Compositions Comprising 4-1BB and OX40 Binding Proteins and Methods of Use

Abstract

The present disclosure provides pharmaceutical compositions comprising bispecific antibodies and antigen-binding fragments thereof that bind 4-1BB and OX40. Methods for using such compositions to treat disorders such as cancer are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international application claims priority to U.S. Provisional Application No. 63 / 150,475, filed February 17, 2021, the entire contents of which are incorporated herein by reference.

[0002] Reference to an Electronically Submitted Sequence Listing The contents of the electronically submitted sequence listing (Name: 4897_0060000_Seqlisting_ST25.txt; Size: 18,030 bytes; and Creation Date: February 16, 2022) are incorporated by reference herein in their entirety.

[0003] Technical Field The present disclosure relates to pharmaceutical compositions comprising bispecific antibodies and antigen-binding fragments thereof that specifically bind to 4-1BB and OX40. The compositions are useful for treating disorders such as solid tumor cancer. Methods for treating disorders such as cancer using such compositions are also provided. [Background technology]

[0004] 4-1BB (CD137) and OX40 are members of the TNF receptor (TNFR) family (Bremer, ISRN Oncol.:371854(2013)). These receptors are not constitutively present on naive T cells or NK cells: their expression is triggered by stimulation of T cells via the T cell receptor (TCR) or by other stimuli in NK cells. 4-1BB is upregulated mainly in CD8 T cells and NK cells, whereas OX40 is upregulated mainly on CD4 T cells. The function of these receptors is to provide costimulatory signals to T cells and NK cells. Activation of these receptors is naturally triggered by trimerization through interaction with 4-1BB ligand (4-1BBL) or OX40 ligand (OX40L) trimers, leading to signal transduction and initiation of specific cellular functions. 4-1BB enhances the effector function of CD8 T cells and NK cells through increased expression of IFN-γ, granzymes, and anti-apoptotic genes, which generates more and better effector CD8 T cells and NK cells. OX40 enhances the effector function of CD4 T cells by enhancing their ability to produce IL-2 and the clonal expansion capacity of memory CD4 T cells.

[0005] 4-1BB and OX40 are often expressed on tumor-infiltrating lymphocytes, and their expression has been used to identify tumor-specific T cells. Human solid tumors are often infiltrated with lymphocytes, mostly CD8+ and CD4+ T cells. The accumulation of tumor-infiltrating lymphocytes is often associated with improved survival in patients with various malignancies. (Ye et al., OncoImmunology 2:e27184(2013);Montler et al., Clin Transl Immunology 5:e70(2016)).

[0006] Preclinical results in various induced and spontaneous tumor models suggest that targeting 4-1BB with agonistic antibodies may result in tumor elimination and sustained antitumor immunity. Urelumab and utomilumab are agonistic anti-4-1BB monoclonal antibodies undergoing clinical trials for indications including the treatment of solid tumors. Despite early indications of efficacy, clinical development of urelumab has been hampered by inflammatory hepatotoxicity at doses above 1 mg / kg. Utomilumab is not as potent as urelumab, but has an improved safety profile compared to urelumab (Chester et al., Blood 131:39-57 (2018)). There is a need for effective therapeutic agents targeting 4-1BB that do not cause hepatotoxicity or other systemic damage as observed with urelumab.

[0007] OX40 agonists have been reported to increase T cell infiltration into tumors. OX40 signaling can also prevent Treg-mediated suppression of antitumor immune responses. In several preclinical mouse cancer models, such as 4T-1 breast cancer, B16 melanoma, Lewis lung carcinoma, and some chemically induced sarcomas, injection of OX40 agonists has resulted in therapeutic responses. (Ohsima et al., J. Immunology 159:3838-3848(1997); Imura et al., J. Exp. Med. 183:2185-2195(1996); Maxwell et al., J. Immunology 164:107-112(2000); Gough et al., J. Immunotherapy 33(8):798-809(2010)).

[0008] In some cases, researchers have generated protein constructs that contain multiple (>2) binding domains to 4-1BB or OX40, or fusions of multiple OX40L and 4-1BBL extracellular domains to induce agonism. In other examples, there are bispecific proteins that contain binding domain(s) to 4-1BB or OX40 and binding domain(s) to tumor-specific antigens. Binding and clustering via tumor antigen binding induces 4-1BB and OX40 clustering and signaling. However, none of these constructs are expected to stimulate the function of tumor-infiltrating lymphocytes, i.e., CD8+ T cells, CD4 T+ cells, and NK cells, and to do so with minimal or no off-target activation of effector cells (i.e., activation by binding to FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb).

[0009] Thus, there is a need for therapeutic options that selectively enhance the activity of tumor-infiltrating lymphocytes (with minimal or no effect on circulating lymphocytes), such as pharmaceutical compositions containing bispecific antibodies that bind to and stimulate both 4-1BB and OX40. Summary of the Invention

[0010] Some embodiments of the present disclosure provide a pharmaceutical composition comprising: (a) a bispecific antibody or antigen-binding fragment thereof that specifically binds 4-1BB and OX40; (b) about 5 mM to about 15 mM of a stability-promoting buffer; (c) about 25 mM to about 50 mM of a stabilizing amino acid; (d) about 2% to about 8% w / v sugar; and (e) about 0.01% to about 0.03% surfactant.

[0011] In some embodiments of the present disclosure, the stability-promoting buffer is selected from the group consisting of succinate, histidine, citrate, and glutamate.In some embodiments, the stability-promoting buffer is present in the composition in an amount of about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or about 15 mM.In some embodiments, the stability-promoting buffer is glutamate.In some embodiments, the glutamate is present in the composition at a concentration of about 5 mM.

[0012] In some embodiments of the present disclosure, the stabilizing amino acid is selected from the group consisting of arginine, methionine, leucine, and glycine.In some embodiments, the stabilizing amino acid is present in the composition at a concentration of about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, or about 50 mM.In some embodiments, the stabilizing amino acid is leucine.In some embodiments, the leucine is present in the composition at a concentration of about 40 mM.

[0013] In some embodiments of the present disclosure, the sugar is selected from the group consisting of sucrose, trehalose, and sorbitol. In some embodiments, the sugar is present in the composition at a concentration of about 2% w / v to about 8% w / v. In some embodiments, the sugar is present in the composition at a concentration of about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, or about 8% w / v. In some embodiments, the sugar is sucrose. In some embodiments, the sucrose is present in the composition at a concentration of about 3% w / v.

[0014] In some embodiments of the present disclosure, the pharmaceutical composition further comprises a surfactant. In some embodiments, the surfactant is present in the composition at a concentration of about 0.01% w / v to about 0.03% w / v. In some embodiments, the surfactant is polysorbate-80. In some embodiments, the polysorbate-80 is present in the composition at a concentration of about 3% w / v.

[0015] In some embodiments of the present disclosure, the pH of the composition ranges from about 4.3 to about 4.7, hi some embodiments, the pH of the composition is about 4.5.

[0016] In some embodiments of the disclosure, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof is present in the composition at a concentration of about 10 mg / mL to 50 mg / mL.

[0017] In some aspects of the disclosure, the pharmaceutical composition does not include sodium chloride (NaCl).

[0018] In some embodiments of the disclosure, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof has a purity of greater than about 90%.

[0019] In some embodiments of the disclosure, the pharmaceutical compositions provided herein comprise: (a) a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof at a concentration of about 10 mg / mL; (b) about 10 mM glutamate; (c) about 40 mM leucine; (d) about 3% w / v sucrose; and (e) about 0.02% polysorbate-80; wherein the pH of the composition is about 4.3 to about 4.7.

[0020] In some aspects of the disclosure, the pharmaceutical compositions provided herein comprise: (a) a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof at a concentration of 10 mg / mL; (b) 10 mM glutamate; (c) 40 mM leucine; (d) 3% w / v sucrose; and (e) 0.02% polysorbate-80; wherein the pH of the composition is 4.5.

[0021] In some aspects of the disclosure, the pharmaceutical compositions provided herein comprise a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof, comprising a single chain Fv (scFv).

[0022] In some aspects of the disclosure, the pharmaceutical compositions provided herein comprise a 4-1BBxOX40 bispecific antibody or an antigen-binding fragment thereof, including a Fab, a Fab', a F(ab')2, a scFv, a disulfide-linked Fv, or a scFv-Fc.

[0023] In some embodiments of the disclosure, the pharmaceutical compositions provided herein comprise a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprising a human 4-1BB antigen-binding domain and a human OX40 antigen-binding domain, wherein (a) the 4-1BB antigen-binding domain comprises: (i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable domain (VL)-CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable domain (VL)-CDR1 comprising the amino acid sequence of SEQ ID NO: 5; (b) the OX40 antigen-binding domain comprises (i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:7; (ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:8; (iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:9; (iv) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:10; (v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:11; and (vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:12.

[0024] In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprises a human 4-1BB antigen-binding domain and a human OX40 antigen-binding domain, wherein (a) the 4-1BB antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15; and (b) the OX40 antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18.

[0025] In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprises a human 4-1BB antigen-binding domain and a human OX40 antigen-binding domain, wherein (a) the 4-1BB antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 16, and (b) the OX40 antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 19.

[0026] In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:13.

[0027] In some embodiments of the disclosure, the pharmaceutical compositions provided herein include (a) 10 mg / mL of a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises (i) a 4-1BB antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light chain variable domain (VL)-CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 7; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 8; a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 10; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 11; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 12. R3; (ii) a 4-1BB antigen binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15; and an OX40 antigen binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18; (iii) a 4-1BB antigen binding domain comprising an scFv comprising the amino acid sequence of SEQ ID NO: 16; and (b) an OX40 antigen-binding domain comprising an scFv comprising the amino acid sequence of SEQ ID NO: 19; or (iv) a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO: 13; (b) about 5 mM to about 15 mM glutamate; (c) about 25 mM to about 50 mM leucine; (d) about 2% to about 8% w / v sucrose; and (e) about 0.01% to about 0.03% polysorbate-80; wherein the pH of the composition is about 4.3 to about 4.7.

[0028] In some embodiments of the disclosure, the pharmaceutical compositions provided herein comprise: (a) 10 mg / mL of a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof; (i) a 4-1BB antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3: a light chain variable domain (VL)-CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 7; and (ii) a 4-1BB antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (b) an antibody or antigen-binding fragment thereof comprising an OX40 antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 8; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 9; a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 10; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 11; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 12; (b) 10 mM glutamate; (c) 40 mM leucine; (d) 3% w / v sucrose; and (e) 0.02% polysorbate-80, wherein the pH of the composition is 4.5.

[0029] In some embodiments, the pharmaceutical composition comprises (a) a 4-1BB antigen binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and (b) an OX40 antigen binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18.

