid position, respectively, with the -V variant showing substantially higher Fc affinity), and a NF AT -responsive luciferase reporter construct, purchased from Promega (USA). For assessing binding to the negative signal receptor FcGRIIb, the Jurkat reporter cell line expresses a chimera of FcGRIIb and FcGRIIa. Secondly, the antibodies were tested for the ability to elicit PBMC -mediated ADCC. For both assays, the MYLA CD4+T cell cancer cell with endogenous expression of 0X40 (Kawana et al., Int. J. Mol. Set. (2021) 22(22): 12576) was used as target cells. Antibodies representing analogues of anti-OX40 clinical candidates (Kirin analogue and Glenmark analogue) in an IgGl-afucosylated or (‘Inmagene’) IgGl- N297A formats were included for comparison and as positive controls (afucosylated formats) to demonstrate an assay window. An IgGl with wildtype Fc (IgGl-wt) targeting an irrelevant antigen was used as a negative control.

[0170] FcGR reporter assays: MYLA CD4+OX40+target cells were seeded in 384-well flat bottom plates in low IgG serum, antibodies added and incubated for 30 minutes.Reporter cells were added at a 1 :2 (targetreporter) ratio and plates were incubated for 16 hours. BioGio (luciferase) reagent was added, and luminescence read in a plate reader after seven minutes of incubation.

[0171] PBMC ADCC assay: MYLA CD4+OX40+target cells were labeled with calcein for 30 minutes. PBMCs were isolated from healthy human donors and added to empty wells together with antibodies to a clear bottom 384-well plate. Calcein loaded cells were added to wells at a 1 :20 (target: effector) ratio and plates incubated for 90 minutes. Calcein levels in supernatants were measured in a fluorescence plate reader. Specific lysis was calculated by subtracting spontaneous lysis (calcein-loaded MYLA cells only, no PBMCs present) and normalizing to maximum lysis (Triton X-100 lysis of calcein-loaded MYLA cells).

[0172] The signaling of the FcGR reporter cells after treatment with the anti-OX40 antibodies is shown in FIGs. 4A-4F. Only the anti-OX40 antibodies Glenmark analogue (titrated) and the Kirin analogue (167 nM), both in the IgGl-afucosylated format, showed activity compared to the untreated control (no antibody) or the IgGl-wt negative control antibody, even in the high-affinity FcGRIIIa-V assay. The Inmagene analogue in the IgGl- N297A Fc attenuated format yielded no signal in the FcGRIIb assay. Consequently, none of the three anti-OX40 antibodies tested (16683, 16706, 16713) elicited any FcGR signaling and may thus be expected to neither bind FcGRs with high affinity nor induce ADCC, which depends on especially FcGRIIIa signaling.

[0173] ADCC activity is exemplified in FIG. 5 using a representative PBMC donor sample from a cohort of ten healthy donors. Killing activity was observed with the Kirin analogue (titrated and constant concentration of 167 nM) and with the Glenmark analogue (constant concentration of 167 nM), both in the IgGl-afucosylated format. All anti-OX40 antibodies (16683, 16706, 16713) in the IgG1.3f format induced no additional killing above the background levels observed with a negative control IgGl-wt antibody or without antibody (‘untreated’).Example 8: Agonistic Activity of Anti-OX40 Antibodies in a Raji + 0X40 Reporter Cell Line Assay

[0174] This example describes in vitro functional evaluation of potential agonistic activity of anti-OX40 antibody 16706 in a dose titration assay. The antibody was evaluated for its ability to induce 0X40 signaling in a bioluminescence-based 0X40 signaling reporter cell line in the presence of FcGR-expressing Raji cells. Other anti-OX40 antibodies (Inmagene analogue and Kirin analogue) were included for comparison.

[0175] 0X40 reporter cells (0X40 Bioassay, Promega, Cat no. JA2191) were seeded out in RPMI 1640 + 5% FBS + 1% Penicillin / Streptomycin and incubated overnight. Subsequently, the cells were incubated for 15 min with a two-fold titration of the indicated antibodies starting from 167nM. Raji cells were added, and the cells were co-cultured for 5 hours. The capacity of the antibodies to induce 0X40 signaling was assessed by measuring the bioluminescence signal.

