A composition comprising a combination of an anti-PD-1 antibody and another antibody

Abstract

The present invention relates to a composition comprising a combination of an anti-PD-1 antibody and an additional antibody. Pharmaceutical compositions are provided, which comprise a combination of anticancer agents that are a first antibody and a second antibody. In some embodiments, the first antibody is an anti-programmed death-1 (PD-1) antibody. In certain embodiments, the composition is a fixed-dose formulation. In certain embodiments, the composition is administered in an absolute dose. The present disclosure also provides a kit for treating a subject having a disease, the kit comprising a dose of any of the compositions disclosed herein and instructions for using the composition to treat a disease in any of the disclosed methods.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680035347.7, "Compositions Comprising Combinations of Anti-PD-1 Antibodies and Additional Antibodies", filed on April 15, 2016.

[0002] In this application, various publications are cited by author name and date in parentheses, or by patent number or patent publication number. The disclosures of these publications are hereby incorporated by reference in their entirety into this application to more fully describe the state of the art known to those skilled in the art as of the date of the invention described and claimed herein. However, the citation of a reference document herein should not be construed as an admission that the reference document is prior art to the present invention. Technical Field

[0003] The present invention relates to pharmaceutical compositions comprising combinations of immune checkpoint antibodies and second antibodies in fixed-dose formulations. Background Art

[0004] Human cancers have numerous genetic and epigenetic alterations that generate neoantigens that can potentially be recognized by the immune system (Sjoblom et al. (2006) Science 314:268 - 74). The adaptive immune system, which includes T and B lymphocytes, has great anti-cancer potential, with a broad repertoire and excellent specificity to respond to a variety of tumor antigens. In addition, the immune system exhibits considerable plasticity and a memory component. Successfully harnessing all of these attributes of the adaptive immune system would make immunotherapy unique among all cancer treatment modalities.

[0005] Recently, several immune checkpoint pathway inhibitors have begun to provide new immunotherapeutic approaches for treating cancer, including the development of antibodies (Abs) that bind and inhibit cytotoxic T-lymphocyte antigen-4 (CTLA-4), ipilimumab for treating patients with advanced melanoma, and the development of antibodies such as nivolumab and pembrolizumab (previously lambrolizumab; USAN Council Statement (2013) Pembrolizumab: Statement of the Nonproprietary Name Adopted by the USAN Council (ZZ-165), November 27, 2013), which specifically bind to the programmed death-1 (PD-1) receptor and block the inhibitory PD-1 / PD-1 ligand pathway.

[0006] Immune checkpoint antibodies can be administered in combination with other antibodies. Nevertheless, due to different dosages and dosing intervals between the two antibodies, administering the two antibodies can be burdensome, entailing multiple intravenous injections at different time points. In addition, the two antibodies may have significantly different stability profiles. Due to the unique nature of each antibody, for example, differences in Fc glycosylation, partial heavy chain C-terminal Lys processing, Fc methionine oxidation, hinge region cleavage, and Lys residue glycosylation, each antibody has different physicochemical and / or thermodynamic properties such as different degradation profiles when exposed to heat, freezing, light, extreme pH values, agitation, sheer stress, some metals, and organic solvents. Thus, although a single formulation containing the two antibodies would improve convenience, the unique nature of each antibody makes such a single formulation difficult to determine. SUMMARY OF THE INVENTION

[0007] The present invention relates to a pharmaceutical composition comprising an amount X of a first antibody or an antigen-binding fragment thereof, which comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and an amount Y of a second antibody or an antigen-binding fragment thereof, wherein the ratio of the amount X to the amount Y is from about 50:1 to about 1:50. In some embodiments, the ratio of X to Y is about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 5:1, about 3:1, about 1:1, about 1:3, about 1:5, about 1:10, about 1:20, about 1:30, about 1:40, or about 1:50.

[0008] In some embodiments, the anti-PD-1 antibody is nivolumab or pembrolizumab. In certain embodiments, the anti-PD-1 antibody is nivolumab.

[0009] In certain embodiments, the amount X of the first antibody or an antigen-binding fragment thereof is at least about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, or about 300 mg. In some embodiments, the amount X of the first antibody is at least about 80 mg, about 160 mg, or about 240 mg. In other embodiments, the amount X of the first antibody or an antigen-binding fragment thereof is about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, or about 300 mg. In certain embodiments, the amount X of the first antibody or an antigen-binding fragment thereof is about 80 mg or about 240 mg. In other embodiments, the amount X of the first antibody or an antigen-binding fragment thereof is greater than at least about 300 mg. In some embodiments, the amount X of the first antibody or an antigen-binding fragment thereof is from at least about 300 mg to at least about 500 mg, from at least about 300 mg to at least about 450 mg, from at least about 300 mg to at least about 400 mg, from at least about 300 mg to at least about 350 mg, from at least about 350 mg to at least about 500 mg, from at least about 400 mg to at least about 500 mg, or from at least about 450 mg to at least about 500 mg. In certain embodiments, the amount X is at least about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg. In one particular embodiment, the amount X of the first antibody or an antigen-binding fragment thereof is about 360 mg. In another embodiment, the amount X of the first antibody or an antigen-binding fragment thereof is about 480 mg.

[0010] On the one hand, the second antibody or its antigen-binding fragment can be an anti-CTLA4 antibody. The ratio of the amount X of the first antibody (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) to the amount Y of the second antibody (e.g., an anti-CTLA-4 antibody) is about 3:1, about 1:1, or about 1:3. In some embodiments, (i) the amount X of the anti-PD-1 antibody is about 240 mg and the amount Y of the anti-CTLA-4 antibody is about 80 mg; (ii) the amount X is about 80 mg and the amount Y is about 80 mg; (iii) the amount X is about 160 mg and the amount Y is about 160 mg; (iv) the amount X is about 240 mg and the amount Y is about 240 mg; or (v) the amount X is about 80 mg and the amount Y is about 240 mg. In certain embodiments, the anti-CTLA4 antibody is tremelimumab or ipilimumab.

[0011] On the other hand, the second antibody can be an anti-LAG3 antibody. The ratio of the amount X of the first antibody (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) to the amount Y of the second antibody (e.g., an anti-LAG-3 antibody) is about 12:1, about 3:1, or about 1:1. In certain embodiments, the anti-LAG3 antibody is BMS-986016.

[0012] In other aspects, the second antibody is an anti-CD137 antibody. In some embodiments, the ratio of the amount X of the first antibody (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) to the amount Y of the second antibody (e.g., an anti-CD-137 antibody) is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 5:1, about 4:1, or about 2:1. In certain embodiments, the anti-CD137 antibody is urelumab.

[0013] In some aspects, the second antibody is an anti-KIR antibody. In some embodiments, the ratio of the amount X of the first antibody (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) to the amount Y of the second antibody (e.g., an anti-KIR antibody) is about 30:1, about 10:1, about 3:1, about 1:1, or about 1:2. In some embodiments, the anti-KIR antibody is 1-7F9 or lirilumab.

[0014] In some aspects, the second antibody can be an anti-GITR antibody. In some embodiments, the anti-GITR antibody is MK4166 or TRX518. In other embodiments, the ratio of the amount X of the first antibody (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) to the amount Y of the second antibody (e.g., an anti-GITR antibody) is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1 or about 10:1.

[0015] In other aspects, the second antibody is selected from anti-TGFβ antibodies, anti-IL-10 antibodies, anti-B7-H4 antibodies, anti-Fas ligand antibodies, anti-CXCR4 antibodies, anti-mesothelin antibodies, anti-CD27 antibodies, anti-CD73 antibodies, and any combination thereof.

[0016] The pharmaceutical composition of the present invention may further comprise one or more other components selected from the following: bulking agents, stabilizers, chelating agents, surfactants, buffers, ionic agents, and any combination thereof.

[0017] In one embodiment, the pharmaceutical composition of the present invention is formulated in one or more of various buffers. For example, the composition of the present invention can be formulated in Tris-Cl, histidine, citrate or Tris-citrate buffer. In one embodiment, the composition is formulated in Tris-Cl buffer, and the concentration of Tris-Cl is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM or about 50 mM. In another embodiment, the concentration of Tris-Cl is about 20 mM. In other embodiments, the composition is formulated in citrate buffer, and the concentration of citrate is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM or about 50 mM. In certain embodiments, the citrate concentration is about 10 mM or about 20 mM. In some embodiments, the composition is formulated in histidine buffer, and the concentration of histidine is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM or about 50 mM. In some embodiments, the histidine concentration is about 20 mM. In other embodiments, the composition is formulated in Tris-citrate buffer, the concentration of Tris-Cl is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM or about 50 mM, and the concentration of citrate is at least about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM or about 50 mM. In certain embodiments, the concentration of Tris-Cl is about 13.3 mM and the concentration of citrate is about 6.7 mM.

[0018] The composition of the present invention can have a pH of about 5 to about 8. For example, the pH of the composition can be at least about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8.0. In some embodiments, the pH of the composition is at least about 6.0, about 6.2, about 6.5, about 6.6 or about 7.0.

[0019] The composition may further comprise bulking agents. In certain embodiments, the bulking agents are selected from NaCl, mannitol, glycine, alanine and any combination thereof.

[0020] In certain embodiments, the composition comprises a stabilizer. The stabilizer is selected from sucrose, trehalose, raffinose, arginine; or any combination thereof.

[0021] In other embodiments, the composition comprises a chelating agent. The chelating agent is selected from diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid, nitrilotriacetic acid, and any combination thereof.

[0022] In certain embodiments, the surfactant is selected from polysorbate 80 (PS80), polysorbate 20 (PS20), and any combination thereof.

[0023] In some embodiments, the composition comprises NaCl at a concentration of at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 110 mM, at least about 120 mM, at least about 130 mM, at least about 140 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, at least about 225 mM, at least about 250 mM, at least about 275 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, or at least about 450 mM. In some embodiments, the concentration of NaCl is about 100 mM, about 96.15 mM, about 83.3 mM, about 78.57 mM, or about 50 mM.

[0024] In some embodiments, the composition comprises mannitol USP at the following concentrations (% w / v): at least about 0.25%, at least about 0.5%, at least about 0.75%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 7.5%, or at least about 10%. In some embodiments, the concentration of mannitol is about 1%, about 1.15%, about 1.67%, about 1.86%, or about 3%.

[0025] In some embodiments, the composition comprises DTPA USP at the following concentrations: at least about 5 μM, at least about 10 μM, at least about 15 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 40 μM, at least about 50 μM, at least about 60 μM, at least about 70 μM, at least about 75 μM, at least about 80 μM, at least about 90 μM, at least about 100 μM, at least about 110 μM, at least about 120 μM, at least about 130 μM, at least about 140 μM, at least about 150 μM, at least about 175 μM or at least about 200 μM. In some embodiments, the concentration of DTPA is about 20 μM, about 50 μM, about 65.71 μM, about 73.3 μM, about 93.85 μM or 100 μM.

[0026] In some embodiments, the composition comprises PS80 (% w / v) at the following concentrations: at least about 0.005%, at least about 0.01%, at least about 0.015%, at least about 0.02%, at least about 0.03%, at least about 0.04%, at least about 0.05%, at least about 0.06%, at least about 0.07%, at least about 0.08%, at least about 0.09% or at least about 0.1%. In some embodiments, the concentration of PS80 is about 0.01%, about 0.012%, about 0.013%, about 0.02%, about 0.23%, about 0.04% or about 0.05%.

[0027] In some embodiments, the composition comprises sucrose (% w / v) at the following concentrations: at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.5%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5%, at least about 10%, at least about 12% or at least about 15%. In some embodiments, the concentration of sucrose is about 6% or about 8.5%.

[0028] In certain embodiments, the present invention includes the following compositions: (i) a pharmaceutical composition comprising nivolumab and ipilimumab in a 1:1 ratio in a buffer containing approximately 13.3 mM Tris, approximately 6.7 mM citrate, approximately 1.67% mannitol, approximately 83.3 mM NaCl, approximately 73.3 μM DTPA, and approximately 0.013% PS80 at a pH of about 6.2; (ii) a pharmaceutical composition comprising nivolumab and ipilimumab in a 3:1 ratio in a Tris-citrate buffer containing approximately 1.15% mannitol, approximately 96.15 mM NaCl, approximately 93.85 μM DTPA, and approximately 0.012% PS80 at a pH of about 6.6; (iii) a pharmaceutical composition comprising nivolumab and ipilimumab in a 1:3 ratio in a Tris-citrate buffer containing approximately 1.86% mannitol, approximately 78.57 mM NaCl, approximately 65.71 μM DTPA, and approximately 0.023% PS80 at a pH of about 6.0; (iv) a pharmaceutical composition comprising nivolumab and ipilimumab in a 3:1 ratio in a 20 mM histidine buffer containing approximately 50 mM NaCl, approximately 50 μM DTPA, approximately 6% sucrose, and approximately 0.05% PS80 at a pH of about 6; (v) a pharmaceutical composition comprising nivolumab and ipilimumab in a 3:1 ratio in a approximately 20 mM histidine buffer containing approximately 50 mM NaCl, approximately 50 μM DTPA, approximately 6% sucrose, and approximately 0.05% PS80 at a pH of about 7; (vi) a pharmaceutical composition comprising nivolumab and ipilimumab in a 3:1 ratio in a approximately 20 mM histidine buffer containing approximately 50 μM DTPA, approximately 8.5% sucrose, and approximately 0.05% PS80 at a pH of about 6; (vii) a pharmaceutical composition comprising nivolumab and ipilimumab in a 3:1 ratio in a approximately 20 mM citrate buffer containing approximately 50 mM NaCl, approximately 50 μM DTPA, approximately 6% sucrose, and approximately 0.05% PS80 at a pH of about 6; (viii) a pharmaceutical composition comprising nivolumab and ipilimumab in a 3:1 ratio in a approximately 20 mM citrate buffer containing approximately 50 mM NaCl, approximately 20 μM DTPA, approximately 3% mannitol, and approximately 0.04% PS80 at a pH of about 6; (ix) a pharmaceutical composition comprising nivolumab and ipilimumab in a 1:1 ratio in a approximately 20 mM citrate buffer containing approximately 50 mM NaCl, approximately 100 μM DTPA, approximately 3% mannitol, and approximately 0.02% PS80 at a pH of about 6; (x) a pharmaceutical composition comprising nivolumab and ipilimumab in a 1:1 ratio in a approximately 20 mM citrate buffer containing approximately 50 mM NaCl, approximately 100 μM DTPA, approximately 3% mannitol, and approximately 0.02% PS80 at a pH of about 6.5; (xi) at a pH of about 6.In about 20 mM citrate buffer containing about 100 mM NaCl, about 100 μM DTPA, about 1.0% mannitol and about 0.02% PS80 at about pH 5, a pharmaceutical composition comprising nivolumab and ipilimumab in a 1:1 ratio; or (xii) in about 20 mM citrate buffer containing about 50 mM NaCl, about 100 μM DTPA, about 6% sucrose and about 0.02% PS80 at about pH 6.0, a pharmaceutical composition comprising nivolumab and ipilimumab in a 1:1 ratio.