[0030] In some aspects of the disclosure, the pharmaceutical compositions provided herein are liquid.

[0031] In some embodiments of the pharmaceutical composition provided herein, human 4-1BB binding domain and human OX40 binding domain are on the same polypeptide.In some embodiments, human 4-1BB binding domain is at the N-terminus of human OX40 binding domain.In some embodiments, human 4-1BB binding domain is at the C-terminus of human OX40 binding domain.

[0032] In some embodiments of the pharmaceutical compositions provided herein, the antibody or antigen-binding fragment thereof comprises an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises the immunoglobulin CH2 and CH3 domains of IgG1. In some embodiments, the antibody does not comprise a CH1 domain. In some embodiments, the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising substitutions E233P, L234A, L235A, G237A, and K322A, and deletion of G236 according to the EU numbering system.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof comprises a linker between the immunoglobulin constant region and the human 4-1BB binding domain, and / or between the immunoglobulin constant region and the human OX40 binding domain. In some embodiments, the linker between the immunoglobulin constant region and the human 4-1BB binding domain, and / or between the immunoglobulin constant region and the human OX40 binding domain comprises 10-30 amino acids, 15-30 amino acids, or 20-30 amino acids. In some embodiments, the linker between the immunoglobulin constant region and the human 4-1BB binding domain, or the linker between the immunoglobulin constant region and the human OX40 binding domain comprises the amino acid sequence (Gly4Ser)n, where n=1-5 (SEQ ID NO:21), optionally n=1. In some embodiments, the antibody comprises a dimer of two polypeptides, each polypeptide comprising, in order from amino terminus to carboxyl terminus, a first scFv, a hinge region, an immunoglobulin constant region, and a second scFv, where (a) the first scFv comprises a human 4-1BB antigen binding domain and the second scFv comprises a human OX40 antigen binding domain, or (b) the first scFv comprises a human OX40 antigen binding domain and the second scFv comprises a human 4-1BB antigen binding domain. In some embodiments, the dimer is a homodimer.

[0034] In some embodiments of the present disclosure, the pharmaceutical compositions provided herein can be stored at -20°C. In some embodiments, the pharmaceutical compositions provided herein have no significant change in pH after about 3 weeks, about 6 weeks, about 3 months, about 6 months, and about 7 months after storage at -20°C. In some embodiments, the pharmaceutical compositions provided herein have no significant change in high molecular weight content (%HMW) after about 3 weeks, about 6 weeks, about 3 months, about 6 months, or about 7 months after storage at -20°C. In some embodiments, the change in %HMW after about 3 weeks, about 6 weeks, about 3 months, about 6 months, or about 7 months after storage at -20°C is less than 5%.

[0035] In some embodiments of the present disclosure, the pharmaceutical compositions provided herein have no significant change in %HMW after 1, 2, or 3 freeze / thaw cycles.In some embodiments, the change in %HMW after 1, 2, or 3 freeze / thaw cycles is less than 5%.In some embodiments, %HMW is measured by size exclusion ultra-high pressure liquid chromatography (SE-UPLC).

[0036] In some aspects of the disclosure, the pharmaceutical compositions provided herein have no significant change in aggregate formation after about 3 weeks, about 6 weeks, about 3 months, about 6 months, or about 7 months of storage at -20°C as measured by size-exclusion ultra-pressure liquid chromatography (SE-UPLC).

[0037] Also provided herein is a method for treating cancer in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is a sarcoma, carcinoma, or lymphoma. In some embodiments, the cancer is selected from the group consisting of melanoma, renal cancer, pancreatic cancer, lung cancer, gastric cancer, colon / intestinal cancer, prostate cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, or blood cancer. In some embodiments, the subject is a human. In some embodiments, the subject expresses 4-1BB and OX40 on tumor-infiltrating lymphocytes.

[0038] In some aspects of the present disclosure, the pharmaceutical compositions provided herein are for use in the treatment of cancer. In some aspects, the pharmaceutical compositions provided herein are for use in the treatment of solid tumor cancer. In some aspects, the solid tumor cancer is a sarcoma, carcinoma, or lymphoma. In some aspects, the cancer is selected from the group consisting of melanoma, renal cancer, pancreatic cancer, lung cancer, colon / intestinal cancer, gastric cancer, prostate cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, or blood cancer. [Brief description of the drawings]

[0039] [Figure 1A] Figure 1 shows Tag analysis of FXX01102 in different protein formulations (see Example 2). [Figure 1B] Figure 1 shows Tag analysis of FXX01102 in different protein formulations (see Example 2). [Diagram 2] FIG. 1 shows the results of a B22 analysis performed on FXX01102 formulated at 10 mg / mL in 10 mM glutamate, 8% w / v sucrose, 0.02% w / v polysorbate-80, with or without 100 mM sodium chloride (see Example 3). [Diagram 3]

[0023] Figure 1 shows the results of a B22 analysis comparing the effect of arginine, methionine, and glycine on 10 mg / mL FXX01102 formulated in 10 mM glutamate, 4% w / v sucrose, pH 4.5 (see Example 4). [Figure 4] 1 shows the results of a B22 experiment to determine the effect of leucine on FXX01102 formulated in 10 mM glutamate, 4% w / v sucrose, pH 4.5 (see Example 4). [Diagram 5] FIG. 1 shows a comparison of Tagg plots of 10 mg / mL FXX01102 formulated in 10 mM glutamate, 4% w / v sucrose, pH 4.5 with or without added 25 mM MgSO4, lysine or leucine (see Example 5). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] To facilitate the understanding of this disclosure, several terms and phrases are defined below. I. Terminology As used herein, the term "4-1BB" refers to mammalian 4-1BB polypeptides, including, but not limited to, naturally occurring 4-1BB polypeptides and isoforms of 4-1BB polypeptides. "4-1BB" encompasses full-length unprocessed 4-1BB polypeptides, as well as forms of 4-1BB polypeptides that result from processing within a cell. As used herein, the term "CD137" is understood to be interchangeable with the term "4-1BB". As used herein, the term "human 4-1BB" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO:1. As used herein, the term "cynomolgus 4-1BB" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO:2. "4-1BB polynucleotide", "4-1BB nucleotide", or "4-1BB nucleic acid" refers to a polynucleotide encoding 4-1BB.

[0041] As used herein, the term "OX40" refers to a mammalian OX40 polypeptide, including, but not limited to, a native OX40 polypeptide and an isoform of the OX40 polypeptide. "OX40" encompasses full-length unprocessed OX40 polypeptides, as well as polypeptide forms of OX40 resulting from processing within a cell. As used herein, the term "human OX40" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO:3. As used herein, the term "cynomolgus OX40" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO:4. "OX40 polynucleotide", "OX40 nucleotide", or "OX40 nucleic acid" refers to a polynucleotide encoding OX40.

[0042] As used herein, the term "tumor infiltrating lymphocyte" or "TIL" refers to lymphocytes that directly fight and / or surround tumor cells.Tumor infiltrating lymphocytes are typically non-circulating lymphocytes, including CD8+ T cells, CD4+ T cells, and NK cells.Tumor infiltrating lymphocytes can express OX40 and 4-1BB.

[0043] As used herein, the term "antibody(s)" is a term of art and may be used interchangeably herein and refers to a molecule or complex of molecules that have at least one antigen-binding site that specifically binds an antigen.

[0044] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (e.g., anti-anti-Id antibodies), bispecific antibodies, and multispecific antibodies. In some embodiments, the antibodies described herein refer to polyclonal antibody populations. An antibody may be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b). In some embodiments, the antibodies described herein are IgG antibodies, or a class (e.g., human IgG1, IgG2, or IgG4) or subclass thereof. In certain embodiments, the antibodies are humanized monoclonal antibodies. In another particular embodiment, the antibodies are human monoclonal antibodies, e.g., immunoglobulins. In some embodiments, the antibodies described herein are IgG1, IgG2, or IgG4 antibodies.

[0045] A "bispecific" antibody is an antibody that has two different antigen-binding sites (excluding the Fc region) that bind to two different antigens. Bispecific antibodies can include, for example, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, heteroconjugate antibodies, linked single chain antibodies or linked single chain Fvs (scFvs), camelized antibodies, affibodies, linked Fab fragments, F(ab')2 fragments, chemically linked Fvs, and disulfide linked Fvs (sdFvs). Bispecific antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG ... 2a or IgG 2b). In some embodiments, the bispecific antibodies described herein are IgG antibodies, or classes (e.g., human IgG1, IgG2, or IgG4) or subclasses thereof. In some embodiments, the bispecific antibodies described herein comprise two polypeptides, optionally identical, each polypeptide comprising, in order from amino terminus to carboxyl terminus, a first scFv antigen-binding domain, a linker (optionally, the linker is a hinge region), an immunoglobulin constant region, and a second scFv antigen-binding domain. This particular type of antibody is exemplified by ADAPTIR™ technology. An exemplary molecule is referred to throughout this disclosure as FXX01102. Bispecific antibodies can be, for example, monovalent for each target (e.g., an IgG molecule with one arm targeted to one antigen and the other arm targeted to a second antigen), or bivalent for each target (e.g., dual variable domain antibodies, IgG-scFv, scFv-Fc-scFv, or ADAPTIR™ antibodies comprising dimers in which each polypeptide of the dimer contains two different antigen-binding domains).

[0046] As used herein, the terms "antigen-binding domain", "antigen-binding region", "antigen-binding site", and similar terms refer to a portion of an antibody molecule (e.g., a complementarity determining region (CDR)) that contains amino acid residues that confer specificity to the antibody molecule for an antigen. The antigen-binding region may be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans. An antigen-binding domain that binds 4-1BB may be referred to herein, for example, as a "4-1BB-binding domain". An antigen-binding domain that binds OX40 may be referred to herein, for example, as an "OX40-binding domain". As used herein, a "human 4-1BB-binding domain" or a "human 4-1BB antigen-binding domain" refers to an antigen-binding domain that specifically binds human 4-1BB but may also bind non-human 4-1BB (e.g., mouse, rodent, or non-human primate 4-1BB).

[0047] Similarly, a "human OX40 binding domain" or a "human OX40 antigen-binding domain" refers to an antigen-binding domain that specifically binds to human OX40.

[0048] As used herein, the term "4-1BB / OX40 antibody," "anti-4-1BB / OX40 antibody," or "4-1BBxOX40 antibody" refers to a bispecific antibody that contains an antigen-binding domain that binds 4-1BB (e.g., human 4-1BB) and an antigen-binding domain that binds OX40 (e.g., human OX40).