[0176] The ability of the anti-OX40 antibodies to induce 0X40 reporter activity in an 0X40 reporter cell line in the presence of Raji cells is shown in FIG. 6. The Kirin analogue antibody initiated the downstream signaling of 0X40 by crosslinking via FcGRs on the surface of Raji cells. On the contrary, 16706 and Inmagene analogue did not bind to FcGRs and did not crosslink, thus failed to activate the downstream signaling of 0X40.Example 9: Functional Activity of Anti-OX40 Antibodies in an 0X40 Reporter Cell Line Assay

[0177] This example describes in vitro functional evaluation of several anti-OX40 antibodies (16706, 16683, 16713) with the purpose of demonstrating a dose-dependent antagonistic activity. The antibodies were evaluated for their ability to inhibit OX40L- induced 0X40 signaling in a bioluminescence-based 0X40 signaling reporter cell line. Otheranti-OX40 antibodies (Kirin analogue and Glenmark analogue) were included for comparison.

[0178] 0X40 reporter cells (0X40 Bioassay, Promega, Cat no. JA2191) were seeded out in RPMI 1640 + 5% FBS + 1% Penicillin / Streptomycin and incubated overnight. The cells were subsequently incubated for 15 min with a two-fold titration of the indicated antibodies starting from 167 nM followed by the addition of soluble OX40L. The capacity of the antibodies to antagonize OX40L-induced signaling was assessed by measuring the bioluminescence signal after 5 hours.

[0179] The ability of the anti-OX40 antibodies to inhibit OX40L-induced 0X40 reporter activity in an 0X40 reporter cell line is shown by the IC50 values in Table 12 below. Anti- 0X40 antibodies 16706, 16683 and 16713 had superior inhibitory activity to the Kirin and Glenmark analogue antibodies.Table 12: IC50 values for anti-OX40 antibodies in an 0X40 reporter cell line assayExample 10: Functional Activity of Anti-OX40 Antagonist Antibodies on a PBMC Assay in the Reversion of IL-2 Production Induced by OX40 / OX40L Engagement

[0180] This example describes in vitro functional evaluation of the anti-OX40 antagonist antibody 16706 with the purpose of demonstrating a dose-dependent activity. The antibody was evaluated for its ability to reverse IL-2 production on PBMC following OX40 / OX40L engagement. The Kirin analogue antibody was included for comparison.

[0181] Human PBMCs were freshly isolated from 3 healthy donor buffy coats. PBMCs were stimulated with a polymeric nanomatrix conjugated to humanized recombinant CD3 and CD28 agonists (TranAct™, Miltenyi) in the presence or absence of antibody 16706, in comparison to the Kirin analogue anti-OX40 antagonist antibody, following an 8 points semilog dose response and incubated 10 min at 37°C before adding OX40L (3.2 pg / mL). After 3 days of incubation at 37°C, supernatants were collected and frozen at -80°C for subsequent IL-2 dosage.

[0182] PBMCs treated with OX40L produced IL-2. In the presence of anti-OX40 antibodies, there was a dose-dependent reversion in the production of IL-2 (FIG. 7). Antibody 16706 showed a better potency (2.3E-10 M) than the Kirin analogue (3.78E-10 M), as reported in Table 13.Table 13: ICso values for anti-OX40 antibodies in a PBMC assay wherein the reversion of IL-2 production was induced by OX40 / OX40L engagementExample 11: Functional Activity of Anti-OX40 Antibodies in a T-cell Activation Assay

[0183] This example describes in vitro functional evaluation of several anti-OX40 antibodies (16706, 16683, 16713). The antibodies were evaluated for their ability to inhibit OX40L-induced interferon-gamma (IFNg) secretion in a T-cell activation assay. Other anti- 0X40 antibodies (Inmagene analogue, Kirin analogue and Glenmark analogue) were included for comparison.

[0184] Human primary CD4+T-cells isolated from healthy donors were seeded out on T- cell-stimulatory aAPC / CHO-Kl cells, seeded out the preceding day in Ham’s F12 + 10% FBS and incubated in RPMI 1640 + 10% FBS + 1% Penicillin / Streptomycin with a two-fold titration of the indicated antibodies starting from 167 nM in addition to IL-2 and soluble OX40L. IFNg levels of the supernatants were assessed by ELISA after three days of culture, and the ability of the anti-OX40 antibodies to inhibit 0X40 signaling was assessed by their capacity to block OX40L-induced IFNg secretion.