[0029] In other embodiments, the present invention includes a pharmaceutical composition comprising nivolumab and ipilimumab in a 1:3 ratio, which at about pH 6.0 contains about 4.62 mg / ml nivolumab, about 1.54 mg / ml ipilimumab, about 18.5 mM Tris hydrochloride, about 1.5 mM sodium citrate dihydrate, about 96.2 mM NaCl, about 1.2% mannitol, about 93.9 μM pentetic acid and about 0.012% PS80, or a pharmaceutical composition comprising nivolumab and ipilimumab in a 1:3 ratio, which at about pH 6.3 contains about 4.61 mg / ml nivolumab, about 1.54 mg / ml ipilimumab, about 18.46 mM Tris hydrochloride, about 1.54 mM sodium citrate dihydrate, about 96.15 mM NaCl, about 1.15% mannitol, about 93.85 μM pentetic acid and about 0.012% PS80.

[0030] The formulated compositions of the present invention can be stable and stored at about 5°C for at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 2 years or at least about 5 years. In some embodiments, the composition is stable at about 40°C and can be stored for at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 2 years or at least about 5 years. In other embodiments, the composition is stable at about 25°C and can be stored for at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 2 years or at least about 5 years.

[0031] The pharmaceutical composition of the present invention can exhibit minimal changes in the acidic peak under stress, for example, after long-term storage at a specific temperature. In one embodiment, the composition exhibits a change in the acidic peak of less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 5°C for about 6 months or about 3 months. In certain embodiments, the composition exhibits a change in the acidic peak of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 25°C for about 3 months. In other embodiments, the composition exhibits a change in the acidic peak of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 40°C for about 3 months.

[0032] The composition of the present invention can also exhibit minimal changes in the high molecular weight peak after long-term storage, for weeks, months, or years. In one embodiment, the composition exhibits a change in the high molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% after storage at about 4°C for about 3 months. In another embodiment, the composition exhibits a change in the high molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% after storage at about 25°C for about 2 months or about 3 months. In other embodiments, the composition exhibits a change in the high molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% after storage at about 40°C for about 2 months or about 3 months.

[0033] In addition, in certain embodiments, the composition may exhibit minimal change in the main peak as determined by capillary isoelectric focusing (cIEF) analysis. In one embodiment, after storage at about 4°C for about 1 month, the composition exhibits a change in the main peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%. In some embodiments, after storage at about 25°C for about 1 month, the composition exhibits a change in the main peak of capillary isoelectric focusing (cIEF) analysis of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%. In some embodiments, after storage at about 40°C for about 1 month, the composition exhibits a change in the main peak of capillary isoelectric focusing (cIEF) analysis of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

[0034] In some embodiments, the composition exhibits minimal change in the low molecular weight peak. In one embodiment, after storage at about 40°C for about 2 months, the composition exhibits a change in the low molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%. In certain embodiments, after storage at about 25°C for about 2 months, the composition exhibits a change in the low molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%. In certain embodiments, after storage at about 4°C for about 2 months, the composition exhibits a change in the low molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

[0035] In some embodiments, the composition is diluted before use. In some embodiments, before use, the composition is diluted with 0.9% Sodium Chloride Injection USP or 5% Dextrose Injection USP. In some embodiments, the composition is diluted to obtain the desired concentrations of the first antibody and the second antibody.

[0036] In some embodiments, the present disclosure relates to a kit comprising the composition disclosed herein.

[0037] In some embodiments, the present disclosure relates to a method of manufacturing the composition disclosed herein. In some embodiments, a formulation comprising an anti-PD-1 antibody drug product is mixed with a formulation comprising a second antibody drug product to obtain the desired ratio in the final drug product without buffer changes. In some embodiments, the formulation comprising the anti-PD-1 antibody drug substance and the formulation comprising the second antibody drug substance are buffer exchanged and / or concentrated and then mixed to obtain the desired ratio in the final drug product.

[0038] In some embodiments, the present disclosure relates to a method of modulating an immune response in a patient in need thereof, comprising administering to the patient the composition disclosed herein.

[0039] In some embodiments, the present disclosure relates to a method of co-administering two antibodies to a patient in need thereof, comprising administering to the patient the composition disclosed herein, wherein the antibodies are capable of treating at least one disease or disorder.

[0040] In some embodiments, the present disclosure relates to a method of treating a disease or disorder, comprising administering to the patient the composition disclosed herein.

[0041] In some embodiments, the disease or disorder is an infectious disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is melanoma cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal area, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer; chronic or acute leukemia, including acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia; childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma; environmentally induced cancers, including cancers induced by asbestos; and any combination thereof.

[0042] In some embodiments, the composition is administered intravenously. In some embodiments, the composition is diluted prior to administration. In some embodiments, the composition is administered at a flat dose. In some embodiments, the amounts of the first antibody and the second antibody administered to the patient in a single dose are the same as an X amount and a Y amount, respectively. In some embodiments, the composition is administered at a weight-based dose. In some embodiments, the amount of the first antibody administered to the patient is at least about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, or about 5 mg / kg. In some embodiments, the amount of the first antibody administered to the patient is at least about 1 mg / kg.

[0043] In some embodiments, the composition is administered at least about weekly, at least about twice a week, at least about every two weeks, at least about every three weeks, or at least about monthly. In some embodiments, the administration continues for at least about 8 weeks, at least about 12 weeks, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 18 months, at least about 2 years, or greater than 2 years. In some embodiments, the patient is also treated with another anti-cancer agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Compositions showing anti-CTLA4 antibody (i.e., ipilimumab) and anti-PD-1 antibody (i.e., nivolumab) drug substance (DS) and drug product (DP) formulations.

[0045] Figure 2AShows the results of size-exclusion chromatography (SEC) of a 1:1 ratio fixed-dose combination (FDRC = fixed-dose ratio combination) formulation of an anti-PD-1 antibody (e.g., nivolumab) and an anti-CTLA-4 antibody (e.g., ipilimumab). Figure 2B Shows imaging capillary isoelectric focusing (cIEF) analysis of a 1:1 ratio fixed-dose combination (FDRC = fixed-dose ratio combination) formulation of an anti-PD-1 antibody (e.g., nivolumab) and an anti-CTLA-4 antibody (e.g., ipilimumab). EC represents a mixture of Tris and citrate buffers. Nivolumab DP and ipilimumab DP results are shown as controls; and the combination of an anti-PD-1 antibody and an anti-CTLA4 antibody (e.g., nivolumab and ipilimumab) is shown as EC Figure 2A -B as EC FDRC. Figure 2A Shows the actual change in high molecular weight (HMW) peak size (%) of the formulation stored at 40 °C for 3 months compared to the Day 0 control. For each prototype formulation, the pH adjusted at 25 °C and the concentrations of polysorbate 80 (PS80), NaCl, and mannitol are shown Figure 2A below the x-axis. Figure 2B Shows the actual change in nivolumab and ipilimumab acidic peak size (%) of the formulation stored at 5 °C for 6 months compared to the Day 0 control. The pH adjusted at 25 °C and the concentrations of NaCl and mannitol for each formulation are shown Figure 2B below the x-axis. Data points are labeled as N (nivolumab), C (combination of nivolumab and ipilimumab), and I (ipilimumab) in Figure 2A -B.

[0046] Figure 3A -B shows the SEC analysis results for 1:3, 1:1, or 3:1 ratio fixed-dose formulations of an anti-PD-1 antibody (e.g., nivolumab) and an anti-CTLA-4 antibody (e.g., ipilimumab). Nivolumab DP and ipilimumab DP results are shown as controls; and the combination of an anti-PD-1 antibody and an anti-CTLA4 antibody (e.g., nivolumab and ipilimumab) is shown as EC( Figure 3A -B). Figure 3A Shows the HMW peak size (%) of each sample and prototype (EC: pH 6.0 (1 ipilimumab: 3 nivolumab); EC: pH 6.2 (1 ipilimumab: 1 nivolumab); and EC: pH 6.6 (3 ipilimumab: 1 nivolumab)) formulation at Day 0 (initial), the HMW peak size after 2 months at 40 °C, and the change in HMW peak size between the Day 0 control and the formulation at 40 °C for 2 months. Figure 3BShows the LMW peak size (%) on day 0 (initial), the LMW peak size after 2 months at 40°C, the change in LMW peak size between each formulation and the control on day 0 and after 2 months of storage at 40°C, and the change in LMW peak size between each formulation and the control on day 0 and after 3 months of storage at 25°C. The pH adjusted at 25°C for each formulation, the concentration of ipilimumab, the concentration of nivolumab, the concentration of PS80, and the concentration of NaCl are shown below the x-axis ( Figure 3A -B).

[0047] Figure 4A -C shows the results of cIEF analysis for fixed-dose formulations of anti-PD-1 antibody (e.g., nivolumab) and anti-CTLA-4 antibody (e.g., ipilimumab) at a ratio of 1:3, 1:1, or 3:1 after 3 months of storage at 25°C ( Figure 4A ), after 3 months of storage at 5°C ( Figure 4B ), and after 1 month of storage at 25°C ( Figure 4C ). The nivolumab DP and ipilimumab DP results are shown as the control ( Figure 4A -C). The actual differences in the acidic peak size (%) of nivolumab (N) and ipilimumab (I) relative to the control on day 0 are shown for the control and each prototype (EC: pH 6.0 (1 ipilimumab: 3 nivolumab); EC: pH 6.2 (1 ipilimumab: 1 nivolumab); and EC: pH 6.6 (3 ipilimumab: 3 nivolumab)) formulation at selected time points ( Figure 4A -C). The theoretical pH value, buffer type, and ratio of ipilimumab to nivolumab at 25°C for each formulation are shown below the x-axis in Figure 4A -B; and the NaCl concentration, theoretical pH at 25°C, and ratio of ipilimumab to nivolumab for each formulation are shown below the x-axis in Figure 4C . The theoretical pH is equal to the stability study pH ( Figure 4A -C).

[0048] Figure 5A -B shows the results of SEC ( Figure 5A ) and cIEF ( Figure 5B ) analysis of a novel experimental design (DoE) 3:1 ratio fixed-dose formulation of anti-PD-1 antibody (e.g., nivolumab) and anti-CTLA-4 antibody (e.g., ipilimumab). The nivolumab DP and ipilimumab DP results are shown as the control ( Figure 5A -B). Figure 5AShows the high molecular weight (HMW) peak size (%) at day 0 (initial) for the control formulation and each prototype formulation (Combo New, Combo 4, Combo 5, Combo 6, and Combo 8), the HMW peak size after 3 months at 40 °C, the change in HMW peak size between the day 0 control and the formulation stored at 40 °C for 3 months, and the change in HMW peak size between the day 0 control and the formulation stored at 25 °C for 3 months. Figure 5B Shows the actual differences in the acidic peak size (%) of nivolumab (N) and ipilimumab (I) relative to the day 0 control for each formulation prototype after storage at 25 °C for 3 months. The ratio of ipilimumab to nivolumab for each formulation; the concentrations of NaCl, mannitol, and sucrose; the theoretical pH at 25 °C, and the buffer type are shown below the x-axis ( Figure 5A -B).

[0049] Figure 6A -B shows the size-exclusion chromatography (SEC) analysis results of fixed-dose formulations of anti-PD-1 antibody (e.g., nivolumab) and anti-CTLA-4 antibody (e.g., ipilimumab) platform combinations (PCs) in 1:1; 1:3; or 3:1 ratios after storage at 40 °C for 3 months ( Figure 6A ) and after storage at 5 °C for 3 months ( Figure 6B ). The nivolumab drug product (DP) and ipilimumab DP results are shown as control ( Figure 6A -B). Figure 6A Shows the actual changes in the HMW peak size (%) between the day 0 control and each control and prototype (PC: pH 6.0 - 1:1; PC: pH 5.5 - 1:3; PC: pH 6.0 - 1:3; PC: pH 6.5 - 1:3; and PC: pH 6.0 - 3:1) formulation after storage at 40 °C for 3 months. Figure 6B Shows the HMW peak size (%) of each formulation at day 0 (initial) and the HMW peak size after 3 months at 5 °C. The buffer type and the ratio of ipilimumab to nivolumab for each formulation are shown below the x-axis ( Figure 6A -B).

[0050] Figure 7A -B shows the capillary isoelectric focusing (cIEF) analysis results of fixed-dose formulations of anti-PD-1 antibody (e.g., nivolumab) and anti-CTLA-4 antibody (e.g., ipilimumab) platform combinations (PCs) in 1:1; 1:3; or 3:1 ratios after storage at 25 °C for 3 months ( Figure 7A ) and after storage at 5 °C for 3 months ( Figure 7B ). The nivolumab DP and ipilimumab DP results are shown as control ( Figure 7A -B). Figure 7AShows the actual differences in the acidic peak size (%) of nivolumab (N) and ipilimumab (I) relative to the control on day 0 for each control and prototype (PC: pH 6.0 - 1:1; PC: pH 5.5 - 1:3; PC: pH 6.0 - 1:3; PC: pH 6.5 - 1:3; and PC: pH 6.0 - 3:1) formulation after storage at 25 °C for 3 months. Figure 7B Shows the actual differences in the acidic peak size (%) of nivolumab (N) and ipilimumab (I) relative to the control on day 0 for each formulation after storage at 5 °C for 3 months. For each formulation, the buffer type and the ratio of ipilimumab to nivolumab are shown below the x-axis ( Figure 7A -B).

[0051] Figure 8 Shows the SEC analysis results of nivolumab-DP-based FDRC (1:1) formulation after storage at 40 °C for 1 month. Nivolumab DP and ipilimumab DP results are shown as control ( Figure 8 ). The actual changes in the HMW peak size (%) between the control on day 0 and the formulation after storage at 40 °C for 1 month are shown for each control and prototype (A, B, C, and D) formulation ( Figure 8 ). The ratio of ipilimumab to nivolumab, buffer type, and theoretical adjusted pH for each formulation are shown below the x-axis ( Figure 8 ).