[0049] A "monoclonal" antibody refers to a homogeneous antibody population that is involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to a polyclonal antibody, which typically contains different antibodies directed against different antigenic determinants. The term "monoclonal" antibody encompasses both intact and full-length immunoglobulin molecules, Fab, Fab', F(ab')2, Fv), single chain (scFv), fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule that contains an antigen recognition site. Furthermore, a "monoclonal" antibody refers to an antibody made in any number of ways, including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0050] The term "chimeric" antibody refers to an antibody whose amino acid sequences are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to the sequences of an antibody derived from another species (usually human) to avoid eliciting an immune response in that species.

[0051] The term "humanized" antibody refers to a form of a non-human (e.g., mouse) antibody that contains minimal non-human (e.g., mouse) sequence. Typically, humanized antibodies are human immunoglobulins in which residues of the complementarity determining regions (CDRs) have been replaced ("CDR-grafted") with residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) that has the desired specificity, affinity, and capacity (Jones et al., Nature 321:522-525 (1986); Riechman et al., Nature 332:323-327 (1988); Verhoeye et al., Science 239:1534-1536 (1988)).

[0052] In some cases, Fv framework region (FR) residues of a human immunoglobulin are replaced with corresponding residues in an antibody from a non-human species having the desired specificity, affinity, and capacity. The humanized antibody can be further modified by substitution of additional residues in the Fv framework regions and / or within the replaced non-human residues to improve and optimize the antibody's specificity, affinity, and / or capacity. Generally, a humanized antibody contains substantially all of at least one, typically two or three, variable domains, including all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, but all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody can also contain at least a portion of an immunoglobulin constant region or domain (Fc), typically a human immunoglobulin constant region or domain (Fc). Examples of methods used to generate humanized antibodies are described in U.S. Pat. No. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996).

[0053] The term "human" antibody means an antibody having an amino acid sequence derived from the human immunoglobulin locus, such an antibody being made using any technique known in the art.

[0054] The variable region typically refers to a portion of an antibody, generally a light or heavy chain, typically about the amino terminal 110-125 amino acids in the mature heavy chain and about the amino terminal 90-115 amino acids in the mature light chain, which vary widely in sequence between antibodies and are used in the binding and specificity of a particular antibody to a particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions within the variable domain are called framework regions (FRs). Without being bound to any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0055] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0056] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0057] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding portion. In some embodiments, the CDRs of an antibody can be determined according to the Kabat numbering system (see, for example, Kabat EA&Wu TT(1971)Ann NY Acad Sci 190:382-391 and Kabat EA et al.,(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,USDepartment of Health and Human Services,NIH Publication No.91-3242). Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acid positions 31-35 (which can optionally include one or two additional amino acids after 35, referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3).

[0058] Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.

[0059] Chothia refers instead to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because in the Kabat numbering scheme, the insertions are placed at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). In certain embodiments, the CDRs of the antibodies described herein are determined according to the Chothia numbering scheme.

[0060] The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. In certain embodiments, the CDRs of the antibodies described herein are determined according to the AbM numbering scheme. [Table 1]

[0061] The IMGT numbering convention is described in Brochet, X, et al, Nucl.Acids Res.36:W503-508 (2008). In certain embodiments, the CDRs of the antibodies described herein are determined according to the IMGT numbering convention. As used herein, unless otherwise specified, the position of amino acid residues in the variable region of an immunoglobulin molecule is numbered according to the IMGT numbering convention.

[0062] As used herein, the terms "constant region" or "constant domain" are interchangeable and have common meanings in the art. A constant region is an antibody portion, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding an antibody to an antigen, but can exhibit various effector functions, such as interacting with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain. An immunoglobulin "constant region" or "constant domain" may include a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, or a subset of these domains, e.g., a CH2 domain and a CH3 domain. In some embodiments provided herein, the immunoglobulin constant region does not include a CH1 domain. In some embodiments provided herein, the immunoglobulin constant region does not include a hinge. In some embodiments provided herein, the immunoglobulin constant region includes a CH2 domain and a CH3 domain.

[0063] "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from the portion of a source antibody responsible for binding to the antibody receptor on cells and the C1q component of complement. Fc stands for "crystal fragment" and refers to a fragment of an antibody that readily forms protein crystals. The individual protein fragments first described by proteolytic digestion can define the overall general structure of an immunoglobulin protein. "Fc region" or "Fc domain" includes the CH2 domain, the CH3 domain, and optionally all or part of the hinge. "Fc region" or "Fc domain" can refer to a single polypeptide or two disulfide-linked polypeptides. For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49- 140; and Padlan, Mol. Immunol. 31:169-217, 1994. As used herein, the term Fc includes variants of the naturally occurring sequence.

[0064] As used herein, an "immunoglobulin dimerization domain" or "immunoglobulin heterodimerization domain" refers to an immunoglobulin domain of a polypeptide chain that preferentially interacts or associates with a different immunoglobulin domain of a second polypeptide chain, where the interaction of the different immunoglobulin heterodimerization domains substantially contributes to or efficiently promotes heterodimerization of the first and second polypeptide chains (i.e., the formation of a dimer between two different polypeptide chains, also called a "heterodimer"). An interaction between heterodimerization domains of immunoglobulins "substantially contributes to or efficiently promotes" heterodimerization of the first and second polypeptide chains if there is a statistically significant decrease in dimerization between the first and second polypeptide chains in the absence of the immunoglobulin heterodimerization domain of the first polypeptide chain and / or the immunoglobulin heterodimerization domain of the second polypeptide chain. In some embodiments, when the first and second polypeptide chains are co-expressed, at least 60%, at least about 60% to about 70%, at least about 70% to about 80%, at least 80% to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the first and second polypeptide chains form heterodimers with each other. Exemplary immunoglobulin heterodimerization domains include an immunoglobulin CH1 domain, an immunoglobulin CL domain (e.g., Cκ or Cλ isotypes), or derivatives thereof, such as wild-type immunoglobulin CH1 and CL domains provided herein, as well as modified (or mutated) immunoglobulin CH1 and CL domains.

[0065] "Wild-type immunoglobulin hinge region" refers to the naturally occurring upper and middle hinge amino acid sequences inserted between and connected to the CH1 and CH2 domains (for IgG, IgA, and IgD) or between and connected to the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of a naturally occurring antibody. In some embodiments, the wild-type immunoglobulin hinge region sequence is human and can include a human IgG hinge region. A "modified wild-type immunoglobulin hinge region" or "modified immunoglobulin hinge region" refers to (a) a wild-type immunoglobulin hinge region having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (b) a length of from about 5 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) up to about 120 amino acids (e.g., from about 10 to about 40 amino acids). "hinge region" refers to a portion of a hinge region of a wild-type immunoglobulin having a length of about 15 to about 30 amino acids, or about 15 to about 20 amino acids, or about 20 to about 25 amino acids, up to about 30% amino acid changes (e.g., up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% amino acid substitutions or deletions, or combinations thereof), and having an IgG core hinge region as disclosed in US 2013 / 0129723 and US 2013 / 0095097. As provided herein, a "hinge region" or "hinge" can be located between an antigen binding domain (e.g., a 4-1BB or OX40 binding domain) and an immunoglobulin constant region.

[0066] As used herein, a "linker" refers to a moiety, e.g., a polypeptide, that can link two compounds, e.g., two polypeptides. Non-limiting examples of linkers include flexible linkers that include glycine-serine (e.g., (Gly4Ser)) repeats, and linkers derived from (a) the interdomain region of a transmembrane protein (e.g., a type I transmembrane protein); (b) the stalk region of a type II C-lectin; or (c) an immunoglobulin hinge. As provided herein, a linker can refer to, for example, (1) the polypeptide region between the VH and VL regions in a single chain Fv (scFv), or (2) the polypeptide region between an immunoglobulin constant region and an antigen-binding domain. In some embodiments, the linker is composed of 5 to about 35 amino acids, e.g., about 15 to about 25 amino acids. In some embodiments, the linker is composed of at least 5 amino acids, at least 7 amino acids, or at least 9 amino acids.

[0067] As used herein, the term "heavy chain," when used in reference to an antibody, can refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant region, which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, e.g., subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4.

[0068] As used herein, the term "light chain" when used in reference to an antibody can refer to any of the different types based on the amino acid sequence of the constant region, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In certain aspects, the light chain is a human light chain.

[0069] As used herein, the term "EU numbering system" refers to the EU numbering rules for the constant region of an antibody as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety. As used herein, unless otherwise specified, the positions of amino acid residues in the constant region of an immunoglobulin molecule are numbered according to the EU nomenclature (Ward et al., 1995 Therap. Immunol. 2:77-94).

[0070] As used herein, the term "dimer" refers to a biological entity consisting of two subunits bound to each other through one or more forms of intramolecular forces, such as covalent bonds (e.g., disulfide bonds) and other interactions (e.g., electrostatic interactions, salt bridges, hydrogen bonds, and hydrophobic interactions), and is stable under appropriate conditions (e.g., under physiological conditions, in aqueous solutions suitable for recombinant protein expression, purification, and / or storage, or under non-denaturing and / or non-reducing electrophoretic conditions). As used herein, a "heterodimer" or "heterodimeric protein" refers to a dimer formed from two different polypeptides. As used herein, a "homodimer" or "homodimeric protein" refers to a dimer formed from two identical polypeptides.

[0071] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the inherent binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for a partner Y is generally determined by the dissociation constant (K DAffinity can be expressed by, but not limited to, the equilibrium dissociation constant (K D ) and the equilibrium binding constant (K A ) can be measured and / or expressed in a number of ways known in the art. D is k off / k on It is calculated from the quotient of K A is k on / k off It is calculated from the quotient of k on refers to the binding rate constant of, for example, an antibody to an antigen, and k off refers to, for example, the dissociation of an antibody from an antigen. on and k off can be determined by techniques known to those skilled in the art, such as BIAcore® or KinExA.

[0072] As used herein, the terms "immunospecifically bind", "immunospecifically recognize", "specifically bind" and "specifically recognize" are similar terms in the context of antibodies. These terms indicate that the antibody binds to an epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. Thus, an antibody that "specifically binds" to human 4-1BB and / or OX40 may also bind to unrelated non-4-1BB and / or OX40 proteins to an extent that is less than about 10% of the antibody's binding to 4-1BB and / or OX40, as measured, for example, by radioimmunoassay (RIA).

[0073] Binding domains can be classified as "high affinity" binding domains and "low affinity" binding domains. -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 K under M D A "low affinity" binding domain refers to a binding domain having a binding affinity of 10 -7 Greater than M, 10 -6 Greater than M or 10-5 "High affinity" and "low affinity" binding domains refer to binding domains that have a KD greater than M. "High affinity" and "low affinity" binding domains bind to a target but do not bind significantly to other components present in the test sample.

[0074] As used herein, an antibody is "capable of binding" if it specifically binds to its target (i.e., human 4-1BB or human OX40) under conditions that would be considered necessary for binding by a person skilled in the art when in close proximity to the target. "Human 4-1BB antigen binding domain" is understood to mean the binding domain that specifically binds to human 4-1BB. "Human OX40 antigen binding domain" is understood to mean the binding domain that specifically binds to OX40.