[0185] The ability of the anti-OX40 antibodies to inhibit the OX40L-induced IFNg secretion in a T-cell activation assay is shown by the IC50 values shown in Table 14. Antibodies 16706, 16683, 16713 had superior inhibitory activity compared to the reference Inmagene, Kirin and Glenmark analogue antibodies.Table 14: IC50 values for anti-OX40 antibodies in a T-cell activation assayExample 12: Functional Activity of Anti-OX40 Antagonist Antibodies in the Restoration of iTreg Differentiation

[0186] This example describes in vitro functional evaluation of anti-OX40 antagonist antibodies 16706 and 16713 with the purpose of demonstrating a dose-dependent activity in induced regulatory T cells (iTreg) positive for CD3, CD4, CD25 and FoxP3 expression. The antibodies were evaluated for their ability to restore iTreg differentiation inhibited by OX40 / OX40L pathway. The Inmagene analogue and Kirin analogue anti-OX40 antagonist antibodies were included for comparison.

[0187] Human PBMCs were freshly isolated from anonymized healthy donor buffy coats provided from EFS. CD4+naive T cells were purified using a commercially available isolation kit according to the manufacturer’s instructions (Miltenyi Biotec) and stimulated with plate-coated anti-CD3 antibody (5 pg / mL, clone OKT3) and soluble anti-CD28 antibody (1 pg / mL, clone CD28.2) in X-VIV015 medium (Lonza) supplemented with recombinant human IL-2 (R&D Systems) at 50 U / mL, recombinant human TGF-beta (Peprotec) at 5 ng / mL. The present anti-OX40 antagonist antibodies (16706 and 16713) in comparison to the Kirin and Inmagene analogues were then added to the cell culture at different concentrations (from 0.1 pg / mL to 300 pg / mL) or the isotype control (IgGl control) and incubated for 10 min at 37°C before adding OX40L (BioLegend) (1.39 pg / mL). After 6 days of incubation at 37°C, cells were harvested and stained with anti-CD4 (BD Biosciences), anti-CD25 (Beckman Coulter), and anti-CD3 (BioLegend) antibodies, fixed and permeabilized (eBioscience) and stained with anti-Foxp3 (BioLegend) antibody.

[0188] Flow cytometry analysis was performed using FlowJo software. Graphs and IC50 calculation were performed on GraphPad-Prism software.

[0189] The ability of anti-OX40 Abs to restore iTreg differentiation inhibited by OX40L is shown in FIG. 8. Anti-OX40 antibodies were able to restore iTreg differentiation, in a dose dependent manner until reaching a plateau at the highest antibody concentrations, at a level comparable to the condition without OX40L. Calculated EC50 showed a better potency of the 16706 antibody (5.5E-9 M) and the 16713 antibody (6.3E-9 M) as compared to Inmagene analogue (1.8 E-8 M) and Kirin analogue (2.8 E-8 M). As expected, the isotype control (IgGl control) showed no effect on iTreg restoration.Example 13: Functional Activity of Anti-OX40 Antagonist Antibodies in the Reversion of CD4+T cell Polarization Induced by OX40 / OX40L Engagement

[0190] This example describes in vitro functional evaluation of the anti-OX40 antagonist antibody 16706 with the purpose of demonstrating a dose-dependent activity. The antibody was evaluated for its ability to reverse the polarization of naive CD4 T cells induced by OX40 / OX40L pathway. The Inmagene analogue and Kirin analogue anti-OX40 antagonist antibodies were included for comparison.

[0191] Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from anonymized healthy donor buffy coats provided from French blood establishment (EFS). CD4+naive T cells were purified using a commercially available isolation kit according to the manufacturer’s instructions (Miltenyi Biotec) and stimulated with plate-coated anti-CD3 antibody (5pg / mL, clone OKT3) and soluble anti-CD28 antibody (1 pg / mL, clone CD28.2) in X-VIV015 medium (Lonza) in the presence of anti-OX40 antagonist antibodies (16706, Inmagene analogue or Kirin analogue) at different concentrations (from 0.1 pg / mL to 300 pg / mL) or the isotype control (IgGl control). After 10 min of incubation at 37°C, 5% CO2, recombinant human OX40L (1.6 pg / mL, BioLegend) was added to the cell culture. The plate was incubated at 37°C, 5% CO2 for 5 days. Supernatants were harvested and frozen for subsequent cytokine dosage. Graphs and IC50 calculation were performed on GraphPad- Prism software.