[0052] Figure 9 Shows the cIEF analysis results of nivolumab-DP-based FDRC (1:1) formulation after storage at 25 °C for 3 months. Nivolumab DP and ipilimumab DP results are shown as control. Figure 9 Shows the actual differences in the acidic peak size (%) of nivolumab (N) and ipilimumab (I) relative to the control on day 0 for each control and prototype (A, B, C, and D) formulation after storage at 25 °C for 3 months. The ratio of ipilimumab to nivolumab, buffer type, and theoretical adjusted pH for each formulation are shown below the x-axis.

[0053] Figure 10 Shows the degradation rate of the acidic peak of ipilimumab for FDRC and commercialized compositions at 25 °C / 60% RH (relative humidity). The FDRC compositions are shown in Table 7.

[0054] Figure 11 Shows the degradation rate of the acidic peak of nivolumab for FDRC and commercialized compositions at 25 °C / 60% RH. The FDRC compositions are shown in Table 7.

[0055] Figure 12 Shows the acidic peak profiles of ipilimumab and nivolumab at 25 °C in the pH range study.

[0056] Figure 13 Shows the size-exclusion chromatography high molecular weight profile of the DP prototype from a ruggedness study. The HMW profile of the FDRC DP remained unchanged after storage at 2-8 °C and 25 °C for 6 months.

[0057] Figure 14 Shows the size-exclusion chromatography monomer profile of the FDRC DP after storage at 2-8 °C and 25 °C for 6 months.

[0058] Figure 15 Shows the ipilimumab acidic peak profile. This evaluation indicates the pH-dependence of deamidation at the accelerated temperature of 25 °C, as shown by the increase in the acidic peak profile in the higher temperature range of pH 7.0.

[0059] Figure 16 Shows the nivolumab acidic peak profile. This evaluation indicates the pH-dependence of deamidation at the accelerated temperature of 25 °C, as shown by the increase in the acidic peak profile in the higher temperature range of pH 7.0.

[0060] Figure 17 Shows the ipilimumab main peak profile. This evaluation indicates the pH-dependence of deamidation at the accelerated temperature of 25 °C, as shown by the increase in the acidic peak profile in the higher temperature range of pH 7.0.

[0061] Figure 18 Shows the nivolumab main peak profile. This evaluation indicates the pH-dependence of deamidation at the accelerated temperature of 25 °C, as shown by the increase in the acidic peak profile in the higher temperature range of pH 7.0.

[0062] Figure 19 Shows the effect of pH on the cIEF profile.

[0063] Figure 20 Shows the iCIEF profile in the pH range of 5.4 - 6.6.

[0064] DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention relates to a pharmaceutical composition comprising an anti-PD-1 antibody and a second antibody. In some embodiments, the composition is a fixed-dose formulation. The advantages of such a single formulation fixed-dose composition can include improved medical compliance or reduced administration burden (e.g., multiple intravenous injections) by reducing the treatment time (for a given composition, e.g., intravenous), and the ability to have a combined drug profile of the two drugs. However, such a single formulation fixed-dose composition can induce an undesired interaction between the two antibodies, thereby reducing the total amount of the active ingredient and limiting the ability of the doctor to customize the dose.

[0066] TERMS

[0067] To facilitate a better understanding of the present disclosure, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, the following terms have the following meanings. Additional definitions are set forth throughout the application.

[0068] The term "and / or" as used herein will be taken to specifically disclose each of the two specific features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to include the following aspects: 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 (alone); B (alone); and C (alone).

[0069] It should be understood that whenever language "comprising" is used to describe an aspect, other similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, revised edition, 2000, Oxford University Press provide a general dictionary of many of the terms used in this disclosure to those skilled in the art.

[0071] Units, prefixes, and symbols are expressed in their accepted form of the Système International de Unites (SI). Numerical ranges include the numbers defining the range. The headings provided herein are not limitations of the aspects of the present disclosure, and the aspects of the present disclosure can be realized by referring to the entire specification. Thus, the terms defined immediately below are more fully defined by reference to the entire specification.

[0072] "Administer" means physically introducing a composition comprising a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Preferred routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" as used herein refers to a mode of administration that is generally by injection and different from enteral and topical administration, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered by a non-parenteral route, preferably oral administration. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or topical. Administration can also be effected, for example, once, multiple times, and / or for one or more extended time periods.

[0073] "Adverse event" (AE) as used herein is any adverse and generally unexpected or undesired sign (including abnormal laboratory findings), symptom or disease associated with the use of a medical treatment. For example, an adverse event may be associated with activation of the immune system or expansion of immune system cells (e.g., T cells) in response to a treatment. A medical treatment can have one or more associated AEs, and each AE can have the same or different severities. Reference to a method capable of "modifying an adverse event" means that the treatment regimen reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0074] "Antibody" (Ab) shall include, but is not limited to, a glycoprotein immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. The heavy chain constant region comprises three constant domains C H1 , C H2 and C H3 . Each light chain comprises a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region comprises one constant domain C L . The V H and V L regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each V H and V LIt contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system.

[0075] The immunoglobulin can be derived from any known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, including but not limited to human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the class or subclass of antibodies encoded by the heavy chain constant region genes (such as IgM or IgG1). The term "antibody" includes, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless otherwise specified, the term "antibody" also includes any antigen-binding fragment or antigen-binding portion of any of the above immunoglobulins, and includes monovalent and divalent fragments or portions, as well as single-chain antibodies.

[0076] The term "monoclonal antibody" ("mAb") refers to a preparation of antibody molecules composed of a single non-naturally occurring molecule, i.e., antibody molecules with substantially the same basic sequence and exhibiting a single binding specificity and affinity for a specific epitope. mAbs are examples of isolated antibodies. MAbs can be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0077] A "human" antibody (HuMAb) refers to an antibody with variable regions in which both the framework regions and the CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutations). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used synonymously.

[0078] "Humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with the corresponding amino acids from a human immunoglobulin. In one embodiment of the humanized form of an Ab, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions have not been altered. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted so long as the ability of the antibody to bind to a particular antigen is not eliminated. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0079] "Chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a murine antibody and the constant region is derived from a human antibody.

[0080] "Anti-antigen" antibody refers to an antibody that specifically binds an antigen. For example, an anti-PD-1 antibody specifically binds PD-1, and an anti-CTLA-4 antibody specifically binds CTLA-4.

[0081] The "antigen-binding portion" (also referred to as the "antigen-binding fragment") of an antibody refers to one or more antibody fragments that retain the ability to specifically bind the antigen bound by the whole antibody.

[0082] "Cancer" refers to a broad variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth (proliferation) results in the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors.

[0083] "CD137", "CD-137", "tumor necrosis factor receptor superfamily member 9 (TNFRSF9)", "4-1BB", and "lymphocyte activation-induced (ILA)" all refer to the same member of the tumor necrosis factor receptor family. One activity associated with CD137 is co-stimulatory activity for activated T cells. (Jang et al. (1998) Biochem. Biophys. Res. Commun. 242(3):613–20). The term "CD137" as used herein includes human CD137 (hCTLA-4), variants, isoforms, and species homologs of hCD137, and analogs having at least one epitope in common with hCD137. The amino acid sequence of hCD137 can be found under GenBank accession number NP_001552.

[0084] "Cytotoxic T-lymphocyte antigen-4" (CTLA-4) refers to an immunosuppressive receptor belonging to the CD28 family. CTLA-4 is expressed in vivo only on T cells and binds two ligands, CD80 and CD86 (also known as B7-1 and B7-2, respectively). The term "CTLA-4" as used herein includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, and analogs having at least one epitope in common with hCTLA-4. The complete hCTLA-4 sequence can be found under GenBank accession number AAB59385.

[0085] "Disease" refers to any disorder of structure or function in an organism, such as a human that is not a direct result of physical injury. "Infectious disease" is a disease caused by an organism such as a bacterium, fungus, parasite, virus, or other pathogen.

[0086] As used herein, "dosing interval" refers to the amount of time elapsed between multiple doses of the formulations disclosed herein that are administered to a subject. The dosing interval can thus be expressed as a range.

[0087] The term "dosing frequency" as used herein refers to the frequency of administering a dose of the formulations disclosed herein over a given period of time. The dosing frequency can be expressed as the number of doses over a given period of time, such as once a week or once in two weeks.

[0088] The term "fixed dose" as used in connection with the compositions of the present invention means that two or more different antibodies in a single composition are present in a specific (fixed) ratio to each other in the composition. In some embodiments, the fixed dose is based on the weight of the antibody (e.g., mg). In certain embodiments, the fixed dose is based on the concentration of the antibody (e.g., mg / ml). In some embodiments, the ratio is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, about 1:200, about 200:1, about 180:1, about 160:1, about 140:1, about 120:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, about 15:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1 or about 2:1 mg of the first antibody to mg of the second antibody. For example, a 3:1 ratio of the first antibody to the second antibody can mean that a vial can contain about 240 mg of the first antibody and 80 mg of the second antibody or about 3 mg / ml of the first antibody and 1 mg / ml of the second antibody.

[0089] The term "absolute dose" as used in connection with the compositions of the present invention means a dose that is administered to a patient without regard to the patient's weight or body surface area (BSA). Thus, the absolute dose is not provided as a mg / kg dose, but rather as an absolute amount of the agent (e.g., anti-CTLA4 antibody and / or anti-PD-1 antibody). For example, a 60 kg person and a 100 kg person will receive the same dose of the composition (e.g., 240 mg of anti-PD-1 antibody and 80 mg of anti-CTLA4 antibody in a single fixed dose formulation vial containing 240 mg of anti-PD-1 antibody and 80 mg of anti-CTLA4 antibody (or two fixed dose formulation vials containing 120 mg of anti-PD-1 antibody and 40 mg of anti-CTLA4 antibody, etc.)).

[0090] The term "weight-based dose" as referred to herein means a dose that is administered to a patient based on the patient's weight. For example, when a patient with a body weight of 60 kg requires a combination of 3 mg / kg of anti-PD-1 antibody and 1 mg / kg of anti-CTLA4 antibody, the individual can obtain an appropriate amount of anti-PD-1 antibody (i.e., 180 mg) and anti-CTLA4 antibody (i.e., 60 mg) at one time from a fixed dose formulation of a 3:1 ratio of anti-PD-1 antibody to anti-CTLA4 antibody.

[0091] As used herein, the term "reference composition" refers to a composition that includes either the first antibody or the second antibody, but not both simultaneously. The reference composition may contain the same components as the composition comprising the first and second antibodies, except for the presence of one of the antibodies. In other embodiments, the reference composition is a commercially available corresponding composition, such as or or for an anti-CTLA-4 antibody

[0092] The terms "GITR", "tumor necrosis factor receptor superfamily member 18", "activation-induced TNFR family receptor", or "glucocorticoid-induced TNFR-related protein" all refer to a protein that is a member of the tumor necrosis factor receptor superfamily. In humans, GITR is encoded by the TNFRSF18 gene. It is a type I transmembrane protein of 241 amino acids, characterized by three cysteine pseudo-repeats in its extracellular domain, and specifically protects against T cell receptor-induced apoptosis, although it does not protect cells against other apoptotic signals, including Fas triggering, dexamethasone treatment, or ultraviolet irradiation (Nocentini, G et al. (1997) Proc. Natl. Acad. Sci, USA 94:6216-622). As used herein, the term GITR includes human GITR (hGITR), variants, isoforms, and species homologs of hGITR, as well as analogs having at least one epitope in common with hGITR. Three isoforms of hGITR have been identified, all having the same extracellular domain except for their C-terminal portions. Variant 1 (accession number NP_004186) consists of 241 amino acids and represents the longest transcript. Compared to variant 2, it contains an additional coding segment that results in a frameshift. Compared to isoform 2, the resulting protein (isoform 1) contains a different and shorter C-terminal. Variant 2 (accession number NP_683699) encodes the longest protein (isoform 2) consisting of 255 amino acids and is soluble. Compared to variant 2, variant 3 (accession number NP_683700) contains an additional coding segment that results in a frameshift. Compared to isoform 2, the resulting protein (isoform 3) contains a different and shorter C-terminal and consists of 234 amino acids.

[0093] The term "immunotherapy" refers to treating a subject having a disease or at risk of contracting or recurring a disease by methods that include inducing, enhancing, suppressing, or otherwise altering an immune response.

[0094] The terms "LAG3", "LAG-3", or "lymphocyte activation gene-3" refer to lymphocyte activation gene-3. The term LAG-3 as used herein includes human LAG-3 (hLAG-3), variants, isoforms, and species homologs of hLAG-3, and analogs having at least one epitope in common with hLAG-3. The term "human LAG-3" refers to the human sequence LAG-3, such as the complete amino acid sequence of human LAG-3 having Genbank accession number NP 002277. The term "mouse LAG-3" refers to the mouse sequence LAG-3, such as the complete amino acid sequence of mouse LAG-3 having Genbank accession number NP_032505. LAG-3 is also known in the art as, for example, CD223. The human LAG-3 sequence may differ from the human LAG-3 of Genbank accession number NP_002277, for example, by having conservative mutations or mutations in non-conserved regions, and LAG-3 has a biological function substantially the same as that of the human LAG-3 of Genbank accession number NP_002277. For example, the biological function of human LAG-3 is to have an epitope in the extracellular domain of LAG-3 that is specifically bound by the antibodies of the present disclosure, or the biological function of human LAG-3 is to bind to MHC class II molecules.

[0095] The term "lyophilized product" as used herein in connection with the formulations according to the invention refers to a formulation produced by a lyophilization method known in the art. The solvent (such as water) is removed by freezing followed by vacuum sublimation and desorption of the remaining water at an elevated temperature. In the pharmaceutical field, a lyophilized product typically has a residual moisture of about 0.1 to 5% (w / w) and exists as a powder or a physically stable cake. A lyophilized product is characterized by rapid dissolution upon addition of a reconstitution medium.