[0075] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified, naturally or by intervention, for example, by formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, such as unnatural amino acids, as well as other modifications known in the art. Because the polypeptides of the present invention are based on antibodies, it is understood that in some embodiments the polypeptides can occur as single chains or linked chains.

[0076] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation in a form such that the biological activity of the active ingredient is effective and does not contain additional ingredients that have unacceptable toxicity to the subject to which the formulation is administered. The formulation may be sterile.

[0077] As used herein, the term "pharmacologically acceptable" refers to molecular entities and compositions that do not generally produce allergic responses or other serious side effects when administered using routes well known in the art. Molecular entities and compositions approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, particularly humans, are considered to be "pharmacologically acceptable."

[0078] As used herein, the terms "administer", "administering", "administration" and the like refer to methods (e.g., intravenous administration) that can be used to allow delivery of a drug, such as a 4-1BB / OX40 antibody or antigen-binding fragment thereof, to a desired site of biological action. Administration techniques that can be used with the agents and methods described herein can be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0079] As used herein, the terms "subject" and "patient" are used interchangeably. A subject may be an animal. In some embodiments, a subject is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate, etc.). In some embodiments, a subject is a human. As used herein, the term "patient in need" or "subject in need" refers to a patient at risk for or suffering from a disease, disorder, or condition that is amenable to treatment or amelioration with a 4-1BB / OX40 antibody or antigen-binding fragment thereof provided herein. A patient in need may be, for example, a patient diagnosed with cancer.

[0080] The term "therapeutically effective amount" refers to an amount of a drug, such as an anti-4-1BB / OX40 antibody, that is effective to treat a disease or disorder in a subject. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size or burden of a tumor; inhibit (i.e., delay to some extent, in some embodiments, stop) the infiltration of cancer cells into peripheral organs; inhibit (i.e., delay to some extent, in some embodiments, stop) the metastasis of a tumor; inhibit tumor growth to some extent; relieve to some extent one or more symptoms associated with cancer; and / or result in a favorable response, such as an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), a complete response (CR), a partial response (PR), or in some cases, stable disease (SD), a decrease in progressive disease (PD), a decrease in time to progression (TTP), or any combination thereof.

[0081] The terms "treat" or "treatment" or "to treat" or "palliating" or "alleviating" and the like refer to a therapeutic measure that cures, slows, relieves symptoms, and / or halts the progression of a diagnosed pathological condition or disorder. Thus, treatment is needed when a patient has already been diagnosed with the disorder or is suspected of having the disorder. In some embodiments, a subject has responded to "treatment" of cancer according to the methods of the invention if the patient exhibits one or more of the following: a reduction in the number or complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell invasion into peripheral organs, e.g., spread of cancer to soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; a reduction in one or more symptoms associated with a particular cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in the tumorigenicity, tumorigenic frequency, or tumorigenic potential of a tumor; a reduction in the number or frequency of cancer stem cells within a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), a complete response (CR), a partial response (PR), stable disease (SD), a reduction in progressive disease (PD), a shortened time to progression (TTP), or any combination thereof.

[0082] The term "cancer" or "cancerous" refers to or describes a physiological condition in a mammal in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, melanoma, renal cancer, pancreatic cancer, lung cancer, intestinal cancer, prostate cancer, breast cancer, liver cancer, brain cancer, and blood cancer. Cancer may be a primary tumor, or an advanced or metastatic cancer.

[0083] The cancer may be a solid tumor cancer. The term "solid tumor" refers to an abnormal mass of tissue that does not usually contain cysts or liquid areas. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (blood cancers) generally do not form solid tumors. Solid tumors may contain tumor-infiltrating lymphocytes that express OX40 and 4-1BB.

[0084] As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the listed components, unless otherwise indicated.

[0085] Unless otherwise stated or clear from the context, as used herein, the term "or" is understood to be inclusive. The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both "A and B", "A or B", "A" and "B". Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0086] Wherever an embodiment is described herein using the language "comprising," it is understood that analogous embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided and are part of the disclosure of this application. In this disclosure, "comprises," "comprising," "containing," and "having" and the like may have the meanings given to them in U.S. and European patent law, and may also mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise have the meanings given to them in U.S. and European patent law. This term is open ended, and more than what is recited may be present, but prior art embodiments are excluded, so long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited. Also, insofar as European patent law is concerned, the use of "consisting essentially of" or "comprising substantially" should be understood to mean that certain additional components may be present, i.e., components that do not substantially affect the essential properties of the compound or composition.

[0087] As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate a deviation of up to 5% above or below that value or range while remaining within the intended meaning of the recited value or range.

[0088] Any domain, component, composition, and / or method provided herein can be combined with one or more of any of the other domains, components, compositions, and / or methods provided herein.

[0089] II. Pharmaceutical Compositions The present disclosure provides novel pharmaceutical compositions comprising 4-1BBxOX40 bispecific antibodies or antigen-binding fragments thereof, which have improved stability compared to known formulations. The novel pharmaceutical compositions provided herein inhibit aggregate formation over time during storage at -20°C and after multiple freeze-thaw cycles. The novel pharmaceutical compositions of the present disclosure comprise 4-1BBxOX40 bispecific antibodies or antigen-binding fragments thereof, which are formulated with specific excipients (i.e., stability-promoting buffers, stabilizing amino acids, sugar moieties, and surfactants) to improve stability. Advantageously, the novel pharmaceutical compositions provide improved stability over a range of pH values.

[0090] For therapeutic formulations, solution conditions that stabilize the secondary and tertiary structure of the protein are desirable to minimize the formation of aggregation or degradation products. In the case of therapeutic proteins used to treat disease, such as the 4-1BBxOX40 bispecific antibody or antigen-binding fragments thereof, product-associated contaminants can have undesirable consequences, such as loss of efficacy, unexpected increase in efficacy, immunogenicity and other undesirable side effects. The formulations may be composed of several components and are typically provided in aqueous solutions. Such components may include, but are not limited to, buffers to maintain a particular pH and resist changes in pH during storage, salts, amino acids, detergents, polymers, sugars, and other chemical excipients such as surfactants that serve to maintain the stability or solubility of the drug or to incorporate other desirable properties into the solution. Such components may have a positive or negative charge, may be zwitterionic, or may be amphipathic, hydrophobic or hydrophilic. Different components may bind to, interact with, and / or stabilize different regions of the protein based on their chemical properties.

[0091] Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, for example in liquid carriers, or suitable for reconstitution into liquid solutions or suspensions for intravenous or subcutaneous administration. Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravenous, intraperitoneal, intramuscular, intrathecal and subcutaneous. In some aspects, the pharmaceutical formulations disclosed herein are lyophilized before administration. In some aspects, the pharmaceutical formulations disclosed herein are not lyophilized before administration.

[0092] The present disclosure provides novel pharmaceutical compositions comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof. In some embodiments, the present disclosure provides (a) a bispecific antibody or antigen-binding fragment thereof that specifically binds 4-1BB and OX40; (b) about 5 mM to about 15 mM of a stability-promoting buffer; (c) about 25 mM to about 50 mM of a stabilizing amino acid; (d) about 2% to about 8% w / v of a sugar; and (e) about 0.01% to about 0.03% of a surfactant.

[0093] In some embodiments, the pharmaceutical composition comprising the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof disclosed herein comprises a stability-promoting buffer selected from the group consisting of succinate, histidine, citrate, and glutamate. In some embodiments, the stability-promoting buffer is present in the composition at a concentration of about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or about 15 mM. In some embodiments, the stability-promoting buffer is glutamate. In some embodiments, the glutamate is present in the composition at a concentration of about 5 mM.

[0094] In some embodiments, the pharmaceutical composition comprising the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof disclosed herein comprises a stabilizing amino acid selected from the group consisting of arginine, methionine, leucine, and glycine. In some embodiments, the stabilizing amino acid is present in the composition at a concentration of about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, or about 50 mM.

[0095] In some embodiments, the stabilizing amino acid is leucine, hi some embodiments, leucine is present in the composition at a concentration of about 40 mM.

[0096] In some embodiments, the pharmaceutical composition comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof disclosed herein comprises a sugar selected from the group consisting of sucrose, trehalose, and sorbitol. In some embodiments, the sugar is present in the composition at a concentration of about 2% w / v to about 8% w / v. In some embodiments, the sugar is present in the composition at a concentration of about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, or about 8% w / v. In some embodiments, the sugar is sucrose. In some embodiments, the sucrose is present in the composition at a concentration of about 3% w / v.

[0097] In some embodiments, the pharmaceutical composition comprising the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof disclosed herein comprises a surfactant. In some embodiments, the surfactant is present in the composition at a concentration of about 0.01% w / v to about 0.03% w / v. In some embodiments, the surfactant is polysorbate-80. In some embodiments, the polysorbate-80 is present in the composition at a concentration of about 3% w / v.

[0098] In some embodiments, the pharmaceutical composition comprising the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof disclosed herein provides enhanced stability within a particular pH range. In some embodiments, the pH of the composition is in the range of about 4.3 to about 4.7. In some embodiments, the pH of the composition is about 4.5. In some embodiments, the pH of the composition is 4.5.

[0099] In some embodiments of the pharmaceutical compositions disclosed herein, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof is present in the composition at a concentration of about 10-50 mg / mL. In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof is present in the composition at a concentration of about 10 mg / mL. In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof is present in the composition at a concentration of about 20 mg / mL. In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof is present in the composition at a concentration of about 30 mg / mL. In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof is present in the composition at a concentration of about 40 mg / mL. In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof is present in the composition at a concentration of about 50 mg / mL.

[0100] The addition of sodium chloride (NaCl) to a pharmaceutical composition comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof formulated with glutamate, sucrose, and polysorbate-80 can have a destabilizing effect on the composition and can promote protein-protein interactions that can cause protein aggregation. Thus, in some embodiments, the pharmaceutical compositions disclosed herein do not contain sodium chloride (NaCl).

[0101] In some embodiments of the pharmaceutical compositions disclosed herein, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof has a purity of greater than about 90%. In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof has a purity of greater than about 91%. In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof has a purity of greater than about 92%. In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof has a purity of greater than about 93%. In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof has a purity of greater than about 94%. In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof has a purity of greater than about 95%.

[0102] In some aspects, provided herein is a pharmaceutical composition comprising: (a) a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof at a concentration of about 10 mg / mL; (b) about 10 mM glutamate; (c) about 40 mM leucine; (d) about 3% w / v sucrose; and (e) about 0.02% polysorbate-80; wherein the pH of the composition is about 4.3 to about 4.7.