[0192] The ability of anti-OX40 antibodies to block T cell polarization induced by OX40 / OX40L is shown in FIGs. 9A and 9B. Polarization induced by OX40 / OX40L pathway leads to IL-2 / IL-21 producing cells. In the presence of anti-OX40 antagonist antibodies, there was a reversion of IL-2 and IL-21 secretion in a dose-dependent manner until reaching a plateau at the highest antibody concentrations, at a level comparable to the condition without OX40L. For IL-2, calculated IC50 showed a better potency of 16706 antibody (2. IE-9 M) as compared to Inmagene analogue antibody (4.2E-9 M) and Kirin analogue antibody (7.3E-8 M). For IL-21, calculated IC50 showed also a better potency of 16706 antibody (1.6E-10 M) as compared to Inmagene analogue (3.5E-10 M) and Kirin analogue (10E-10 M). As expected, the isotype control (IgGl control) showed no effect on polarization reversion.Example 14: In Vivo Suppression of Graft-Versus-Host Disease (GvHD)

[0193] This example demonstrates in vivo efficacy of 16706 antibody in suppressing development of GvHD.

[0194] Human PBMCs were engrafted into severely immunodeficient NOD Rag2‘ 'yc' ' (NRG) mice. One day prior PBMC engraftment, mice were sub-lethally irradiated to remove remaining endogenous murine immune cells. Fourteen days after PBMC engraftment, mice started treatment by intraperitoneal injection of vehicle or antibody 16706 dosed at 0.1, 1 or lO mg / kg. Mice were treated two times weekly for a total of ten treatments. One group of irradiated mice not engrafted with PBMCs and treated with vehicle were included as control. Mice were weighed and scored based on clinical signs such as anemia, weight loss, posture, activity and hair loss three times weekly throughout the study period. Mice were euthanized when reaching a global score of 6 and / or 20% weight loss, sacrifice corresponded to a maximum score of 8.

[0195] As shown in FIGs. 10A-C, antibody 16706 reduced GvHD symptoms in PBMC engrafted NRG mice at all doses, and maintained better the animals’ body weight and survival over 45 weeks.

Claims

1. CLAIMS1. An isolated anti-OX40 antibody or an antigen-binding portion thereof, wherein the antibody binds specifically to amino acids 80-104 and / or amino acids 110-122 of SEQ ID NO: 106.

2. The anti-OX40 antibody or antigen-binding portion of claim 1, wherein the antibody further binds specifically to amino acids 70-72 and / or amino acids 86-92 of SEQ ID NO: 106.

3. The anti-OX40 antibody or antigen-binding portion of claim 1 or 2, wherein the antibody further binds specifically to one or more of amino acids 74, 82, 116, and 124 of SEQ ID NO: 106.

4. An isolated anti-OX40 antibody or an antigen-binding portion thereof, wherein the antibody binds to the same epitope as a reference antibody having a heavy chain variable domain (VH) and a light chain variable domain (VL) havingSEQ ID NOs:5 and 6, respectively,SEQ ID NOs: 1 and 2, respectively,SEQ ID NOs: 3 and 4, respectively,SEQ ID NOs: 7 and 8, respectively,SEQ ID NOs: 9 and 10, respectively, or SEQ ID NOs: 11 and 12, respectively.

5. The anti-OX40 antibody or antigen-binding portion of claim 4, wherein a) the heavy chain of the anti-OX40 antibody comprises: i) heavy chain complementarity determining regions (H-CDR)-l-3 comprising SEQ ID NOs:37-39, respectively; ii) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90% identical to SEQ ID NO: 5; iii) a VH comprising SEQ ID NO:5; or iv) SEQ ID NOs: 5 and 97; and b) the light chain of the anti-OX40 antibody comprises: i) light chain complementarity determining regions (L-CDR)-l-3 comprising SEQ ID NOs:40-42, respectively;52ii) a light chain variable domain (VL) comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 6; iii) a VL comprising SEQ ID NO:6; or iv) SEQ ID NOs: 6 and 98.

6. The anti-OX40 antibody or antigen-binding portion of claim 4, wherein a) the heavy chain of the anti-OX40 antibody comprises: i) H-CDR1-3 comprising SEQ ID NOs: 13-15, respectively; ii) a VH comprising an amino acid sequence at least 90% identical to SEQ ID NON; iii) a VH comprising SEQ ID NO: 1; or iv) SEQ ID NOs: 1 and 97; and b) the light chain of the anti-OX40 antibody comprises: i) L-CDR-1-3 comprising SEQ ID NOs: 16-18, respectively; ii) a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2; iii) a VL comprising SEQ ID NO:2; or iv) SEQ ID NOs:2 and 98.