[0096] The term "killer Ig-like receptor", "killer inhibitory receptor", or "KIR" refers to a protein or polypeptide encoded by a member gene of the KIR gene family or by a cDNA prepared from such a gene. A detailed review of the KIR gene family, including the nomenclature of KIR genes and KIR gene products, as well as Genbank accession numbers for exemplary KIRs, is "The KIR Gene Cluster" by M. Carrington and P. Norman, available from the NCBI website called Bookshelf (accessible at ncbi.nlm.nih.gov / books). The term KIR as used herein includes human KIR (hKIR), variants, isoforms, and species homologs of hKIR, as well as analogs having at least one epitope in common with hKIR. Sequences of human KIR genes and cDNAs and their protein products are available from public databases, including GenBank. Non-limiting exemplary GenBank entries for human KIRs have the following accession numbers: KIR2DL1: Genbank accession number U24076, NM_014218, AAR16197, or L41267; KIR2DL2: Genbank accession number U24075 or L76669; KIR2DL3: Genbank accession number U24074 or L41268; KIR2DL4: Genbank accession number X97229; KIR2DS1: Genbank accession number X89892; KIR2DS2: Genbank accession number L76667; KIR2DS3: Genbank accession number NM_012312 or L76670 (splice variant); KIR3DL1: Genbank accession number L41269; and KIR2DS4: Genbank accession number AAR26325. KIR can contain 1 to 3 extracellular domains and can have a long (i.e., more than 40 amino acids) or short (i.e., less than 40 amino acids) cytoplasmic tail region. As previously described herein, these features define the nomenclature of KIR. KIR is further described in International Publication No. WO / 2014 / 055648, the entire contents of which are incorporated herein by reference.

[0097] "Programmed death-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed mainly on previously activated T cells in vivo and binds two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs having at least one epitope in common with hPD-1. The complete hPD-1 sequence can be found under GenBank accession number U64863. "PD-1" and "PD-1 receptor" are used interchangeably herein.

[0098] "Programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as analogs having at least one epitope in common with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

[0099] The term "reconstituted formulation" as used herein refers to a formulation that has been lyophilized and re-dissolved by the addition of a diluent. The diluent can contain, for example, 0.9% Sodium Chloride Injection USP or 5% Dextrose Injection USP.

[0100] "Subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0101] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes the regression of a disease (as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or the prevention of injury or disability resulting from the disease). The ability of a therapeutic agent to promote the regression of a disease can be evaluated using a variety of methods known to those of skill in the art, such as evaluating in human subjects during clinical trials, evaluating in animal model systems predictive of efficacy in humans, or evaluating the activity of the agent in in vitro assays.

[0102] As used herein, a "sub-therapeutic dose" refers to a dose of a therapeutic compound (e.g., an antibody) that is lower than the usual or typical dose of the therapeutic compound when administered alone to treat a proliferative disease (such as cancer).

[0103] "Treatment" or "therapy" of a subject refers to any type of intervention, method, or administration of an active agent to a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or conditions or biochemical markers associated with a disease.

[0104] The use of alternatives (e.g., "or") is to be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" shall be understood to mean "one or more" of any of the components so described or recited.

[0105] The terms "about" or "substantially consists of" refer to a value or a component within an acceptable error range of a specific value or component determined by a person of ordinary skill in the art, which will depend in part on how the value or component is measured or determined, i.e., the limitations of the measuring system. For example, in accordance with the practice in the art, "about" or "substantially consists of" can mean within 1 or more standard deviations. Alternatively, "about" or "substantially consists of" can mean a range up to 10% or 20% (i.e., ±10% or ±20%). For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (10%) or between 2.4 mg and 3.6 mg (20%). In addition, particularly with respect to biological systems or methods, these terms can mean up to an order of magnitude or up to 5 times the value. When a specific value or component is provided in the application and claims, unless otherwise stated, the meaning of "about" or "substantially consists of" should be assumed to be within the acceptable error range of that specific value or component.

[0106] As used herein, the terms "about once a week", "about once every two weeks", or any other similar dosing interval term mean approximate numbers. "About once a week" can include ± one day every seven days, i.e., every six days to every eight days. "About once every two weeks" can include ± three days every fourteen days, i.e., every eleven days to every seventeen days. Similar approximations apply to, for example, about once every three weeks, about once every four weeks, about once every five weeks, about once every six weeks, and about once every twelve weeks. In some embodiments, a dosing interval of about once every six weeks or about once every twelve weeks means that the first dose can be administered on any day of the first week, and then the next dose can be administered on any day of the sixth or twelfth week, respectively. In other embodiments, a dosing interval of about once every six weeks or about once every twelve weeks means that the first dose is administered on a specific day of the first week (e.g., Monday), and then the next dose is administered on the same day (i.e., Monday) of the sixth or twelfth week, respectively.

[0107] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range, and, where appropriate, its fractions (such as tenths and hundredths of an integer), unless otherwise stated.

[0108] Various aspects of the present invention will be described in further detail in the following subsections.

[0109] Anti-PD-1 and anti-PD-L1 antibodies

[0110] The composition of the present invention comprises a first antibody and a second antibody in a ratio of 1:100 to 100:1. On the one hand, the first antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. PD-1 is a key immune checkpoint receptor expressed by activated T cells and B cells and mediates immunosuppression. PD-1 is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1 and BTLA. Two cell surface glycoprotein ligands of PD-1, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), have been identified, which are expressed on antigen presenting cells and many human cancers, and have been shown to downregulate T cell activation and cytokine secretion after binding to PD-1. Inhibition of PD-1 / PD-L1 interaction in preclinical models mediates effective anti-tumor activity.

[0111] HuMAbs that specifically bind to PD-1 with high affinity have been disclosed in US Patent Nos. 8,008,449 and 8,779,105. Other anti-PD-1 mAbs have been described, for example, in US Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, and PCT Publication No. WO 2012 / 145493. Each anti-PD-1 HuMAb disclosed in US Patent No. 8,008,449 has been shown to exhibit one or more of the following characteristics: (a) high affinity at 1 x 10 -7 M or smaller K D Binds to human PD-1 as determined by surface plasmon resonance using a Biacore biosensor system; (b) does not significantly bind to human CD28, CTLA-4 or ICOS; (c) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (d) increases interferon-γ production in an MLR assay; (e) increases IL-2 secretion in an MLR assay; (f) binds to human PD-1 and macaque PD-1; (g) inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (h) stimulates antigen-specific memory responses; (i) stimulates Ab responses; and (j) inhibits tumor cell growth in vivo. Anti-PD-1 antibodies useful in the present invention include mAbs that specifically bind to human PD-1 and exhibit at least one, preferably at least five, of the aforementioned characteristics.

[0112] In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab (also known as Previously known as 5C4, BMS-936558, MDX-1106, or ONO-4538) is a fully human IgG4 (S228P) PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the downregulation of anti-tumor T cell function (U.S. Patent No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56). In another embodiment, the anti-PD-1 antibody or fragment thereof cross-competes with nivolumab. In other embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as nivolumab. In certain embodiments, the anti-PD-1 antibody has the same CDRs as nivolumab.

[0113] In another embodiment, the anti-PD-1 antibody or fragment thereof cross-competes with pembrolizumab. In some embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as pembrolizumab. In certain embodiments, the anti-PD-1 antibody has the same CDRs as pembrolizumab. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as lambrolizumab and MK-3475) is a humanized monoclonal IgG4 antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587; see also http: / / www.cancer.gov / drugdictionary?cdrid=695789 (most recent access date: December 14, 2014). Pembrolizumab has been approved by the FDA for the treatment of recurrent or refractory melanoma.

[0114] In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with MEDI0608. In other embodiments, the anti-PD-1 antibody or fragment thereof binds to the same epitope as MEDI0608. In certain embodiments, the anti-PD-1 antibody has the same CDRs as MEDI0608. In other embodiments, the anti-PD-1 antibody is MEDI0608 (previously known as AMP-514), which is a monoclonal antibody. MEDI0608 is described, for example, in US Patent No. 8,609,089 B2 or http: / / www.cancer.gov / drugdictionary?cdrid=756047 (most recent access date: December 14, 2014).

[0115] In certain embodiments, the first antibody is an anti-PD-1 antagonist. An example of an anti-PD-1 antagonist is AMP-224, which is a B7-DC Fc fusion protein. AMP-224 is discussed in U.S. Publication No. 2013 / 0017199 or http: / / www.cancer.gov / publications / dictionaries / cancer-drug?cdrid=700595 (most recently accessed on July 8, 2015).

[0116] In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with BGB-A317. In some embodiments, the anti-PD-1 antibody or fragment thereof binds the same epitope as BGB-A317. In certain embodiments, the anti-PD-1 antibody has the same CDRs as BGB-A317. In certain embodiments, the anti-PD-1 antibody is BGB-A317, which is a humanized monoclonal antibody. BGB-A317 is described in U.S. Publication No. 2015 / 0079109.

[0117] Anti-PD-1 antibodies useful in the disclosed compositions also include isolated antibodies that specifically bind to human PD-1 and cross-compete with nivolumab for binding to human PD-1 (see, e.g., U.S. Patent Nos. 8,008,449 and 8,779,105; WO2013 / 173223). The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have very similar functional properties to nivolumab, due to their binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO 2013 / 173223).

[0118] In certain embodiments, the antibody that cross-competes with nivolumab for binding to human PD-1 or binds to the same epitope region of human PD-1 as nivolumab is an mAb. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, or humanized or human antibodies. Such chimeric, humanized, or human mAbs can be prepared and isolated by methods well known in the art.

[0119] Anti-PD-1 antibodies useful in the compositions of the disclosed invention also include antigen-binding portions of the above-described antibodies. It has been well demonstrated that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include (i) Fab fragments, consisting of V L 、VH , C L and C H1 domain - containing monovalent fragment; (ii) F(ab’) 2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of V H and C H1 domains; and (iv) an Fv fragment consisting of the V L and V H domains of a single arm of the antibody.

[0120] An anti - PD - 1 antibody suitable for the disclosed compositions is an antibody that binds to PD - 1 with high specificity and affinity, blocks the binding of PD - L1 and / or PD - L2, and inhibits the immunosuppressive effect of the PD - 1 signaling pathway. In any of the compositions or methods disclosed herein, an anti - PD - 1 “antibody” includes an antigen - binding portion or fragment that binds to the PD - 1 receptor and exhibits functional properties similar to those of a full - length antibody in inhibiting ligand binding and upregulating the immune system. In certain embodiments, the anti - PD - 1 antibody or its antigen - binding portion cross - competes with nivolumab for binding to human PD - 1. In other embodiments, the anti - PD - 1 antibody or its antigen - binding portion is a chimeric, humanized, or human monoclonal antibody or a portion thereof. In certain embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is a human antibody. Antibodies of the IgG1, IgG2, IgG3, or IgG4 isotype can be used.

[0121] In certain embodiments, the anti-PD-1 antibody or antigen-binding portion thereof comprises a heavy chain constant region of the human IgG1 or IgG4 isotype. In certain other embodiments, the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody or antigen-binding portion thereof contains an S228P mutation that replaces the serine residue in the hinge region with a proline residue (the position corresponding to that typically found in IgG1 isotype antibodies). This mutation, present in nivolumab, prevents Fab arm exchange with endogenous IgG4 antibodies while maintaining low affinity for activating Fc receptors associated with wild-type IgG4 antibodies (Wang et al., 2014). In other embodiments, the antibody comprises a light chain constant region that is a human κ or λ constant region. In other embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is an mAb or antigen-binding portion thereof. In certain embodiments of any of the treatment methods described herein that include administration of an anti-PD-1 antibody, the anti-PD-1 antibody is nivolumab. In other embodiments, the anti-PD-1 antibody is pembrolizumab. In other embodiments, the anti-PD-1 antibody is selected from the human antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in U.S. Patent No. 8,008,449. In other embodiments, the anti-PD-1 antibody is MEDI0608 (previously designated AMP-514), AMP-224, or pidilizumab (CT-011).

[0122] In certain embodiments, the first antibody of the disclosed compositions is an anti-PD-L1 antibody. Because anti-PD-1 and anti-PD-L1 target the same signaling pathway and have been shown in clinical trials to exhibit similar levels of efficacy in various cancers, an anti-PD-L1 antibody can replace the anti-PD-1 antibody in any of the therapeutic methods or compositions disclosed herein. In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (previously known as 12A4 or MDX-1105) (see, e.g., U.S. Patent No. 7,943,743; WO 2013 / 173223). In other embodiments, the anti-PD-L1 antibody is MPDL3280A (also known as RG7446 and atezolizumab) (see, e.g., Herbst et al 2013 J Clin Oncol 31(suppl):3000; U.S. Patent No. 8,217,149), MEDI4736 (Khleif, 2013, In: Proceedings from the European Cancer Congress 2013 (see Proceedings of the European Cancer Congress 2013); September 27 - October 1, 2013; Amsterdam, The Netherlands. Abstract 802) or MSB0010718C (also known as Avelumab; see US 2014 / 0341917). In certain embodiments, an antibody that cross-competes with the PD-L1 antibodies mentioned above for binding to human PD-L1 or that binds to the same epitope region of human PD-L1 as the PD-L1 antibodies mentioned above is an mAb. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, or can be humanized or human antibodies. Such chimeric, humanized or human mAbs can be prepared and isolated by methods well known in the art.

[0123] Anti-CTLA-4 antibody

[0124] The anti-CTLA-4 antibody for use in the present invention binds to human CTLA-4 to disrupt the interaction of CTLA-4 with the human B7 receptor. Because the interaction of CTLA-4 with B7 transduces a signal that results in the inactivation of T cells carrying the CTLA-4 receptor, disruption of this interaction effectively induces, enhances or prolongs the activation of such T cells, thereby inducing, enhancing or prolonging the immune response.

[0125] HuMAbs that specifically bind CTLA-4 with high affinity have been disclosed in US Patent Nos. 6,984,720 and 7,605,238. Other anti-CTLA-4 mAbs have been described, for example, in US Patent Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121. The anti-CTLA-4 HuMAbs disclosed in US Patent Nos. 6,984,720 and 7,605,238 have been shown to exhibit one or more of the following characteristics: (a) a binding affinity of at least about 10 as determined by Biacore analysis; 7 M -1 , or about 10 9 M -1 , or about 10 10 M -1 Up to 10 11 M -1 or higher equilibrium association constant (K a ) specifically binds to human CTLA-4; (b) at least about 10 3 , about 10 4 , or about 10 5 m -1 s -1 The kinetic association constant (k a ); (c) at least about 10 3 , about 10 4 , or about 10 5 m -1 s -1 The kinetic dissociation constant (k d ); and (d) inhibiting the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies useful in the present invention include mAbs that specifically bind to human CTLA-4 and exhibit at least one, at least two, or at least three of the foregoing characteristics. An exemplary clinical anti-CTLA-4 antibody is human mAb 10D1 (now known as ipilimumab and marketed as ipilimumab) disclosed in US Patent No. 6,984,720. Commercially available).