[0103] In some aspects, provided herein is a pharmaceutical composition comprising: (a) a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof at a concentration of 10 mg / mL; (b) 10 mM glutamate; (c) 40 mM leucine; (d) 3% w / v sucrose; and (e) 0.02% polysorbate-80; wherein the pH of the composition is 4.5.

[0104] In general, such compositions can typically further comprise a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a government regulatory agency for use in animals, particularly humans, or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias. Such pharmaceutical carriers include, but are not limited to, sterile liquids such as water and oils, for example, those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, and the like. Water, or aqueous saline solutions, dextrose and glycerol solutions can be used as carriers, particularly for injectable solutions.

[0105] III.4-1BBxOX40 bispecific antibody Provided herein is a novel pharmaceutical composition comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof, which comprises an antigen-binding domain that specifically binds to human 4-1BB (i.e., human 4-1BB antigen-binding domain) and an antigen-binding domain that binds to human OX40 (i.e., human OX40 antigen-binding domain). The 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein is bivalent for both target proteins, i.e., the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprises two 4-1BB binding domains and two OX40 binding domains. Such a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof is exemplified by ADAPTIR™ technology. An exemplary molecule is referred to as FXX01102 throughout this disclosure. Bispecific antibodies or antigen-binding fragments thereof that are bivalent for each target include, but are not limited to, dual variable domain antibodies, IgG-scFv, scFv-Fc-scFv, or ADAPTIR™ antibodies, including dimers, in which each polypeptide of the dimer includes two different antigen-binding domains. In some embodiments, the 4-1BBxOX40 bispecific antibodies or antigen-binding fragments thereof described herein include single chain Fvs (scFvs). In some embodiments, the 4-1BBxOX40 bispecific antibodies or antigen-binding fragments thereof described herein include Fab, Fab', F(ab')2, scFv, disulfide-linked Fv, or scFv-Fc.

[0106] A.4-1BB binding domain Provided herein is an antigen-binding domain (i.e., 4-1BB binding domain) that binds to human 4-1BB, which can be used to assemble a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof. In addition to binding to human 4-1BB, the 4-1BB binding domain can bind to 4-1BB from other species, such as cynomolgus monkeys, and / or mouse 4-1BB. In some embodiments, the 4-1BB binding domain binds to human 4-1BB and cynomolgus monkey 4-1BB.

[0107] The 4-1BB binding domain can include six complementarity determining regions (CDRs): variable heavy chain (VH) CDR1, VH CDR2, VH CDR3, variable light chain (VL) CDR1, VL CDR2, and VL CDR3. The 4-1BB binding domain can include a variable heavy chain (VH) and a variable light chain (VL). The VH and VL can be separate polypeptides or can be present on the same polypeptide (e.g., in an scFv).

[0108] In some embodiments, the 4-1BB binding domain described herein comprises a combination of the six CDRs provided in Table A (eg, SEQ ID NOs: 1-6). [Table 2] [Table 3]

[0109] A 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof that is bivalent for 4-1BB can comprise two 4-1BB binding domains, each of which comprises the six CDRs listed in Tables A and B above.

[0110] In some embodiments of the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein, the 4-1BB binding domain comprises a VH sequence provided in Table C. [Table 4]

[0111] A 4-1BB binding domain as described herein may comprise a VH that includes the CDRs of a VH sequence in Table C, e.g., the IMGT-defined CDRs, the Kabat-defined CDRs, the Chothia-defined CDRs, or the AbM-defined CDRs.

[0112] In some embodiments of the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein, the 4-1BB binding domain comprises a VL sequence provided in Table D. [Table 5]

[0113] A 4-1BB binding domain described herein may comprise a VL that includes the CDRs of a VL sequence in Table D, for example the IMGT-defined CDRs, the Kabat-defined CDRs, the Chothia-defined CDRs, or the AbM-defined CDRs.

[0114] In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein can comprise two 4-1BB binding domains, each comprising a VH provided in Table C and a VL provided in Table D. The VH provided in Table C and the VL provided in Table D can be on different polypeptides or on the same polypeptide. When the VH and VL are on the same polypeptide, they can be in either orientation (i.e., VH-VL or VL-VH) and can be connected by a linker (e.g., a glycine-serine linker). In some embodiments, the VH and VL are connected by a glycine-serine linker that is at least 15 amino acids in length (e.g., 15-50 amino acids, 15-40 amino acids, 15-30 amino acids, 15-25 amino acids, or 15-20 amino acids). In some embodiments, the VH and VL are connected by a glycine-serine linker that is at least 20 amino acids in length (eg, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, or 20 to 25 amino acids).

[0115] In some embodiments of the 4-1BBxOX40 bispecific antibodies or antigen-binding fragments described herein, the 4-1BB binding domain can comprise a VH that includes the CDRs of a VH sequence of Table C, e.g., the IMGT-defined CDRs, the Kabat-defined CDRs, the Chothia-defined CDRs, or the AbM-defined CDRs, and a VL that includes the CDRs of a VL sequence of Table D, e.g., the IMGT-defined CDRs, the Kabat-defined CDRs, the Chothia-defined CDRs, or the AbM-defined CDRs.

[0116] B. OX40-binding domain Provided herein is an antigen-binding domain (i.e., OX40-binding domain) that can be used to assemble 4-1BBxOX40 bispecific antibody or its antigen-binding fragment and bind to human OX40.In addition to binding to human OX40, the OX40-binding domain can also bind to OX40 from other species, such as cynomolgus monkey and / or mouse OX40.In some embodiments, the OX40-binding domain binds to human OX40 and cynomolgus monkey OX40.

[0117] The OX40 binding domain can include six complementarity determining regions (CDRs): variable heavy chain (VH) CDR1, VH CDR2, VH CDR3, variable light chain (VL) CDR1, VL CDR2, and VL CDR3.The OX40 binding domain can include a variable heavy chain (VH) and a variable light chain (VL).VH and VL can be separate polypeptides or can be present on the same polypeptide (e.g., in scFv).

[0118] In some embodiments, the OX40 binding domain described herein comprises the six CDRs listed in Tables E and F. [Table 6] [Table 7]

[0119] A 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof that is bivalent for OX40 can comprise two OX40 binding domains, each of which comprises the six CDRs listed in Tables E and F above.

[0120] In some embodiments of the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein, the OX40 binding domain comprises a VH sequence shown in Table G. [Table 8]

[0121] An OX40 binding domain as described herein can comprise a VH that includes the CDRs of a VH sequence in Table G, for example, the IMGT-defined CDRs, the Kabat-defined CDRs, the Chothia-defined CDRs.

[0122] In some embodiments of the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein, the OX40 binding domain comprises a VL sequence shown in Table H. [Table 9]

[0123] An OX40 binding domain described herein can comprise a VL that includes the CDRs of a VL sequence in Table I, for example, the IMGT-defined CDRs, the Kabat-defined CDRs, the Chothia-defined CDRs, or the AbM-defined CDRs.

[0124] In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein can comprise two OX40 binding domains, each comprising a VH provided in Table G and a VL provided in Table H. The VH provided in Table G and the VL provided in Table H can be on different polypeptides or on the same polypeptide. When the VH and VL are on the same polypeptide, they can be in either orientation (i.e., VH-VL or VL-VH) and can be connected by a linker (e.g., a glycine-serine linker). In some embodiments, the VH and VL are connected by a glycine-serine linker that is at least 15 amino acids in length (e.g., 15-50 amino acids, 15-40 amino acids, 15-30 amino acids, 15-25 amino acids, or 15-20 amino acids). In some embodiments, the VH and VL are connected by a glycine-serine linker that is at least 20 amino acids in length (eg, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, or 20 to 25 amino acids).

[0125] In some embodiments of the 4-1BBxOX40 bispecific antibodies or antigen-binding fragments described herein, the OX40 binding domain can comprise a VH that includes the CDRs of a VH sequence of Table G, e.g., the IMGT-defined CDRs, the Kabat-defined CDRs, the Chothia-defined CDRs, or the AbM-defined CDRs, and a VL that includes the CDRs of a VL sequence of Table H, e.g., the IMGT-defined CDRs, the Kabat-defined CDRs, the Chothia-defined CDRs, or the AbM-defined CDRs.

[0126] C.4-1BB and / or OX40 binding domain In the 4-1BB or OX40 binding domain, the VH CDR or VH and the VL CDR or VL may be separate polypeptides or may be on the same polypeptide. If the VH CDR or VH and the VL CDR or VL are on the same polypeptide, they may be in either orientation (i.e., VH-VL or VL-VH).

[0127] When the VH CDR or VH and the VL CDR or VL are on the same polypeptide, they can be connected by a linker (e.g., a glycine-serine linker). The VH can be located at the N-terminus of the linker sequence, and the VL can be located at the C-terminus of the linker sequence. Alternatively, the VL can be located at the N-terminus of the linker sequence, and the VH can be located at the C-terminus of the linker sequence.

[0128] The use of peptide linkers to join VH and VL regions is well known in the art, and there are numerous publications in this particular field. In some embodiments, the peptide linker is a 15-mer consisting of three repeats of the amino acid sequence Gly-Gly-Gly-Gly-Ser ((Gly4Ser)3) (SEQ ID NO: 20). In some embodiments, a suitable linker can be obtained by optimizing a simple linker (e.g., (Gly4Ser)n) (n=1-5 (SEQ ID NO: 21)) by random mutagenesis.

[0129] The 4-1BB and / or OX40 binding domains may be humanized binding domains. The 4-1BB and / or OX40 binding domain may be a rat binding domain. The 4-1BB and / or OX40 binding domain may be a mouse binding domain. In some embodiments, the 4-1BBxOX40 bispecific antibody comprises a humanized 4-1BB binding domain and a rat OX40 binding domain. In some embodiments, the 4-1BBxOX40 bispecific antibody comprises a humanized 4-1BB binding domain and a mouse OX40 binding domain. In some embodiments, the 4-1BBxOX40 bispecific antibody comprises a humanized 4-1BB binding domain and a humanized OX40 binding domain.

[0130] In some embodiments of the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein, the 4-1BB and / or OX40 binding domain can be scFv. In some embodiments, all of the 4-1BB and OX40 binding domains in the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof are scFv. In some embodiments, at least one of the 4-1BB or OX40 binding domains in the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof is scFv. In some embodiments of the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein, the polypeptide comprises a 4-1BB binding domain (e.g., scFv) and an OX40 binding domain (e.g., scFv).

[0131] In some embodiments of the 4-1BBxOX40 bispecific antibodies or antigen-binding fragments described herein, the 4-1BB scFv comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments of the 4-1BBxOX40 bispecific antibodies or antigen-binding fragments described herein, the OX40 scFv comprises the amino acid sequence of SEQ ID NO: 19.