7. The anti-OX40 antibody or antigen-binding portion of claim 4, wherein a) the heavy chain of the anti-OX40 antibody comprises: i) H-CDR1-3 comprising SEQ ID NOs:25-27, respectively; ii) a VH comprising an amino acid sequence at least 90% identical to SEQ ID NON; iii) a VH comprising SEQ ID NO:3; or iv) SEQ ID NOs:3 and 97; and b) the light chain of the anti-OX40 antibody comprises: i) L-CDR1-3 comprising SEQ ID NOs:28-30, respectively; ii) a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NON; iii) a VL comprising SEQ ID NON; or iv) SEQ ID NOsN and 98.

8. The anti-OX40 antibody or antigen-binding portion of claim 4, whereina) the heavy chain of the anti-OX40 antibody comprises: i) H-CDR1-3 comprising SEQ ID NOs:49-51, respectively; ii) a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 7; iii) a VH comprising SEQ ID NO:7; or iv) SEQ ID NOs:7 and 97; and b) the light chain of the anti-OX40 antibody comprises: i) L-CDR1-3 comprising SEQ ID NOs: 52-54, respectively; ii) a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:8; iii) a VL comprising SEQ ID NO:8; or iv) SEQ ID NOs: 8 and 98.

9. The anti-OX40 antibody or antigen-binding portion of claim 4, wherein a) the heavy chain of the anti-OX40 antibody comprises: i) H-CDR1-3 comprising SEQ ID NOs:61-63, respectively; ii) a VH comprising an amino acid sequence at least 90% identical to SEQ ID NOV; iii) a VH comprising SEQ ID NOV; or iv) SEQ ID NOsV and 97; and b) the light chain of the anti-OX40 antibody comprises: i) L-CDR1-3 comprising SEQ ID NOs: 64-66, respectively; ii) a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 10; iii) a VL comprising SEQ ID NO: 10; or iv) SEQ ID NOs: 10 and 98.

10. The anti-OX40 antibody or antigen-binding portion of claim 4, wherein a) the heavy chain of the anti-OX40 antibody comprises: i) H-CDR1-3 comprising SEQ ID NOs:73-75, respectively; ii) a VH comprising an amino acid sequence at least 90% identical to SEQ ID NO: 11; iii) a VH comprising SEQ ID NO: 11; or iv) SEQ ID NOs: 11 and 97; andb) the light chain of the anti-OX40 antibody comprises: i) L-CDR1-3 comprising SEQ ID NOs:76-78, respectively; ii) a VL comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 12; iii) a VL comprising SEQ ID NO: 12; or iv) SEQ ID NOs: 12 and 98.

11. An anti-OX40 antibody or an antigen-binding portion thereof, wherein the antibody comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of: a) SEQ ID NOs:37-42, respectively; b) SEQ ID NOs: 13-18, respectively; c) SEQ ID NOs:25-30, respectively; d) SEQ ID NOs:49-54, respectively; e) SEQ ID NOs:61-66, respectively; or f) SEQ ID NOs:73-78, respectively.

12. The anti-OX40 antibody or antigen-binding portion of claim 11, wherein the antibody comprises a heavy chain variable domain (VH) amino acid sequence and a light chain variable domain (VL) amino acid sequence that are at least 90% identical to the amino acid sequences of: a) SEQ ID NOs: 5 and 6, respectively; b) SEQ ID NOs: 1 and 2, respectively; c) SEQ ID NOs:3 and 4, respectively; d) SEQ ID NOs:7 and 8, respectively; e) SEQ ID NOs: 9 and 10, respectively; or f) SEQ ID NOs: 11 and 12, respectively.

13. The anti-OX40 antibody or antigen-binding portion of claim 12, wherein the antibody comprises a VH and a VL comprising: a) SEQ ID NOs: 5 and 6, respectively; b) SEQ ID NOs: 1 and 2, respectively; c) SEQ ID NOs:3 and 4, respectively; d) SEQ ID NOs:7 and 8, respectively; e) SEQ ID NOs: 9 and 10, respectively; orf) SEQ ID NOs: 11 and 12, respectively.

14. The anti-OX40 antibody of any one of claims 1-13, wherein the antibody is of isotype subtype IgGi.

15. The anti-OX40 antibody of claim 14, wherein the antibody comprises a mutant Fc domain with reduced binding affinity for an Fc gamma receptor.

16. The anti-OX40 antibody of claim 15, wherein the antibody comprises a heavy chain constant region comprising SEQ ID NO:97.