[0126] An exemplary clinical anti-CTLA-4 antibody is human mAb 10D1 (now known as ipilimumab and marketed as Commercially available). Ipilimumab is an anti-CTLA-4 antibody used in the methods disclosed herein. Ipilimumab is a fully human IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation and improving overall survival (OS) in patients with advanced melanoma.

[0127] Another anti-CTLA-4 antibody useful in the methods of the present invention is tremelimumab (also known as CP-675,206). Tremelimumab is a human IgG2 monoclonal anti-CTLA-4 antibody. Tremelimumab is described in WO / 2012 / 122444, U.S. Publication No. 2012 / 263677, or WO Publication No. 2007 / 113648A2.

[0128] Anti-CTLA-4 antibodies useful in the disclosed compositions also include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with ipilimumab or tremelimumab for binding to human CTLA-4 or bind to the same epitope region of human CTLA-4 as ipilimumab or tremelimumab. In certain embodiments, the antibodies that cross-compete with ipilimumab or tremelimumab for binding to human CTLA-4 or bind to the same epitope region of human CTLA-4 as ipilimumab or tremelimumab are antibodies that comprise a heavy chain of the human IgG1 isotype. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, or humanized or human antibodies. Useful anti-CTLA-4 antibodies also include antigen-binding portions of the above antibodies, such as Fab, F(ab’)2, Fd, or Fv fragments.

[0129] Anti-LAG-3 antibodies

[0130] The anti-LAG-3 antibodies of the present invention bind to human LAG-3. Antibodies that bind LAG-3 have been disclosed in International Publication No. WO / 2015 / 042246 and U.S. Publication Nos. 2014 / 0093511 and 2011 / 0150892.

[0131] An exemplary LAG-3 antibody useful in the present invention is 25F7 (described in U.S. Publication No. 2011 / 0150892). Another exemplary LAG-3 antibody useful in the present invention is BMS-986016. In one embodiment, the anti-LAG-3 antibody useful in the composition cross-competes with 25F7 or BMS-986016. In another embodiment, the anti-LAG-3 antibody useful in the composition binds to the same epitope as 25F7 or BMS-986016. In other embodiments, the anti-LAG-3 antibody comprises the 6 CDRs of 25F7 or BMS-986016.

[0132] Anti-CD137 antibodies

[0133] Anti-CD137 antibodies specifically bind to and activate immune cells expressing CD137, stimulating an immune response against tumor cells, particularly a cytotoxic T cell response. Antibodies that bind CD137 are disclosed in U.S. Publication No. 2005 / 0095244 and U.S. Patents No. 7,288,638, 6,887,673, 7,214,493, 6,303,121, 6,569,997, 6,905,685, 6,355,476, 6,362,325, 6,974,863, and 6,210,669.

[0134] In some embodiments, the anti-CD137 antibody is urelumab (BMS-663513), described in U.S. Patent No. 7,288,638 (20H4.9-IgG4 [10C7 or BMS-663513]). In some embodiments, the anti-CD137 antibody is BMS-663031 (20H4.9-IgG1), described in U.S. Patent No. 7,288,638. In some embodiments, the anti-CD137 antibody is 4E9 or BMS-554271, described in U.S. Patent No. 6,887,673. In some embodiments, the anti-CD137 antibody is an antibody disclosed in U.S. Patent No. 7,214,493; 6,303,121; 6,569,997; 6,905,685; or 6,355,476. In some embodiments, the anti-CD137 antibody is 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1, described in U.S. Patent No. 6,362,325. In some embodiments, the anti-CD137 antibody is an antibody disclosed in the issued U.S. Patent No. 6,974,863 (e.g., 53A2). In some embodiments, the anti-CD137 antibody is an antibody disclosed in the issued U.S. Patent No. 6,210,669 (e.g., 1D8, 3B8, or 3E1). In some embodiments, the antibody is Pfizer's PF-05082566 (PF-2566). In other embodiments, the anti-CD137 antibodies useful in the present invention cross-compete with the anti-CD137 antibodies disclosed herein. In some embodiments, the anti-CD137 antibody binds the same epitope as the anti-CD137 antibodies disclosed herein. In other embodiments, the anti-CD137 antibodies useful in the present invention comprise the six CDRs of the anti-CD137 antibodies disclosed herein.

[0135] Anti-KIR antibody

[0136] Antibodies that specifically bind to KIR block the interaction of killer cell immunoglobulin-like receptors (KIR) on NK cells with their ligands. Blocking these receptors promotes the activation of NK cells and potentially promotes the destruction of tumor cells by the latter. Examples of anti-KIR antibodies have been disclosed in International Publication Nos. WO / 2014 / 055648, WO 2005 / 003168, WO 2005 / 009465, WO 2006 / 072625, WO 2006 / 072626, WO 2007 / 042573, WO 2008 / 084106, WO 2010 / 065939, WO 2012 / 071411, and WO / 2012 / 160448.

[0137] An anti-KIR antibody useful in the present invention is lirilumab (also known as the S241P variant of BMS-986015, IPH2102, or 1-7F9), which was first described in International Publication No. WO 2008 / 084106. Another anti-KIR antibody useful in the present invention is 1-7F9 (also known as IPH2101), described in International Publication No. WO 2006 / 003179. In one embodiment, the anti-KIR antibody for use in the compositions of the present invention cross-competes with lirilumab or 1-7F9 for binding to KIR. In another embodiment, the anti-KIR antibody binds to the same epitope as lirilumab or 1-7F9. In other embodiments, the anti-KIR antibody comprises the six CDRs of lirilumab or 1-7F9.

[0138] Anti-GITR antibody

[0139] An anti-GITR antibody for combination with an anti-PD-1 antibody in a fixed dose can be any anti-GITR antibody that specifically binds to the human GITR target and activates glucocorticoid-induced tumor necrosis factor receptor (GITR). GITR is a member of the TNF receptor superfamily expressed on the surface of multiple types of immune cells, including regulatory T cells, effector T cells, B cells, natural killer (NK) cells, and activated dendritic cells (“anti-GITR agonist antibody”). Specifically, GITR activation increases the proliferation and function of effector T cells and abrogates the inhibition induced by activated T regulatory cells. In addition, GITR stimulation promotes anti-tumor immunity by increasing the activity of other immune cells such as NK cells, antigen-presenting cells, and B cells. Examples of anti-GITR antibodies have been disclosed in International Publication Nos. WO / 2015 / 031667, WO2015 / 184,099, WO2015 / 026,684, WO11 / 028683, and WO / 2006 / 105021, U.S. Patent Nos. 7,812,135 and 8,388,967, and U.S. Publication Nos. 2009 / 0136494, 2014 / 0220002, 2013 / 0183321, and 2014 / 0348841.

[0140] In one embodiment, the anti-GITR antibody useful in the present invention is TRX518 (described in, for example, Schaer et al Curr Opin Immunol. (2012) Apr; 24(2):217–224, and WO / 2006 / 105021). In another embodiment, the anti-GITR antibody useful in the present invention is MK4166 or MK1248, and the antibodies described in WO11 / 028683 and U.S. 8,709,424, including, for example, the VH chain comprising SEQ ID NO:104 and the VL chain comprising SEQ ID NO:105, wherein the SEQ ID NOs are from WO11 / 028683 or U.S. 8,709,424). In certain embodiments, the anti-GITR antibody is the anti-GITR antibody disclosed in WO2015 / 031667, such as the antibody comprising VH CDR 1-3 (comprising SEQ ID NO:31, 71 and 63 of WO2015 / 031667, respectively) and VL CDR1-3 (comprising SEQ ID NO:5, 14 and 30 of WO2015 / 031667). In certain embodiments, the anti-GITR antibody is the anti-GITR antibody disclosed in WO2015 / 184099, such as the antibody Hum231#1 or Hum231#2 or their CDRs, or their derivatives (e.g., pab1967, pab1975 or pab1979). In certain embodiments, the anti-GITR antibody is the anti-GITR antibody disclosed in JP2008278814, WO09 / 009116, WO2013 / 039954, US20140072566, US20140072565, US20140065152, or WO2015 / 026684, or is INBRX-110 (INHIBRx), LKZ-145 (Novartis), or MEDI-1873 (MedImmune). In certain embodiments, the anti-GITR antibody is the anti-GITR antibody described in PCT / US2015 / 033991 (e.g., the antibody comprising the variable regions of 28F3, 18E10 or 19D3). For example, the anti-GITR antibody can be an antibody comprising the following VH and VL chains or their CDRs:

[0141]

[0142] In certain embodiments, the antibody comprising a pair of the above VH and VL light chains or their CDRs comprises a heavy chain constant region of the wild-type or mutant (e.g., thus effectorless) IgG1 isotype. In one embodiment, the anti-GITR antibody comprises the following heavy and light chain amino acid sequences:

[0143] Heavy chain:

[0144]

[0145] Light chain:

[0146]

[0147] Heavy chain:

[0148]

[0149] Light chain:

[0150]

[0151] In certain embodiments, the anti-GITR antibody for use in the compositions of the invention cross-competes with the anti-GITR antibodies described herein (e.g., TRX518, MK4166 or an antibody comprising the amino acid sequences of the VH and VL domains described herein). In some embodiments, the anti-GITR antibody for use in the compositions of the invention binds to the same epitope as the anti-GITR antibodies described herein (e.g., TRX518, MK4166 or an antibody comprising the amino acid sequences of the VH and VL domains described herein). In certain embodiments, the anti-GITR antibody comprises six CDRs of TRX518, MK4166 or an antibody comprising the amino acid sequences of the VH and VL domains described herein. Exemplary pharmaceutical compositions comprise an anti-PD-1 antibody, such as nivolumab, MK-3475 (pembrolizumab) or atezolizumab, and an anti-GITR agonist antibody, such as TRX518, MK4166 or an antibody comprising the amino acid sequences of the VH and VL domains described herein, wherein the ratio of the amount of the anti-PD-1 antibody (e.g., concentration (e.g., mg / ml) or weight (e.g., mg)) to the amount of the anti-GITR antibody (respectively e.g., concentration (e.g., mg / ml) or weight (e.g., mg)) is about 1:1-20; about 1:1-10; about 1:1-5; about 1:2-5; about 1:2-3; about 1:3-5; about 1-20:1; about 1-10:1; about 1-5:1; about 2-5:1; about 2-3:1; or about 3-5:1. For example, the ratio of (i) an anti-PD-1 or anti-PD-L1 antibody to (2) an anti-GITR antibody can be 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1. ":" means "ratio", for example, "1:1-20" refers to a ratio of 1 to a number selected from 1-20. The combination can be administered weekly, bi-weekly, every three weeks or monthly.

[0152] In certain embodiments, an anti-PD-1 antibody or an anti-PD-L1 antibody, such as nivolumab, pembrolizumab or atezolizumab, is co-formulated with an anti-GITR antibody, wherein the anti-GITR antibody is at a dose such as an absolute dose: 0.1 to 1000 mg, such as 0.1 to 100 mg, 0.5 to 100 mg, 1 to 100 mg, 5 to 100 mg, 10 to 100 mg, 50 to 100 mg, 0.1 to 300 mg, 0.5 to 300 mg, 1 to 300 mg, 5 to 300 mg, 10 to 300 mg, 50 to 300 mg, 100 to 300 mg or 200 to 300 mg. Exemplary amounts of the anti-GITR antibody that can be co-formulated with the anti-PD-1 or anti-PD-L1 antibody include about 0.1 mg, about 0.3 mg, about 0.5 mg, about 1 mg, about 3 mg, about 10 mg, about 30 mg, about 100 mg, about 200 mg, about 240 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg or about 1000 mg. In certain embodiments, an anti-PD-1 or anti-PD-L1 antibody is co-formulated with an anti-GITR antibody, wherein the dose of the anti-PD-1 or PD-L1 antibody is the following dose (e.g., absolute dose): 100 - 300 mg, such as 200 - 300 mg, 220 - 260 mg, 230 - 250 mg or 240 mg, such as about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 or about 300 mg.

[0153] In an exemplary embodiment, an anti-PD-1 antibody or an anti-PD-L1 antibody, such as nivolumab, pembrolizumab or atezolizumab, is co-formulated with an anti-GITR antibody, such as an antibody comprising: (i) VH and VL domains comprising the amino acid sequences of SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, or SEQ ID NO: 5 and 6, respectively, or any of these pairs of VH CDR1, CDR2, CDR3 and VL CDR1, CDR2 and CDR3 of the variable regions, or (ii) heavy and light chains comprising the amino acid sequences of SEQ ID NO: 7 and 8, or SEQ ID NO: 7 and 9, respectively, at the following fixed dose ratios: 80 - 300 mg of the anti-PD-1 or anti-PD-L1 antibody to 1 - 1000 mg of the anti-GITR antibody; 80 - 300 mg of the anti-PD-1 or anti-PD-L1 antibody to 1 - 100 mg of the anti-GITR antibody; 80 - 300 mg of the anti-PD-1 or anti-PD-L1 antibody to 10 - 100 mg of the anti-GITR antibody; 80 - 300 mg of the anti-PD-1 or anti-PD-L1 antibody to 10 - 300 mg of the anti-GITR antibody or 80 - 300 mg of the anti-PD-1 or anti-PD-L1 antibody to 100 - 300 mg of the anti-GITR antibody. In an exemplary embodiment, nivolumab is co-formulated with the anti-GITR antibody, wherein the dose of nivolumab is about 80 mg or about 240 mg. The fixed dose combination can be administered by intravenous infusion, for example, by intravenous infusion for about 30, about 30 - 60, about 60 or about 60 - 90 minutes every about 1, about 2, about 3 or about 4 weeks.