[0132] In some embodiments, the 4-1BB and / or OX40 binding domains can comprise VH and VL on separate polypeptide chains. In some embodiments, all of the 4-1BB and OX40 binding domains in the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprise VH and VL on separate polypeptide chains. In some embodiments, the 4-1BB and / or OX40 binding domains can comprise VH and VL on the same polypeptide chain. In some embodiments, all of the 4-1BB or OX40 binding domains in the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprise VH and VL on the same polypeptide chain.

[0133] In some embodiments of the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprises two polypeptides, each polypeptide comprising, in order from amino terminus to carboxyl terminus, a first antigen-binding domain, a linker (e.g., the linker is a hinge region), an immunoglobulin constant region, and a second antigen-binding domain. This configuration is also referred to herein as ADAPTIR™ format. An exemplary molecule is referred to throughout this disclosure as FXX01102.

[0134] In such a format, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprises a dimer of two polypeptides, each polypeptide comprising, in order from amino terminus to carboxyl terminus, a first scFv, a hinge region, an immunoglobulin constant region, and a second scFv, where (a) the first scFv comprises a human 4-1BB antigen-binding domain and the second scFv comprises a human OX40 antigen-binding domain, or (b) the first scFv comprises a human OX40 antigen-binding domain and the second scFv comprises a human 4-1BB antigen-binding domain.

[0135] The antibody or antigen-binding fragment thereof provided herein, or a polypeptide comprising any of the CDRs, VH, VL, scFv, and / or hinges provided herein, may further comprise an immunoglobulin constant region. The presence of the constant region increases the half-life of the bispecific antibody compared to a similar bispecific antibody without a constant region. The immunoglobulin constant region may be located, for example, between the hinge and the 4-1BB binding domain (e.g., 4-1BB binding scFv). The immunoglobulin constant region may also be located between the hinge and the OX40 binding domain (e.g., OX-40 binding scFv). In some embodiments, the polypeptide comprises, in order from amino terminus to carboxyl terminus, a hinge region, an immunoglobulin constant region, and an antigen-binding domain (e.g., scFv).

[0136] In some embodiments, the immunoglobulin constant region comprises the immunoglobulin CH2 and CH3 domains of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgD, and optionally, the IgG is human. In some cases, the immunoglobulin constant region comprises the immunoglobulin CH2 and CH3 domains of IgG1 (e.g., human IgG1). In some embodiments, the polypeptide does not comprise a CH1 domain.

[0137] In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein comprises 4-1BB VH CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 1-3, respectively, 4-1BB VL CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 4-6, respectively, OX40 VH CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 7-9, respectively, and OX40 VL CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 10-12, respectively.

[0138] In some embodiments, the human 4-1BB binding domain and the human OX40 binding domain are on the same polypeptide.In some embodiments, the human 4-1BB binding domain is at the N-terminus of the human OX40 binding domain.In some embodiments, the human 4-1BB binding domain is at the C-terminus of the human OX40 binding domain.In some embodiments, the immunoglobulin constant region comprises the immunoglobulin CH2 and CH3 domains of IgG1.

[0139] In some embodiments, the antibody does not comprise a CH1 domain.

[0140] In some embodiments, the immunoglobulin constant region comprises a human IgG1 CH2 domain comprising substitutions E233P, L234A, L235A, G237A, and K322A, and a deletion of G236 according to the EU numbering system. In some embodiments, the antibody or antigen-binding fragment thereof comprises a linker between the immunoglobulin constant region and the human 4-1BB binding domain and / or between the immunoglobulin constant region and the human OX40 binding domain. In some embodiments, the linker between the immunoglobulin constant region and the human 4-1BB binding domain and / or between the immunoglobulin constant region and the human OX40 binding domain comprises 10-30 amino acids, 15-30 amino acids, or 20-30 amino acids. In some embodiments, the linker between the immunoglobulin constant region and the human 4-1BB binding domain, or the linker between the immunoglobulin constant region and the human OX40 binding domain, comprises the amino acid sequence (Gly4Ser)n, where n=1 to 5 (SEQ ID NO: 21), optionally n=1.

[0141] D. Compositions Comprising 4-1BB and OX40 Binding Domains In some embodiments, the pharmaceutical compositions described herein include a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprising a dimer of two polypeptides, each polypeptide comprising, in order from amino terminus to carboxyl terminus, a first scFv, a hinge region, an immunoglobulin constant region, and a second scFv, wherein (a) the first scFv comprises a human 4-1BB antigen-binding domain and the second scFv comprises a human OX40 antigen-binding domain, or (b) the first scFv comprises a human OX40 antigen-binding domain and the second scFv comprises a human 4-1BB antigen-binding domain. In some embodiments, the dimer is a homodimer.

[0142] In some embodiments, the 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof described herein comprises the amino acid sequence of SEQ ID NO:13.

[0143] Some embodiments of the present disclosure provide pharmaceutical compositions comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprising a human 4-1BB antigen-binding domain and a human OX40 antigen-binding domain, wherein (a) the 4-1BB antigen-binding domain comprises: (i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable domain (VL)-CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable domain (VL)-CDR1 comprising the amino acid sequence of SEQ ID NO: 5; (b) the OX40 antigen-binding domain comprises (i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:7; (ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:8; (iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:9; (iv) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:10; (v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:11; and (vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:12.

[0144] Some embodiments of the present disclosure provide pharmaceutical compositions comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprising a human 4-1BB antigen-binding domain and a human OX40 antigen-binding domain, wherein (a) the 4-1BB antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15; and (b) the OX40 antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18.

[0145] Some embodiments of the present disclosure provide pharmaceutical compositions comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprising a human 4-1BB antigen-binding domain and a human OX40 antigen-binding domain, wherein (a) the 4-1BB antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 16; and (b) the OX40 antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 19.

[0146] Some embodiments of the present disclosure provide pharmaceutical compositions comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof comprising a human 4-1BB antigen-binding domain comprising the amino acid sequence of SEQ ID NO:13 and a human OX40 antigen-binding domain.

[0147] Some embodiments of the present disclosure provide a pharmaceutical composition comprising: (a) 10 to 50 mg / mL of a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (i) a 4-1BB antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light chain variable domain (VL)-CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 7; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 8; a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 10; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 11; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 12; DR3; (ii) a 4-1BB antigen binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15; and an OX40 antigen binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18; (iii) a 4-1BB antigen binding domain comprising an scFv comprising the amino acid sequence of SEQ ID NO: 16; and the sequence (b) about 5 mM to about 15 mM glutamate; (c) about 25 mM to about 50 mM leucine; (d) about 2% to about 8% w / v sucrose; and (e) about 0.01% to about 0.03% polysorbate-80; wherein the pH of the composition is about 4.3 to about 4.7.

[0148] Some embodiments of the disclosure provide a pharmaceutical composition comprising: (a) 10 mg / mL of a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof; (i) a 4-1BB antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light chain variable domain (VL)-CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 7; (ii) a 4-1BB antigen-binding domain comprising the amino acid sequence of SEQ ID NO: 7; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 12; (b) an antibody or antigen-binding fragment thereof comprising an OX40 antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 8; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 9; a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 10; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 11; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 12; (b) 10 mM glutamate; (c) 40 mM leucine; (d) 3% w / v sucrose; and (e) 0.02% polysorbate-80, wherein the pH of the composition is 4.5.

[0149] In some embodiments, the pharmaceutical composition comprises (a) a 4-1BB antigen binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and (b) an OX40 antigen binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18.

[0150] IV. METHODS AND USES The present disclosure provides pharmaceutical compositions comprising antibodies or antigen-binding fragments that bind to 4-1BB and / or OX40, which are useful in a variety of applications, including, but not limited to, therapeutic treatments, such as cancer treatments. In some embodiments, the pharmaceutical compositions are useful for inhibiting tumor growth and / or reducing tumor volume. The method of use can be an in vitro method or an in vivo method. The pharmaceutical compositions described herein can include the use of any of the disclosed antibodies or antigen-binding fragments for use in treatments.

[0151] The present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising: (a) a bispecific antibody or antigen-binding fragment thereof that specifically binds 4-1BB and OX40; (b) about 5 mM to about 15 mM of a stability-promoting buffer; (c) about 25 mM to about 50 mM of a stabilizing amino acid; (d) about 2% to about 8% w / v sugar; and (e) about 0.01% to about 0.03% surfactant.

[0152] In some embodiments, the cancer includes, but is not limited to, melanoma, renal cancer, pancreatic cancer, lung cancer, colon / intestinal cancer, gastric cancer, prostate cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, and blood cancer. The cancer may be a primary tumor, or an advanced or metastatic cancer. In some embodiments, the cancer is a solid tumor. For example, the present disclosure includes the use of bispecific antibodies for the treatment of sarcoma, carcinoma, and lymphoma. The present disclosure provides a method for treating a human subject with sarcoma, carcinoma, or lymphoma by administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein.

[0153] In some embodiments, the present disclosure provides a method of treating a human subject having a tumor or cancerous tissue comprising tumor-infiltrating lymphocytes. The present disclosure provides treating a human subject having a tumor comprising lymphocytes expressing 4-1BB and OX40. In some embodiments, the present disclosure provides administering to a human subject having a solid tumor a therapeutically effective amount of a pharmaceutical composition comprising: (a) a bispecific antibody or antigen-binding fragment thereof that specifically binds 4-1BB and OX40; (b) about 5 mM to about 15 mM of a stability-promoting buffer; (c) about 25 mM to about 50 mM of a stabilizing amino acid; (d) about 2% to about 8% w / v of a sugar; and (e) about 0.01% to about 0.03% of a surfactant.

[0154] Some aspects of the present disclosure provide a method of enhancing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0155] Some embodiments of the present disclosure provide a method of stimulating a T cell costimulatory pathway in a subject, the method comprising administering to the subject a pharmaceutical composition comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0156] Some embodiments of the present disclosure provide a method for increasing proliferation of NK cells and / or T cells (e.g., CD4+ T cells and / or CD8+ T cells) in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0157] Some aspects of the present disclosure provide a method for increasing the number of tumor-infiltrating lymphocytes in a subject by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0158] Some aspects of the present disclosure provide a method for increasing expression of granzymes by tumor-infiltrating lymphocytes in a subject by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof disclosed herein.

[0159] In some embodiments, the subject is a human.

[0160] In some embodiments, provided herein is a pharmaceutical composition comprising a 4-1BBxOX40 bispecific antibody or an antigen-binding fragment thereof for use as a medicament. In some embodiments, provided herein is a pharmaceutical composition comprising a 4-1BBxOX40 bispecific antibody or an antigen-binding fragment thereof for use in a method for treating cancer.

[0161] V. Kits and Containers The present disclosure also provides a container comprising any of the pharmaceutical formulations described and exemplified herein.