17. The anti-OX40 antibody of claim 16, wherein the antibody comprises: a) a heavy chain (HC) comprising SEQ ID NOs:5 and 97, and a light chain (LC) comprising SEQ ID NOs:6 and 98; b) an HC comprising SEQ ID NOs: 1 and 97, and an LC comprising SEQ ID NOs:2 and 98; c) an HC comprising SEQ ID NOs:3 and 97, and an LC comprising SEQ ID NOs:4 and 98; d) an HC comprising SEQ ID NOs:7 and 97, and an LC comprising SEQ ID NOs:8 and 98; e) an HC comprising SEQ ID NOs:9 and 97, and an LC comprising SEQ ID NOs: 10 and 98; or f) an HC comprising SEQ ID NOs: 11 and 97, and an LC comprising SEQ ID NOs: 12 and 98.

18. A pharmaceutical composition comprising the anti-OX40 antibody or antigen-binding portion of any one of claims 1-17 and a pharmaceutically acceptable excipient.

19. Isolated nucleic acid molecule(s) comprising a nucleotide sequence that encodes the heavy chain, or a nucleotide sequence that encodes the light chain, or both, of the anti-OX40 antibody or antigen-binding portion of any one of claims 1-17.

20. The isolated nucleic acid molecule(s) of claim 19, wherein the nucleic acid molecule(s) comprise any one of SEQ ID NOs: 85-96.

21. Vector(s) comprising the isolated nucleic acid molecule(s) of claim 19 or 20, wherein the vector(s) further comprise an expression control sequence.

22. A host cell comprising a nucleotide sequence that encodes the heavy chain, and a nucleotide sequence that encodes the light chain, of the anti-OX40 antibody or antigenbinding portion of any one of claims 1-17.

23. A method for producing an anti-OX40 antibody or an antigen-binding portion thereof, comprising providing a host cell of claim 22, culturing said host cell under conditions suitable for expression of the antibody or antigen-binding portion, and isolating the resulting antibody or antigen-binding portion.

24. A method of treating an autoimmune or inflammatory disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the anti- 0X40 antibody or antigen-binding portion of any one of claims 1-17, or the pharmaceutical composition of claim 18.

25. The anti-OX40 antibody or antigen-binding portion of any one of claims 1-17, or the pharmaceutical composition of claim 18, for use in treating an autoimmune or inflammatory disease in a patient in need thereof.

26. Use of the anti-OX40 antibody or antigen-binding portion of any one of claims 1-17, or the pharmaceutical composition of claim 18, in the manufacture of a medicament for treating an autoimmune or inflammatory disease in a patient in need thereof.

27. The method of claim 24, the anti-OX40 antibody or antigen-binding portion or the pharmaceutical composition for use of claim 25, or the use of claim 26, wherein the disease is selected from the group consisting of asthma, allergic asthma, chronic spontaneous urticaria (CSU), diabetes (optionally type 1 diabetes or latent autoimmune diabetes), lupus (optionally systemic lupus erythematosus or lupus nephritis), arthritis (optionally rheumatoid arthritis), allergy, antibody-mediated rejection, organ graft rejection, graft-versus-host disease (GvHD),Addison’s disease, ankylosing spondylitis, anti -glomerular basement membrane disease, autoimmune hepatitis, celiac disease, autoimmune alopecia, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, juvenile myositis, Kawasaki disease, inflammatory bowel diseases (optionally Crohn’s disease and ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, pemphigus vulgaris, thrombocytopenic purpura, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis, vitiligo, and Vogt-Koyanagi-Harada Disease.

28. The method of claim 24, the anti-OX40 antibody or antigen-binding portion or the pharmaceutical composition for use of claim 25, or the use of claim 26, wherein the disease is chronic spontaneous urticaria, atopic dermatitis, pemphigus vulgaris, bullous pemphigoid, Sjogren’s syndrome, lupus (optionally systemic lupus erythematosus or lupus nephritis), arthritis (optionally rheumatoid arthritis), Graves’ disease, inflammatory bowel diseases (optionally Crohn’s disease and ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, psoriasis, psoriatic arthritis, Sjogren’s syndrome, systemic scleroderma, systemic sclerosis, uveitis, autoimmune uveitis, thyroid eye disease, vasculitis or vitiligo.

29. The method of claim 24, the anti-OX40 antibody or antigen-binding portion or the pharmaceutical composition for use of claim 25, or the use of claim 26, wherein the disease is chronic spontaneous urticaria, atopic dermatitis or pemphigus vulgaris.

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