[0154] In certain embodiments, about 3 mg / kg of an anti-PD-1 antibody, such as nivolumab, may be administered in combination with an antibody such as the following in a fixed-dose combination: about 0.1 - 10 mg / kg, about 0.1 - 5 mg / kg, about 0.5 - 10 mg / kg, about 0.5 - 5 mg / kg, about 0.5 - 2 mg / kg, about 1 - 2 mg / kg, or about 2 - 5 mg / kg of an anti-GITR antibody, such as TRX518, MK4166, or an antibody comprising the heavy and light chains or variable regions or CDRs described herein, such as by intravenous infusion, such as by intravenous infusion for about 30, about 30 - 60, about 60, or about 60 - 90 minutes every about 1, about 2, about 3, or about 4 weeks. In certain embodiments, about 2 mg / kg of an anti-PD-1 antibody, such as nivolumab or MK-3475, is administered in combination with an antibody such as the following in a fixed-dose combination: about 0.1 - 10 mg / kg, about 0.1 - 5 mg / kg, about 0.5 - 10 mg / kg, about 0.5 - 5 mg / kg, about 0.5 - 2 mg / kg, about 1 - 2 mg / kg, or about 2 - 5 mg / kg of an anti-GITR antibody, such as MK4166 or an antibody comprising the heavy and light chains or variable regions or CDRs described herein, such as by intravenous infusion, such as by intravenous infusion for about 30, about 30 - 60, or about 60 minutes every about 1, about 2, about 3, or about 4 weeks. The amount of antibody in mg / kg can be calculated to determine the weight (mg) or concentration (mg / ml) of antibody required for a fixed-dose ratio formulation. In certain embodiments, the anti-PD-1 antibody and the anti-GITR antibody are provided as a lyophilized composition, such as in a vial or dual-chamber syringe. The lyophilized composition can comprise, for example, about 50 mg of an anti-PD-1 or anti-PD-L1 antibody, such as nivolumab, MK3475, or atezolizumab, and about 5 - 250 mg, about 10 - 250, about 30 - 100 mg, about 30 - 70 mg, or about 50 mg of an anti-GITR antibody, such as TRX-518, MK4166, or an antibody comprising the heavy and light chains or variable regions or CDRs described herein.

[0155] Other antibodies

[0156] In some embodiments, the second antibody combined with the first antibody is an anti-TGFβ antibody, as disclosed in International Publication No. WO / 2009 / 073533. In some embodiments, the second antibody is an anti-IL-10 antibody, as disclosed in International Publication No. WO / 2009 / 073533. In some other embodiments, the second antibody is an anti-B7-H4 antibody, as disclosed in International Publication No. WO / 2009 / 073533. In certain embodiments, the second antibody is an anti-Fas ligand antibody, as disclosed in International Publication No. WO / 2009 / 073533. In some embodiments, the second antibody is an anti-CXCR4 antibody, as disclosed in U.S. Publication No. 2014 / 0322208 (e.g., Ulocuplumab (BMS-936564)). In some embodiments, the second antibody is an anti-mesothelin antibody, as disclosed in U.S. Patent No. 8,399,623. In some embodiments, the second antibody is an anti-HER2 antibody, such as Herceptin (U.S. Patent No. 5,821,337), trastuzumab, or ado-trastuzumab emtansine (Kadcyla, e.g., WO / 2001 / 000244). In an embodiment, the second antibody combined with the first antibody is an anti-CD27 antibody. In an embodiment, the anti-CD-27 antibody is Varlilumab (also known as "CDX-1127" and "1F5"), which is a human IgG1 antibody that acts as an agonist of human CD27 and is disclosed, for example, in U.S. Patent No. 9,169,325. In some embodiments, the second antibody combined with the first antibody is an anti-CD73 antibody. In certain embodiments, the anti-CD73 antibody is CD73.4.IgG2C219S.IgG1.1f.

[0157] Formulations, pharmaceutical compositions, and dosages

[0158] In the formulations of the present invention, the first antibody and the second antibody are formulated in a single composition of the present invention, such as a pharmaceutical composition containing the first antibody, the second antibody, and a pharmaceutically acceptable carrier. In one embodiment, the first antibody is an anti-PD-1 antibody. In another embodiment, the first antibody is an anti-PD-L1 antibody. The anti-PD-L1 antibody can be used in place of the anti-PD-1 antibody in any of the compositions or methods described herein.

[0159] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In certain embodiments, the carrier of the composition containing the antibody is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). The pharmaceutical compositions of the invention may include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

[0160] In one embodiment, a composition comprising a first antibody and a second antibody is provided in a single-use vial. In another embodiment, a composition comprising a first antibody and a second antibody is provided in a multi-use vial.

[0161] In other embodiments, the first antibody (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) is formulated with any known second antibody. In some embodiments, the second antibody is an anti-CTLA4 antibody. In certain embodiments, the anti-CTLA4 antibody is tremelimumab or ipilimumab. In some embodiments, the second antibody is an anti-CD137 antibody. In some embodiments, the anti-CD137 antibody is urelumab. In some embodiments, the second antibody is an anti-LAG3 antibody. In some embodiments, the anti-LAG3 antibody is 25F7. In some embodiments, the second antibody is an anti-GITR antibody. In some embodiments, the anti-GITR antibody is MK4166, TRX518, an antibody comprising the CDRs, variable chains or heavy and light chains of the anti-GITR antibody described in PCT / US2015 / 033991 (e.g., those of 28F3, 18E10 or 19D3), or any other anti-GITR antibody described herein. In some embodiments, the second antibody is an anti-KIR antibody. In some embodiments, the anti-KIR antibody is 1-7F9 or lirilumab. In some embodiments, the second antibody is an anti-TGFβ antibody, an anti-IL-10 antibody, an anti-B7-H4 antibody, an anti-Fas ligand antibody, an anti-CXCR4 antibody, an anti-mesothelin antibody, an anti-CD27 antibody, an anti-CD73 antibody or any combination thereof.

[0162] In some embodiments, the first antibody and the second antibody are present in the composition at a fixed dose (i.e., a fixed ratio). In other embodiments, the fixed dose is at least about 1:200 to at least about 200:1, at least about 1:150 to at least about 150:1, at least about 1:100 to at least about 100:1, at least about 1:75 to at least about 75:1, at least about 1:50 to at least about 50:1, at least about 1:25 to at least about 25:1, at least about 1:10 to at least about 10:1, at least about 1:5 to at least about 5:1, at least about 1:4 to at least about 4:1, at least about 1:3 to at least about 3:1, or at least about 1:2 to at least about 2:1 mg anti-PD-1 antibody (or anti-PD-L1 antibody) per mg of the second antibody. In some embodiments, the fixed dose is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1:100, about 1:120, about 1:140, about 1:160, about 1:180, or about 1:200 anti-PD-1 antibody (or anti-PD-L1 antibody) per second antibody. In some embodiments, the fixed dose is at least about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 120:1, about 140:1, about 160:1, about 180:1, or about 200:1 mg of the first antibody per mg of the second antibody.

[0163] In other embodiments, the composition comprises a ratio (e.g., 200:1 to 1:200, 100:1 to 1:100, 20-1:1 to 1:1-20, or any ratio disclosed herein) of a first antibody and a second antibody, wherein the composition has one or more of the following characteristics: (i) after storage at 2°C to 8°C for 6 months, the aggregation in the composition is comparable to the aggregation in a reference composition (i.e., a composition comprising the first antibody or the second antibody); (ii) after storage at 2°C to 8°C for 6 months, the fragmentation in the composition is comparable to the aggregation in a reference composition (i.e., a composition comprising the first antibody or the second antibody); (iii) after storage at 2°C to 8°C for 6 months, the deamidation of the first antibody or the second antibody in the composition is comparable to the deamidation of the antibody in a reference composition (i.e., a composition comprising the first antibody or the second antibody); (iv) after storage at 2°C to 8°C for 6 months, the level of particulate matter in the composition is comparable to the level of a specific substance in a reference composition (i.e., a composition comprising the first antibody or the second antibody); and (v) any combination thereof.

[0164] In other embodiments, the composition comprises a ratio (e.g., 200:1 to 1:200, 100:1 to 1:100, 20-1:1 to 1:1-20, or any ratio disclosed herein) of a first antibody and a second antibody, wherein the composition has one or more of the following characteristics: (i) after storage at 25°C for 6 months, the aggregation in the composition is comparable to the aggregation in a reference composition (i.e., a composition comprising the first antibody or the second antibody); (ii) after storage at 25°C for 6 months, the fragmentation in the composition is comparable to the aggregation in a reference composition (i.e., a composition comprising the first antibody or the second antibody); (iii) after storage at 25°C for 6 months, the deamidation of the first antibody or the second antibody in the composition is comparable to the deamidation of the antibody in a reference composition (i.e., a composition comprising the first antibody or the second antibody); (iv) after storage at 25°C for 6 months, the level of particulate matter in the composition is comparable to the level of a specific substance in a reference composition (i.e., a composition comprising the first antibody or the second antibody); and (v) any combination thereof.

[0165] In some embodiments, the aggregation of the composition is measured by the level of high molecular weight (HMW) substances in the composition, which can be detected by size exclusion high performance liquid chromatography (SE-HPLC). In some embodiments, the fragmentation of the composition is measured by the level of low molecular weight (LMW) substances in the composition, which is detected by SE-HPLC. In some embodiments, the deamidation of the composition is measured by the level of acidic charge variants in the composition, which is detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing (iCIEF).

[0166] In some embodiments, the amount of the anti-PD-1 antibody in the composition is at least about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, or about 300 mg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is at least about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, or about 500 mg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is from about 60 mg to about 300 mg, from about 60 mg to about 100 mg, from about 100 mg to about 200 mg, or from about 200 mg to about 300 mg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is from about 300 mg to about 500 mg, from about 300 mg to about 450 mg, from about 300 mg to about 400 mg, from about 300 mg to about 350 mg, from about 350 mg to about 500 mg, from about 400 mg to about 500 mg, or from about 450 mg to about 500 mg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is at least about 80 mg, about 160 mg, or about 240 mg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is at least about 240 mg or at least about 80 mg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is at least about 360 mg or at least about 480 mg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg or at least about 5 mg / kg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is from about 0.5 mg / kg to about 5 mg / kg, from about 0.5 mg / kg to about 5 mg / kg, from about 0.5 mg / kg to about 3 mg / kg or from about 0.5 mg / kg to about 2 mg / kg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is at least about 1 mg / kg. In some embodiments, the anti-PD-1 antibody is nivolumab or pembrolizumab.

[0167] In some embodiments, the anti-PD-1 antibody is pembrolizumab, and the amount of the anti-PD-1 antibody in the composition is at least about 50 mg, at least about 75 mg, at least about 100 mg, at least about 150 mg, at least about 200 mg, at least about 250 mg, or at least about 300 mg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is at least about 100 mg or at least about 200 mg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is at least about 300 mg, at least about 350 mg, at least about 400 mg, at least about 450 mg, or at least about 500 mg. In some embodiments, the anti-PD-1 antibody is pembrolizumab, and the amount of the anti-PD-1 antibody for treating a disease or disorder can be a weight-based dose, such as at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 15 mg / kg or at least about 20 mg / kg. In some embodiments, the amount of the anti-PD-1 antibody that can be used to treat a disease or disorder is a weight-based dose, such as at least about 1 mg / kg, at least about 2 mg / kg or at least about 10 mg / kg. In some embodiments, the second antibody is an anti-CTLA4 antibody, and the fixed dose is about 1:1, about 3:1 or about 1:3 mg anti-PD-1 antibody to mg anti-CTLA4 antibody. In some embodiments, the amount of the anti-CTLA4 antibody in the composition is at least about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg or about 300 mg. In some embodiments, the amount of the anti-CTLA4 antibody in the composition is from about 60 mg to about 300 mg, from about 60 mg to about 100 mg, from about 100 mg to about 200 mg, or from about 200 mg to about 300 mg. In some embodiments, the amount of the anti-CTLA4 antibody in the composition is at least about 80 mg, about 160 mg or about 240 mg. In some embodiments, the amount of the anti-CTLA4 antibody in the composition is at least about 240 mg. In some embodiments, the amount of the anti-CTLA4 antibody in the composition is at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg or at least about 5 mg / kg. In some embodiments, the amount of the anti-CTLA4 antibody in the composition is used as a weight-based dose, such as from about 1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 5 mg / kg or from about 2 mg / kg to about 5 mg / kg. In some embodiments, the amount of the anti-CTLA4 antibody in the composition is at least about 3 mg / kg.In some embodiments, (i) the amount of X is about 240 mg and the amount of Y is about 80 mg; (ii) the amount of X is about 80 mg and the amount of Y is about 80 mg; (iii) the amount of X is about 160 mg and the amount of Y is about 160 mg; (iv) the amount of X is about 240 mg and the amount of Y is about 240 mg; or (v) the amount of X is about 80 mg and the amount of Y is about 240 mg.

[0168] In some embodiments, the second antibody is an anti-KIR antibody and the fixed dose is about 30:1, about 10:1, about 3:1, about 1:1, about 1:2 or about 3:10 mg anti-PD-1 antibody per mg anti-KIR antibody.

[0169] In some embodiments, the second antibody is an anti-LAG3 antibody and the fixed dose is about 80:3, about 80:1, about 12:1, about 3:1 or about 1:1 mg anti-PD-1 antibody per mg anti-LAG3 antibody. In some embodiments, the amount of anti-LAG3 antibody in the composition is at least about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg or about 350 mg. In some embodiments, the amount of anti-LAG3 antibody in the composition is about 60 to about 350 mg, about 60 to about 300 mg, about 100 to about 300 mg, or about 150 to about 250 mg. In some embodiments, the amount of anti-LAG3 antibody is at least about 240 mg.

[0170] In some embodiments, the second antibody is an anti-CD137 antibody and the fixed dose is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 10:1, about 5:1, about 4:1 or about 2:1 mg anti-PD-1 antibody per mg anti-CD137 antibody. In some embodiments, the amount of anti-CD137 antibody in the composition is at least about 1 mg, at least about 2 mg, at least about 3 mg, at least about 4 mg, at least about 5 mg, at least about 6 mg, at least about 7 mg, at least about 8 mg, at least about 9 mg, at least about 10 mg, at least about 12 mg, at least about 15 mg, or at least about 20 mg. In some embodiments, the amount of anti-CD137 antibody in the composition is about 1 mg to about 20 mg, about 1 mg to about 15 mg, about 5 mg to about 12 mg or about 5 mg to about 10 mg. In some embodiments, the amount of anti-CD137 antibody in the composition is at least about 8 mg.

[0171] In some embodiments, the second antibody is an anti-CD73 antibody, and the fixed dose is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 10:1, about 5:1, about 4:1 or about 2:1 mg of anti-PD-1 antibody per mg of anti-CD73 antibody. In some embodiments, the amount of anti-CD73 antibody in the composition is from about 100 mg to about 2000 mg or from about 150 mg to about 1600 mg. In some embodiments, the amount of anti-CD73 antibody in the composition is at least about 100 mg, 150 mg, 200 mg, 300 mg, 500 mg, 600 mg, 800 mg, 1000 mg, 1200 mg or 1600 mg.