[0162] In some embodiments, the present disclosure provides a kit comprising any of the pharmaceutical preparations and / or bispecific antibodies or antigen-binding fragments thereof described and exemplified herein. The kit can be used to provide pharmaceutical preparations, antibodies, antigen-binding fragments thereof, and other agents for use in diagnostic, basic research, or therapeutic methods, among others. In some embodiments, the kit comprises any one or more of the pharmaceutical preparations, antibodies or antigen-binding fragments thereof, and / or bispecific antibodies described and exemplified herein, and instructions for using the one or more pharmaceutical preparations, antibodies, or antigen-binding fragments thereof in a method for treating cancer. In some embodiments, the kit comprises any one or more of the pharmaceutical preparations, antibodies or antigen-binding fragments thereof, and / or bispecific antibodies described and exemplified herein, and instructions for using the one or more pharmaceutical preparations, antibodies, or antigen-binding fragments thereof in a method for treating solid tumor cancer in a subject.

[0163] Working Example It should be understood that the examples described herein are for illustrative purposes only, and that various modifications or changes in view thereof may be suggested to one skilled in the art and are intended to be included within the spirit and scope of the present application.

[0164] Example 1. Various excipients are used to stabilize proteins and minimize aggregation and / or degradation. Different components and excipients may bind to, interact with and / or stabilize different regions of a protein based on their chemical characteristics. Therefore, it is useful to screen a variety of different types of excipients, such as the subset listed in Table 1 below, to determine their effect on protein formulations.

[0165] The impact of different formulations can be measured by various means. One approach is to store the formulated protein at various temperatures, including both potential storage conditions after the drug is manufactured (the "intended storage conditions") and accelerated storage conditions (temperatures higher than the intended storage conditions). When performing storage stability experiments, samples are removed at various time points and analyzed by various techniques, such as analytical size-exclusion high performance liquid chromatography (SE-HPLC). Measuring changes in purity over time using SE-HPLC can be useful to evaluate whether different formulations inhibit or promote aggregation or degradation. Analytical cation exchange chromatography (CEX-HPLC) and / or capillary isoelectric focusing (cIEF) can be used to monitor changes in the charge variation profile of the protein to determine whether different formulation conditions inhibit or promote charge-related changes in protein structure.

[0166] In addition to using storage stability experiments to evaluate different formulations, other methods can be used to characterize the biophysical effects on protein stability. For example, differential scanning fluorometry (DSF) or differential scanning calorimetry (DSC) can be used to determine the melting point (T M The thermal stability of the protein was evaluated by measuring the thermal melting transition midpoint (TM (T M Another biophysical method that can be used to evaluate the stabilizing effect of a formulation is the particle interaction parameter or second virial coefficient, B 22 Using light scattering measurements at various protein concentrations in a particular formulation, the slope of the line can be used to determine whether the solution is beneficial (increasing, positive slope) or unfavorable (decreasing, negative slope). Dynamic light scattering (DLS) monitoring at 266 nm can be used to determine the aggregation temperature (T agg ) can also be determined, which is useful when comparing different formulations. [Table 10]

[0167] Example 2. Evaluation of different buffers, pH, and sucrose concentrations The FXX01102 bispecific protein was expressed by CHO cells in a bioreactor and purified to near homogeneity using column chromatography (>95% purity by SE-HPLC). It was concentrated, buffer exchanged, and formulated into a series of different compositions shown in Table 2 below. All buffers were used at 10 mM and the FXX01102 protein concentration was 10 mg / mL in each solution. The purpose of this study was to measure the effect of various buffers, pH, and sucrose concentrations on the stability of the FXX01102 bispecific antibody. The onset of aggregation was measured using an Uncle apparatus (Unchained Labs) at temperature (T agg ) and T m (midpoint of the melting curve). agg was measured by light scattering measurements at 266 nm in response to increasing temperatures.

[0168] T in the sample set m A comparison of the values ​​revealed that the pH, buffer, and sucrose concentration were sufficient to induce the formation of the unfolded first domain (T m1) was suggested to have no significant effect on the T. However, comparison of dynamic light scattering (DLS) data at 266 nm showed differences in the formation of insoluble particulates in the samples (Figure 1A and Figure 1B). Because sucrose concentration caused little change in the buffer and pH effects, only the 2% w / v sucrose data was plotted for succinate, histidine, citrate and 8% w / v glutamate buffers. As shown in Figure 1A, the two citrate buffers at pH 5.8 and 6.6 had very similar T agg The curves show that the pH 6.6 trace is shifted slightly to higher temperatures, reflecting more stable solution conditions for FXX01102. agg The data suggested that citrate was the lowest stability enhancing buffer among the buffers tested, followed by succinate. The low pH succinate formulation at pH 5.2 had a higher T than the pH 6.0 solution. agg The histidine and glutamate buffers all showed minimal increases in light scattering and were identified as the preferred formulations in the set. The plots for the His and Glu buffers (Figure 1B) showed that glutamate had the lowest light scattering signal among those evaluated. The histidine formulations showed the same trend as the succinate formulations, with the low pH 5.8 formulation performing better than the high pH 6.2 solution. As similar pH values ​​were used in the different buffers, T agg The difference in pH may not be a result of pH alone, but rather a combination of pH and charge properties of the buffer. Based in part on these results, glutamate became the primary buffer in subsequent experiments. [Table 11]

[0169] Example 3. Evaluating the effect of NaCl on low pH formulations FXX01102 bispecific protein was concentrated and exchanged into a solution of 10 mM glutamate, 8% weight / volume sucrose, and 0.02% weight / volume polysorbate-80 with and without 100 mM sodium chloride (NaCl) at pH 4.5 and a final protein concentration of 8 mg / mL. A dilution series was generated at protein concentrations of 0, 2, 4, 6, and 8 mg / mL. These solutions were analyzed on an Unchained Laboratories "Uncle" instrument. The goal was to determine if the inclusion of sodium chloride would improve the stabilization properties of the glutamate / sucrose / polysorbate-80 formulation.

[0170] For these two solutions, 22 When the effect of NaCl on the protein-protein interactions was examined, it was clear that the addition of NaCl to the formulations resulted in a significant decrease in the slope, corresponding to a destabilizing effect and promoting protein-protein interactions that may lead to aggregation (Figure 2). Therefore, sodium chloride was not further evaluated in these studies.

[0171] Example 4. Evaluation of the effect of arginine, methionine, leucine, and glycine on B22 levels of FXX01102 formulations The amino acids methionine, arginine, and glycine were also investigated for the stabilizing properties they may exert on FXX01102. A 10 mg / mL solution of FXX01102 was prepared in 10 mM glutamate, 4% w / v sucrose, pH 4.5, with no additional excipients and with either 100 mM arginine, 50 mM methionine, or 50 mM glycine. A dilution series of 0, 2, 4, 6, 8, and 10 mg / mL was analyzed by Uncle and B. 22 The effect of 22 Comparison of the slopes (Figure 3) shows that both glycine and methionine have a higher B 22The addition of additional amino acids appeared to increase the slope value and reduced the slope compared to formulations in which no additional amino acids were added, suggesting that uncharged or more hydrophobic amino acids may have a more beneficial effect on the stability of FXX01102 compared to the addition of salt or other charged amino acids.

[0172] To further investigate the effect of hydrophobic amino acids on the stability of FXX01102, the B 22 The slopes were compared for a dilution series of FXX01102 in 10 mM glutamate, 4% w / v sucrose at pH 4.5 with or without leucine. The slope of the leucine-containing formulation was slightly higher than that of the protein solution without leucine, suggesting that leucine may exert a stabilizing effect on the protein, reducing its self-interacting properties (Figure 4).

[0173] Example 5. Aggregation Temperature (T agg Evaluation of the effects of magnesium sulfate, lysine, and leucine on For a subset of formulations, the onset of aggregation (T agg ) was determined using an Uncle instrument (Unchained Labs). agg was measured by light scattering measurements at 266 nm in response to increasing temperatures.

[0174] FXX01102 in 10 mM glutamate, 4% w / v sucrose, pH 4.5, containing 25 mM leucine, MgSO4 and lysine. Comparison of plots of scattering vs. temperature at 266 nm showed that both MgSO4 and lysine caused an increase in scattering at much lower temperatures compared to the formulation without additional excipients (Figure 5). Samples containing leucine did not show any increase in scattering over the entire range of temperatures evaluated. This is consistent with the B obtained with leucine. 22 These results demonstrate that the improvements in β-amyloid ...

[0175] Example 6. Stability evaluation of FXX01102 formulations at -20°C Experiments were conducted to evaluate the stability of FXX01102 when stored at -20°C. All formulations, shown in Table 3 below, contained 10 mM glutamate and 0.02% polysorbate as buffering agents. In addition, all formulations contained a sugar component, 3% w / v or 8% w / v sucrose, 3% trehalose, or 3% sorbitol. Based on previous data, the addition of Leu, Met, or Gly amino acids was investigated to improve the stability of FXX01102. Formulations with improved stability will show less aggregate formation as measured by size exclusion ultra-pressure liquid chromatography (SE-UPLC).

[0176] FXX01102 was purified from the clarified bioreactor supernatant using two chromatography columns to achieve a starting purity of >95%. The material was buffer exchanged into glutamate using a UF / DF system and at least 10x the volume of buffer and concentrated to 10mg / mL. After the various formulations were prepared, 200μL aliquots were made into 0.5mL Sarstedt vials. The pH was measured at the beginning of the study. Most samples have a pH of around 4.6. SE-UPLC was used to determine the initial % high molecular weight content (%HMW). The vials were then placed at -20°C and measured (for a subset of formulations) at 3 weeks, 6 weeks, 3 months, 6 months, and 7 months after storage. At the appropriate time frame, the vials were removed from the freezer and analyzed. Larger 3.75mL aliquots were prepared and subjected to three freeze / thaw (3xF / T) cycles. This was used as an additional stress test to compare the various formulations. This data is summarized below in Table 3. After evaluation of the three month data, only a subset of samples were analyzed at six and seven months in order to focus on specific formulations of interest.

[0177] When comparing sorbitol and trehalose containing formulations (ID#5, 6) with other formulations in the set, they were shown to contribute to aggregate formation. These two formulations were the poorest performing conditions analyzed based on %HMW for 3xF / T samples, 3 and 6 months. Thus, sucrose was identified as the preferred sugar component. Aggregation data for 3% and 8% sucrose (ID#2 and #3, respectively) showed that higher concentrations of sucrose did not have a significant advantage for long-term storage, although the %HMW for these formulations at 3 and 6 months was quite similar. However, it was observed that fewer aggregates were present after 3xF / T with the higher sucrose concentrations (6.3 vs. 8.8%).