[0172] In certain embodiments, the anti-CD73 antibody CD73.4.IgG2C219S.IgG1.1f and nivolumab are administered at a fixed dose in one of the following combined doses: 50 mg of anti-CD73 antibody and 240 mg of nivolumab every two weeks; 50 mg of anti-CD73 antibody and 360 mg of nivolumab every three weeks; 150 mg of anti-CD73 antibody and 240 mg of nivolumab every two weeks; 150 mg of anti-CD73 antibody and 360 mg of nivolumab every three weeks; 300 mg of anti-CD73 antibody and 240 mg of nivolumab every two weeks; 300 mg of anti-CD73 antibody and 360 mg of nivolumab every three weeks; 600 mg of anti-CD73 antibody and 240 mg of nivolumab every two weeks; 600 mg of anti-CD73 antibody and 360 mg of nivolumab every three weeks; 1200 mg of anti-CD73 antibody and 240 mg of nivolumab every two weeks; 1200 mg of anti-CD73 antibody and 360 mg of nivolumab every three weeks; 1600 mg of anti-CD73 antibody and 240 mg of nivolumab every two weeks; 1600 mg of anti-CD73 antibody and 360 mg of nivolumab every three weeks; 2000 mg of anti-CD73 antibody and 240 mg of nivolumab every two weeks; 2000 mg of anti-CD73 antibody and 360 mg of nivolumab every three weeks.

[0173] In some embodiments, the PD-1 antibody and the second antibody are combined using the current formulations of the two antibodies (e.g., combining 2 ml of anti-PD-1 antibody in a citrate-based buffer with 2 ml of anti-CTLA4 antibody in a Tris-based buffer without buffer exchange).

[0174] In some embodiments, the composition comprises one or more additional components selected from the group consisting of bulking agents, stabilizers, chelating agents, surfactants, buffers, and any combination thereof. In some embodiments, the buffer comprises a citrate buffer, Tris buffer, Tris-Cl buffer, histidine buffer, TAE buffer, HEPES buffer, TBE buffer, sodium phosphate buffer, MES buffer, ammonium sulfate buffer, potassium phosphate buffer, potassium thiocyanate buffer, succinate buffer, tartrate buffer, DIPSO buffer, HEPPSO buffer, POPSO buffer, PIPES buffer, PBS buffer, MOPS buffer, acetate buffer, phosphate buffer, cacodylate buffer, glycine buffer, sulfate buffer, imidazole buffer, guanidine hydrochloride buffer, phosphate-citrate buffer, borate buffer, malonate buffer, 3-methylpyridine buffer, 2-methylpyridine buffer, 4-methylpyridine buffer, 3,5-dimethylpyridine buffer, 3,4-dimethylpyridine buffer, 2,4-dimethylpyridine buffer, Aces, diethylmalonate buffer, N-methylimidazole buffer, 1,2-dimethylimidazole buffer, TAPS buffer, bis-Tris buffer, L-arginine buffer, lactate buffer, glycolate buffer.

[0175] In some embodiments, the PD-1 antibody and the second antibody are formulated in a buffer based on the buffer conditions of one of the two separate antibody formulations. In some embodiments, the buffer conditions used are the buffer conditions of the anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is nivolumab, and the two antibodies are formulated in the citrate-based buffer system of nivolumab. In some embodiments, the buffer is a citrate buffer.

[0176] In some embodiments, the PD-1 antibody and the second antibody are formulated in buffer conditions different from the buffer conditions of either of the two antibodies themselves. In some embodiments, the buffer is a citrate-based buffer. In some embodiments, the concentration of citrate in the buffer is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 50 mM. In some embodiments, the concentration of citrate is from about 5 mM to about 50 mM, in some embodiments from about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 15 mM, about 10 mM to about 30 mM, or about 15 mM to about 25 mM. In some embodiments, the concentration of citrate is about 10 mM. In some embodiments, the concentration of citrate is about 20 mM.

[0177] In some embodiments, the buffer used is a Tris-based buffer. In some embodiments, the Tris buffer is a Tris-Cl buffer. In some embodiments, the concentration of Tris-Cl in the buffer is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM or about 50 mM. In some embodiments, the concentration of Tris-Cl is from about 5 mM to about 50 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 10 mM to about 30 mM or from about 15 mM to about 25 mM. In some embodiments, the concentration of Tris-Cl is about 20 mM.

[0178] In some embodiments, the buffer used is a histidine-based buffer. In some embodiments, the concentration of histidine is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM or about 50 mM. In some embodiments, the concentration of histidine is from about 5 mM to about 50 mM, from about 5 mM to about 40 mM, from about 5 mM to about 30 mM, from about 5 mM to about 25 mM or from about 10 mM to about 15 mM. In some embodiments, the concentration of histidine is about 20 mM.

[0179] In some embodiments, the buffer used is a Tris-citrate buffer. In some embodiments, the concentration of Tris-Cl is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM or about 50 mM, and the concentration of citrate is at least about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM or about 50 mM. In some embodiments, the concentration of Tris-Cl is from about 5 to about 20 mM, from about 5 to about 15 mM, or from about 10 to about 15 mM, and the concentration of citrate is from about 1 mM to about 15 mM, from about 1 mM to about 10 mM, or from about 5 mM to about 10 mM. In some embodiments, the concentration of Tris-Cl is about 13.3 mM and the concentration of citrate is about 6.7 mM.

[0180] In some embodiments, the pH of the composition is at least about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. In some embodiments, the pH of the composition is from about 5.0 to about 8.0, from about 5.5 to about 6.5, from about 6.0 to about 7.0, or from about 6.5 to about 7.5. In some embodiments, the pH is about 6.0, in other embodiments, the pH is about 7.0. In other embodiments, the pH is about 6.2. In other embodiments, the pH is about 6.5. In other embodiments, the pH is about 6.6. In other embodiments, the pH is about 5.5.

[0181] In some embodiments, the composition of the present invention further comprises a bulking agent. The bulking agent can be selected from NaCl, mannitol, glycine, alanine, and any combination thereof. In other embodiments, the composition of the present invention comprises a stabilizer. The stabilizer can be selected from sucrose, trehalose, raffinose, arginine; or any combination thereof. In other embodiments, the composition of the present invention comprises a surfactant. The surfactant can be selected from polysorbate 80 (PS80), polysorbate 20 (PS20), and any combination thereof. In certain embodiments, the composition further comprises a chelating agent. The chelating agent can be selected from diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid, nitrilotriacetic acid, and any combination thereof.

[0182] In one embodiment, the composition comprises NaCl, mannitol, pentetic acid (DTPA), sucrose, PS80, and any combination thereof. In another embodiment, the composition comprises NaCl at the following concentrations: at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 110 mM, at least about 120 mM, at least about 130 mM, at least about 140 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, at least about 225 mM, at least about 250 mM, at least about 275 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM, or at least about 450 mM. In other embodiments, the composition comprises from about 10 to about 200 mM NaCl, from about 25 to about 150 mM NaCl, from about 40 to about 125 mM NaCl, from about 25 to about 75 mM NaCl, from about 50 to about 100 mM NaCl, or from about 75 to 125 mM NaCl. In some embodiments, the composition comprises about 100 mM NaCl. In certain embodiments, the composition comprises about 50 mM NaCl. In other embodiments, the composition comprises about 83.3 mM NaCl. In other embodiments, the composition comprises about 96.15 mM NaCl. In a particular embodiment, the composition comprises about 78.57 mM NaCl.

[0183] In certain embodiments, the composition comprises mannitol ( % w / v) USP at the following concentrations: at least about 0.25%, at least about 0.5%, at least about 0.75%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 7.5%, or at least about 10%. In other embodiments, the composition comprises from about 0.5% to about 5% mannitol, from about 0.5% to about 4% mannitol, from about 0.5% to about 1.5% mannitol, from about 1% to about 2% mannitol, or from about 2.5% to about 3.5% mannitol. In other embodiments, the composition comprises about 1% mannitol. In still further other embodiments, the composition comprises about 3.0% mannitol. In some embodiments, the composition comprises about 1.67% mannitol. In certain embodiments, the composition comprises about 1.15% mannitol. In a particular embodiment, the composition comprises about 1.86% mannitol.

[0184] In other embodiments, the composition comprises pentetic acid (DTPA) USP at the following concentrations: at least about 5 μM, at least about 10 μM, at least about 15 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 40 μM, at least about 50 μM, at least about 60 μM, at least about 70 μM, at least about 75 μM, at least about 80 μM, at least about 90 μM, at least about 100 μM, at least about 110 μM, at least about 120 μM, at least about 130 μM, at least about 140 μM, at least about 150 μM, at least about 175 μM or at least about 200 μM. In some embodiments, the composition comprises from about 10 μM to about 200 μM DTPA, from about 10 μM to about 150 μM DTPA, from about 10 μM to about 100 μM DTPA, from about 10 μM to about 30 μM DTPA, from about 50 μM to about 100 μM DTPA, or from about 75 μM to about 125 μM DTPA. In other embodiments, the composition comprises about 100 μM of DTPA. In certain embodiments, the composition comprises about 20 μM of DTPA. In other embodiments, the composition comprises about 73.3 μM of DTPA. In particular embodiments, the composition comprises about 50 μM of DTPA. In specific embodiments, the composition comprises about 93.85 μM of DTPA. In certain embodiments, the composition comprises about 65.71 μM of DTPA.

[0185] In some embodiments, the composition comprises polysorbate 80, NF (PS80) at the following concentrations (% w / v): at least about 0.005%, at least about 0.01%, at least about 0.015%, at least about 0.02%, at least about 0.03%, at least about 0.04%, at least about 0.05%, at least about 0.06%, at least about 0.07%, at least about 0.08%, at least about 0.09%, or at least about 0.1%. In other embodiments, the composition comprises from about 0.005% to about 0.1% PS80, from about 0.005% to about 0.02% PS80, from about 0.005% to about 0.05% PS80, from about 0.01% to about 0.02% PS80, from about 0.02% to about 0.1% PS80 or from about 0.01% to about 0.03% PS80. In other embodiments, the composition comprises PS80 at a concentration of about 0.01%. In other embodiments, the composition comprises PS80 at a concentration of about 0.04%. In some embodiments, the composition comprises PS80 at a concentration of about 0.013%. In certain embodiments, the composition comprises PS80 at a concentration of about 0.05%. In some embodiments, the composition comprises PS80 at a concentration of about 0.02%. In other embodiments, the composition comprises PS80 at a concentration of about 0.012%. In specific embodiments, the composition comprises PS80 at a concentration of about 0.23%.

[0186] In certain embodiments, the composition comprises sucrose at the following concentrations (% w / v): at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.5%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5%, at least about 10%, at least about 12% or at least about 15% sucrose. In other embodiments, the composition comprises from about 1% to about 10%, from about 2% to about 10%, from about 5% to about 10%, from about 5% to about 7%, or from about 7.5% to about 10% sucrose. In other embodiments, the composition comprises about 6% sucrose. In other embodiments, the composition comprises about 8.5% sucrose. In other embodiments, the composition comprises about 8.0% sucrose.

[0187] In certain embodiments, the composition comprises nivolumab and ipilimumab in Tris-citrate buffer. In some embodiments, the composition comprises nivolumab and ipilimumab in a 1:1 ratio in a buffer comprising about 13.3 mM Tris (or 13.3 mM Tris ± 10%, 20%, 30%, 40%, or 50%), about 6.7 mM citrate (or 6.7 mM citrate ± 10%, 20%, 30%, 40% or 50%), about 1.67% mannitol (1.67% mannitol ± 10%, 20%, 30%, 40% or 50%), about 83.3 mM NaCl (or 83.3 mM NaCl ± 10%, 20%, 30%, 40% or 50%), about 73.3 μM DTPA (or 73.3 μM DTPA ± 10%, 20%, 30%, 40% or 50%) and about 0.013% PS80 (or 0.013% PS80 ± 10%, 20%, 30%, 40% or 50%), with a pH of about 6.2. In some embodiments, the composition comprises nivolumab and ipilimumab in a 3:1 ratio in Tris-citrate buffer, the Tris-citrate buffer comprising about 1.15% mannitol (or 1.15% mannitol ± 10%, 20%, 30%, 40% or 50%), about 96.15 mM NaCl (or 96.15 mM NaCl ± 10%, 20%, 30%, 40% or 50%), about 93.85 μM DTPA (or 93.85 μM DTPA ± 10%, 20%, 30%, 40% or 50%) and about 0.012% PS80 (or 0.012% PS80 ± 10%, 20%, 30%, 40% or 50%), with a pH of about 6.6. In some embodiments, the composition comprises nivolumab and ipilimumab in a 1:3 ratio in Tris-citrate buffer, the Tris-citrate buffer comprising about 1.86% mannitol (or 1.86% mannitol ± 10%, 20%, 30%, 40%, or 50%), about 78.57 mM NaCl (or 78.57 mM NaCl ± 10%, 20%, 30%, 40% or 50%), about 65.71 μM DTPA (or 65.71 μM DTPA ± 10%, 20%, 30%, 40% or 50%) and about 0.023% PS80 (or 0.023% PS80 ± 10%, 20%, 30%, 40% or 50%), with a pH of about 6.0.