[0178] B implemented above 22 Based on the analysis, it was predicted that Met, Gly, and Leu would have a positive impact on the storage stability of FXX01102. Three concentrations of each amino acid were analyzed for their impact on stability at -20°C. For each, increasing concentrations reduced aggregate formation over time. There was a clear trend for Met (ID#10, 11, 12), Gly (ID#13, 14, 15), and Leu (ID#7, 8, 9) to decrease in %HMW over time, corresponding to the concentration of added amino acid excipient. Leucine appears to have the greatest stabilizing impact, as formulation ID#9 had the lowest aggregate formation in the 3xF / T analysis and over time. Formulations containing combinations of amino acids were also investigated (ID#16, 17, 18), and this approach showed some merit. However, when the %HMW of this combination was compared to formulations containing equivalent concentrations of Leu (i.e., #16 vs. #8, or #18 vs. #8), it was suggested that Leu was the main driver of this effect. m Value and T agg The T values ​​were determined to see if increasing the leucine concentration from 10 mM to 40 mM would have a significant effect on either of these two parameters. The differences were relatively small, with T values ​​for all three buffers being 0.01 and 0.02. mThe temperature was 61°C, and there was no significant change in light scattering measured at 266 nm. [Table 12] TIFF2024508746000013.tif155162

[0179] Example 7 - Evaluation of Additional Glutamate Formulations of FXX01102 at 20°C Experiments were conducted to evaluate the stability of FXX01102 when stored at -20°C. All formulations shown in Table 4 below contained 5 mM glutamate as a buffering agent. In addition, all formulations contained a sugar component (either 0, 2, 3, or 8% w / v sucrose). Based on previous data, the effect of Leu concentration and pH variation on the stability of FXX01102 was further investigated. A similar method was performed as described in Example 6. Formulations with improved stability will show less aggregate formation as measured by size exclusion ultra-pressure liquid chromatography (SE-UPLC). Data was analyzed by %HMW difference between the initial and 6 week time points, and 3xF / T stress test. Data showed that lower pH values ​​and higher leucine concentrations inhibited the formation of aggregates over time and even during multiple freeze-thaw cycles. The inclusion of sucrose in the formulation had a positive effect with 10 mM glutamate and 0.02% polysorbate as buffering agents. Additionally, all formulations contained a sugar component, 3% w / v or 8% w / v sucrose, 3% trehalose, or 3% sorbitol. Based on previous data, the addition of Leu, Met, or Gly amino acids was also examined, but in the absence of sucrose, a higher %HMW was obtained than in the other formulations (formulation #6, Table 4). As with the previous experiment, higher levels of sucrose had a nominal impact on the 6-week storage stability data, but had a greater impact on inhibiting aggregate formation in 3XF / T. [Table 13] TIFF2024508746000015.tif242159TIFF2024508746000016.tif74162

[0180] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0181] All references (e.g., publications, patents, or patent applications) cited in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual reference (e.g., publication, patent, or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0182] Other aspects are within the scope of the following claims. array SEQ ID NO:1 GYTFTSYW SEQ ID NO:2 IYPGSSTT SEQ ID NO:3 ASFSDGYYAYAMDY SEQ ID NO:4 QDISNY SEQ ID NO:5 YTS SEQ ID NO:6 QQGYTLPYT SEQ ID NO:7 GFTLSYYG SEQ ID NO:8 ISHDGSDK SEQ ID NO:9 SNDQFDP SEQ ID NO:10 NIGSKS SEQ ID NO:11 DDS SEQ ID NO:12 QVWDSSSDHVV SEQ ID NO:13 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGNIYPSGGS TNYAQKFQGRVTMTVDTSTSTVYMELSSLRSEDTAVYYCASFSDGYYAYAMDYWGQG TLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQSVSSYL NWYQQKPGQAPRLLIYYASRRHTGIPARFSGSGTDFTLTISSLQPEDFAVYYCQQGYN LPYTFGQGTKVEIKEPKSSDKTHTCPPCPAPPAAAPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGGGGSPSSYVLTQPPSVSVAPGKTARITCGGNNIGSKSVNWFQQKPGQAPVLV VYDDDSGRPSGIPERFSGSTSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKL TVLGGGGSGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTLSYYGM HWVRQAPGKGLEWVAAISHDGSDKYYADSVKGRFTISRDNSKNRLYLQMNSLRAEDT AVYYCSNDQFDPWGQGTLVTVSSR SEQ ID NO:14 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGNIYPSGGS TNYAQKFQGRVTMTVDTSTSTVYMELSSLRSEDTAVYYCASFSDGYYAYAMDYWGQG TLVTVSS SEQ ID NO:15 EIVMTQSPATLSLSPGERATLSCRASQSVSSYLNWYQQKPGQAPRLLIYYASRRHTGIPA RFSGSGSGTDFTLTISSLQPEDFAVYYCQQGYNLPYTFGQGTKVEIK SEQ ID NO:16 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGNIYPSGGS TNYAQKFQGRVTMTVDTSTSTVYMELSSLRSEDTAVYYCASFSDGYYAYAMDYWGQG TLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRASQSVSSYL NWYQQKPGQAPRLLIYYASRRHTGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQGYN LPYTFGQGTKVEIK SEQ ID NO:17 QVQLVESGGGVVQPGRSLRLSCAASGFTLSYYGMHWVRQAPGKGLEWVAAISHDGSD KYYADSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCSNDQFDPWGQGTLVTVSS SEQ ID NO:18 SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVNWFQQKPGQAPVLVVYDDSGRPSGIPER FSGSTSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVL SEQ ID NO:19 SYVLTQPPSVSVAPGKTARITCGGNNIGSKSVNWFQQKPGQAPVLVVYDDSGRPSGIPER FSGSTSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGGGGSGGGS GGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTLSYYGMHWVRQAPGKGLEW VAAISHDGSDKYYADSVKGRFTISRDNSKNRLYLQMNSLRAEDTAVYYCSNDQFDPWG QGTLVTVSSR SEQ ID NO:20 GGGGSGGGGSGGGGS SEQ ID NO:21 GGGGS

Claims

1. (a) a bispecific antibody or antigen-binding fragment thereof that specifically binds to 4-1BB and OX40; (b) about 5 mM to about 15 mM of a stability-promoting buffer; (c) about 25 mM to about 50 mM of a stabilizing amino acid; (d) about 2% to about 8% w / v sugar; (e) about 0.01% to about 0.03% of a surfactant; and Including, Pharmaceutical compositions.

2. 2. The pharmaceutical composition of claim 1, wherein the stability-promoting buffer is selected from the group consisting of succinate, histidine, citrate, and glutamate.

3. 2. The pharmaceutical composition of claim 1, wherein the stabilizing amino acid is selected from the group consisting of arginine, methionine, leucine, and glycine.

4. 2. The pharmaceutical composition of claim 1, wherein the sugar is selected from the group consisting of sucrose, trehalose, and sorbitol.

5. 2. The pharmaceutical composition of claim 1, wherein the surfactant is polysorbate-80.

6. The pharmaceutical composition of claim 1, wherein the composition further comprises polysorbate-80 at a concentration of about 3% w / v.

7. 10. The pharmaceutical composition of claim 1, wherein the pH of the composition ranges from about 4.3 to about 4.

7.

8. 2. The pharmaceutical composition of claim 1, wherein the bispecific antibody or antigen-binding fragment thereof is present in the composition at a concentration of about 10 mg / mL to about 50 mg / mL.

9. 10. The pharmaceutical composition of claim 1, which does not contain sodium chloride (NaCl).

10. 2. The pharmaceutical composition of claim 1, wherein the bispecific antibody or antigen-binding fragment thereof has a purity of greater than about 90%.

11. 2. The pharmaceutical composition of claim 1, (a) a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof at a concentration of about 10 mg / mL; (b) about 10 mM glutamate; (c) about 40 mM leucine; (d) about 3% w / v sucrose; (e) about 0.02% polysorbate-80; and Including, A pharmaceutical composition, wherein the pH of said composition is from about 4.3 to about 4.

7.

12. 2. The pharmaceutical composition of claim 1, (a) 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof at a concentration of 10 mg / mL; (b) 10 mM glutamate; (c) 40 mM leucine; (d) 3% w / v sucrose; (e) 0.02% polysorbate-80; and Including, A pharmaceutical composition, wherein the pH of the composition is 4.

5.

13. the bispecific antibody or antigen-binding fragment thereof comprises a human 4-1BB antigen-binding domain and a human OX40 antigen-binding domain; (a) the 4-1BB antigen-binding domain comprises: (i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable domain (VL)-CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (b) the OX40 antigen-binding domain comprises: (i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 7; (ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 8; (iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; (iv) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 10; (v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 11; and (vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:

12. The pharmaceutical composition of claim 1.

14. the bispecific antibody or antigen-binding fragment thereof comprises a human 4-1BB antigen-binding domain and a human OX40 antigen-binding domain; (a) the 4-1BB antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15; (b) the OX40 antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18; The pharmaceutical composition of claim 1.

15. A 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof, comprising a human 4-1BB antigen-binding domain and a human OX40 antigen-binding domain; (a) the 4-1BB antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 16; (b) the OX40 antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 19; The pharmaceutical composition of claim 1.

16. 2. The pharmaceutical composition of claim 1, wherein the bispecific antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:

13.

17. A pharmaceutical composition comprising: (a) 10 mg / mL of a 4-1BBxOX40 bispecific antibody or antigen-binding fragment thereof: (i) a 4-1BB antigen-binding domain comprising: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light chain variable domain (VL)-CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and an OX40 antigen-binding domain comprising: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 7; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 8; a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 9; a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 10; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 11; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 12; (ii) a 4-1BB antigen-binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and an OX40 antigen-binding domain comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18; (iii) a 4-1BB antigen-binding domain comprising an scFv comprising the amino acid sequence of SEQ ID NO: 16, and an OX40 antigen-binding domain comprising an scFv comprising the amino acid sequence of SEQ ID NO: 19; or (iv) the amino acid sequence of SEQ ID NO: 13; a 4-1BBxOX40 bispecific antibody or an antigen-binding fragment thereof; (b) about 5 mM to about 15 mM glutamate; (c) about 25 mM to about 50 mM leucine; (d) about 2% to about 8% w / v sucrose; (e) about 0.01% to about 0.03% polysorbate-80; Including, the pH of the composition is from about 4.3 to about 4.7; Pharmaceutical compositions.

18. The pharmaceutical composition according to claim 1, wherein the pharmaceutical formulation can be stored at -20°C.

19. 2. The pharmaceutical composition of claim 1, having no significant change in aggregate formation after about 3 weeks, about 6 weeks, about 3 months, about 6 months, or about 7 months of storage at -20°C as measured by size-exclusion ultra-pressure liquid chromatography (SE-UPLC).

20. 13. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 1.

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