[0188] In other embodiments, the composition comprises nivolumab and ipilimumab in a histidine buffer. In some embodiments, the composition comprises nivolumab and ipilimumab in a 3:1 ratio in a 20 mM histidine buffer (or 20 mM histidine buffer ± 10%, 20%, 30%, 40%, or 50%), the histidine buffer comprising approximately 50 mM NaCl (or 50 mM NaCl ± 10%, 20%, 30%, 40%, or 50%), approximately 50 μM DTPA (or 50 μM DTPA ± 10%, 20%, 30%, 40%, or 50%), approximately 6% sucrose (or 6% sucrose ± 10%, 20%, 30%, 40%, or 50%), and approximately 0.05% PS80 (or 0.05% PS80 ± 10%, 20%, 30%, 40%, or 50%), with a pH of approximately 6. In some embodiments, the composition comprises nivolumab and ipilimumab in a 3:1 ratio in an approximately 20 mM histidine buffer, the histidine buffer comprising approximately 50 mM NaCl (or 50 mM NaCl ± 10%, 20%, 30%, 40%, or 50%), approximately 50 μM DTPA (or 50 μM DTPA ± 10%, 20%, 30%, 40%, or 50%), approximately 6% sucrose (or 6% sucrose ± 10%, 20%, 30%, 40%, or 50%), and approximately 0.05% PS80 (or 0.05% PS80 ± 10%, 20%, 30%, 40%, or 50%), with a pH of approximately 7. In some embodiments, the composition comprises nivolumab and ipilimumab in a 3:1 ratio in an approximately 20 mM histidine buffer (or 20 mM histidine buffer ± 10%, 20%, 30%, 40%, or 50%), the histidine buffer comprising approximately 50 μM DTPA (or 50 μM DTPA ± 10%, 20%, 30%, 40%, or 50%), approximately 8.5% sucrose (or 8.5% sucrose ± 10%, 20%, 30%, 40%, or 50%), and approximately 0.05% PS80 (or 0.05% PS80 ± 10%, 20%, 30%, 40%, or 50%), with a pH of approximately 6. In some embodiments, the composition comprises nivolumab and ipilimumab in a 1:1, 3:1, or 1:3 ratio in a histidine buffer (20 mM ± 10%, 20%, 30%, 40%, or 50%), the histidine buffer comprising 5 μM DTPA (or 50 μM DTPA ± 10%, 20%, 30%, 40%, or 50%), 0.05% PS80 (or 0.05% PS80 ± 10%, 20%, 30%, 40%, or 50%), and 8.0% sucrose (or 8.0% sucrose ± 10%, 20%, 30%, 40%, or 50%), with a pH of 5.5, 6.0, or 6.5. In one embodiment, the composition comprises nivolumab and ipilimumab in a citrate buffer.In another embodiment, the composition comprises nivolumab and ipilimumab in a 3:1 ratio in about 20 mM citrate buffer (or 20 mM citrate buffer ± 10%, 20%, 30%, 40%, or 50%), the citrate buffer comprising about 50 mM NaCl (or 50 mM NaCl ± 10%, 20%, 30%, 40% or 50%), about 50 μM DTPA (or 50 μM DTPA ± 10%, 20%, 30%, 40% or 50%), about 6% sucrose (or 6% sucrose ± 10%, 20%, 30%, 40% or 50%) and about 0.05% PS80 (or 0.05% PS80 ± 10%, 20%, 30%, 40% or 50%), with a pH of about 6. In other embodiments, the composition comprises nivolumab and ipilimumab in a 3:1 ratio in about 20 mM citrate buffer (or 20 mM citrate buffer ± 10%, 20%, 30%, 40%, or 50%), the citrate buffer comprising about 50 mM NaCl (or 50 mM NaCl ± 10%, 20%, 30%, 40% or 50%), about 20 μM DTPA (or 20 μM DTPA ± 10%, 20%, 30%, 40% or 50%), about 3% mannitol (or 3% mannitol ± 10%, 20%, 30%, 40% or 50%) and about 0.04% PS80 (or 0.04% PS80 ± 10%, 20%, 30%, 40% or 50%), with a pH of about 6. In other embodiments, the composition comprises nivolumab and ipilimumab in a 1:1 ratio in about 20 mM citrate buffer (or 20 mM citrate buffer ± 10%, 20%, 30%, 40%, or 50%), the citrate buffer comprising about 50 mM NaCl (or 50 mM NaCl ± 10%, 20%, 30%, 40% or 50%), about 100 μM DTPA (or 100 μM DTPA ± 10%, 20%, 30%, 40% or 50%), about 3% mannitol (or 3% mannitol ± 10%, 20%, 30%, 40% or 50%) and about 0.02% PS80 (or 0.02% PS80 ± 10%, 20%, 30%, 40% or 50%), with a pH of about 6.In certain embodiments, the composition comprises nivolumab and ipilimumab in a 1:1 ratio in about 20 mM citrate buffer (or 20 mM citrate buffer ± 10%, 20%, 30%, 40% or 50%), the citrate buffer comprising about 50 mM NaCl (or 50 mM NaCl ± 10%, 20%, 30%, 40% or 50%), about 100 μM DTPA (or 100 μM DTPA ± 10%, 20%, 30%, 40% or 50%), about 3% mannitol (or 3% mannitol ± 10%, 20%, 30%, 40% or 50%) and about 0.02% PS80 (or 0.02% PS80 ± 10%, 20%, 30%, 40% or 50%), with a pH of about 6.5. In some embodiments, the composition comprises nivolumab and ipilimumab in a 1:1 ratio in about 20 mM citrate buffer (or 20 mM citrate buffer ± 10%, 20%, 30%, 40% or 50%), the citrate buffer comprising about 100 mM NaCl (or 100 mM NaCl ± 10%, 20%, 30%, 40% or 50%), about 100 μM DTPA (or 100 μM DTPA ± 10%, 20%, 30%, 40% or 50%), about 1.0% mannitol (or 1.0% mannitol ± 10%, 20%, 30%, 40% or 50%) and about 0.02% PS80 (or 0.02% PS80 ± 10%, 20%, 30%, 40% or 50%), with a pH of about 6.5. In other embodiments, the composition comprises nivolumab and ipilimumab in a 1:1 ratio in about 20 mM citrate buffer (or 20 mM citrate buffer ± 10%, 20%, 30%, 40% or 50%), the citrate buffer comprising about 50 mM NaCl (or 50 mM NaCl ± 10%, 20%, 30%, 40% or 50%), about 100 μM DTPA (or 100 μM DTPA ± 10%, 20%, 30%, 40% or 50%), about 6% sucrose (or 6% sucrose ± 10%, 20%, 30%, 40% or 50%) and about 0.02% PS80 (or 0.02% PS80 ± 10%, 20%, 30%, 40% or 50%), with a pH of about 6.0.

[0189] In some embodiments, the composition comprises nivolumab and ipilimumab in a 1:3 ratio, which comprises about 4.62 mg / ml nivolumab, about 1.54 mg / ml ipilimumab, about 18.5 mM Tris hydrochloride, about 1.5 mM sodium citrate dihydrate, about 96.2 mM NaCl, about 1.2% mannitol, about 93.9 μM diethylenetriaminepentaacetic acid, and about 0.012% PS80, with a pH of about 6.0.

[0190] In some embodiments, the composition comprises nivolumab and ipilimumab in a 1:3 ratio, which comprises about 4.61 mg / ml nivolumab, about 1.54 mg / ml ipilimumab, about 18.46 mM Tris hydrochloride, about 1.54 mM sodium citrate dihydrate, about 96.15 mM NaCl, about 1.15% mannitol, about 93.85 μM pentetic acid, and about 0.012% PS80, with a pH of about 6.3.

[0191] In some embodiments, the pharmaceutical composition comprises 30 mg nivolumab and 90 mg ipilimumab per vial. In other embodiments, the composition comprises 40 mg nivolumab and 120 mg ipilimumab per vial.

[0192] In other embodiments, the composition comprises a third antibody. In some embodiments, the third antibody is any antibody disclosed herein.

[0193] Stability of the composition

[0194] In one embodiment, the composition disclosed herein is stable at about 0 °C, about 5 °C, about 10 °C, about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, or about 55 °C for at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 2 years, or at least about 5 years.

[0195] In another embodiment, the composition shows a change in acidic peaks (e.g., deamidation) of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 5 °C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In other embodiments, the composition shows a change in acidic peaks of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 25 °C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, the composition shows a change in acidic peaks of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 40 °C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, the acidic peaks are measured using imaging capillary isoelectric focusing assay (cIEF).

[0196] In some embodiments, the deamidation of the compositions of the invention is comparable to the deamidation of a reference composition (a composition comprising a first antibody or a second antibody) if the compositions exhibit a change in acidic peaks (e.g., deamidation) of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% compared to the acidic peaks of the reference composition.

[0197] In certain embodiments, the composition exhibits a change in high molecular weight (HMW) peaks (e.g., aggregation) of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 5°C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, the composition exhibits a change in HMW peaks of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 25°C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, the composition exhibits a change in HMW peaks of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 40°C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, the composition exhibits a change in HMW peaks of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, or less than about 0.1%. In certain embodiments, the composition exhibits HMW peaks of about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% after storage at about 5°C, about 25°C, or about 40°C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, high molecular weight peaks are measured using chromatography. In some embodiments, the chromatography is size exclusion chromatography.

[0198] In some embodiments, the aggregation (e.g., the level of HMW species) of the compositions of the invention is comparable to that of a reference composition (a composition comprising a first antibody or a second antibody) if the compositions exhibit a change in the HMW species peak of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% compared to the HMW species peak of the reference composition.

[0199] In some embodiments, the composition exhibits a change in the main peak of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 5 °C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, the composition exhibits a change in the main peak of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 25 °C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, the composition exhibits a change in the main peak of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 40 °C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, the composition exhibits a change in the main peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%. In some embodiments, the main peak is measured using imaging capillary isoelectric focusing assay (cIEF).

[0200] In some embodiments, the composition exhibits a change in low molecular weight (LMW) peaks (e.g., fragmentation) of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 5 °C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, the composition exhibits a change in LMW peaks of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 25 °C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, the composition exhibits a change in LMW peaks of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at about 40 °C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, the composition exhibits a change in LMW peaks of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%. In certain embodiments, the composition exhibits LMW peaks of about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% after storage at about 5 °C, about 25 °C, or about 40 °C for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months, or about 1 year. In some embodiments, low molecular weight peaks are measured using chromatography. In some embodiments, the chromatography is size exclusion chromatography.

[0201] In some embodiments, if a composition comprising a first and a second antibody exhibits a change in LMW species peaks of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% compared to the LMW species peaks of a reference composition, then the fragmentation (e.g., the level of LMW species) of the composition of the invention is comparable to the fragmentation of the reference composition (a composition comprising the first antibody or the second antibody).

[0202] Methods for preparing the compositions disclosed herein

[0203] In one embodiment, the present invention relates to a method for manufacturing any of the compositions disclosed herein. In another embodiment, a formulation comprising an anti-PD-1 antibody drug product is mixed with a formulation comprising a second antibody drug product to obtain the desired ratio in the final drug product, and there is no buffer change. In other embodiments, the final composition is in Tris-citrate buffer.

[0204] In some embodiments, a formulation comprising an anti-PD-1 antibody drug substance and a formulation comprising a second antibody drug substance are subjected to buffer exchange and / or concentration and then mixed to obtain the desired ratio in the final drug product.

[0205] In other embodiments, the composition is diluted before use. In certain embodiments, the composition is diluted with 0.9% Sodium Chloride Injection USP or 5% Dextrose Injection USP before use. In other embodiments, the composition is diluted to obtain an infusion having the desired concentrations of the first and second antibodies. In other embodiments, the final concentrations of the first and second antibodies are from about 1 mg / ml to about 500 mg / ml, from about 1 mg / ml to about 450 mg / ml, from about 1 mg / ml to about 400 mg / ml, from about 1 mg / ml to about 350 mg / ml, from about 1 mg / ml to about 300 mg / ml, from about 1 mg / ml to about 250 mg / ml, from about 1 mg / ml to about 200 mg / ml, from about 1 mg / ml to about 150 mg / ml, from about 1 mg / ml to about 100 mg / ml, from about 1 mg / ml to about 90 mg / ml, from about 1 mg / ml to about 80 mg / ml, from about 1 mg / ml to about 70 mg / ml, from about 1 mg / ml to about 60 mg / ml, from about 1 mg / ml to about 50 mg / ml, from about 1 mg / ml to about 40 mg / ml, from about 1 mg / ml to about 30 mg / ml, from about 1 mg / ml to about 20 mg / ml, from about 1 mg / ml to about 15 mg / ml, from about 1 mg / ml to about 10 mg / ml, from about 1 mg / ml to about 9 mg / ml, from about 1 mg / ml to about 8 mg / ml, from about 1 mg / ml to about 7 mg / ml, from about 1 mg / ml to about 6 mg / ml, from about 1 mg / ml to about 5 mg / ml, from about 1 mg / ml to about 4 mg / ml, from about 1 mg / ml to about 3 mg / ml, from about 1 mg / ml to about 2 mg / ml, from about 0.5 mg / ml to about 3 mg / ml, from about 50 mg / ml to about 400 mg / ml, or from about 100 mg / ml to about 300 mg / ml.

[0206] In certain embodiments, the diluted infusion is stored at room temperature for no more than about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 hour after dilution. In some embodiments, the diluted infusion is stored under refrigeration (about 2°C to about 8°C) for no more than about 1 week, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, or about 12 hours.

[0207] The method of the present invention

[0208] The present disclosure provides a method of treating a subject having a disease or disorder with any of the compositions disclosed herein. In one embodiment, the method involves administering a pharmaceutical composition comprising an amount X of a first antibody and an amount Y of a second antibody, wherein the first antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody, and wherein the ratio of the amount of the first antibody to the amount of the second antibody is present in the composition at a fixed dose ratio of from about 100:1 to about 1:100.

[0209] In some embodiments, the disease or disorder is an infectious disease. In other embodiments, the disease or disorder is cancer. In other embodiments, the cancer is melanoma cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer; chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia; childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma; environmentally induced cancers, including cancers induced by asbestos; and any combination thereof. In other embodiments, the cancer is lung cancer, metastatic melanoma, glioblastoma, or renal cell carcinoma.

[0210] In certain embodiments, the cancer is squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, kidney cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostatic adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer (or cancers), gastric cancer, germ cell tumors, pediatric sarcomas, sinonasal natural killer, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal area cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvic cancer, central nervous system tumors (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancers (including asbestos-induced cancers), virus-related cancers (e.g., human papillomavirus (HPV)-related tumors); and hematological malignancies, the hematological malignancies arising from either of two major blood cell lineages, namely the myeloid lineage (which gives rise to granulocytes, erythrocytes, platelets, macrophages, and mast cells) or the lymphoid lineage (which gives rise to B, T, NK, and plasma cells), such as all types of leukemia, lymphoma, and myeloma, such as acute, chronic, lymphocytic, and / or myelogenous leukemia, such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), undifferentiated AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant, with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroleukemia; lymphomas, such as Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL);B cell hematological malignancies such as B cell lymphoma, T cell lymphoma, lymphoplasmacytic lymphoma, monocytoid B cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T cell lymphoma, angiocentric lymphoma, enteropathy-type T cell lymphoma, primary mediastinal B cell lymphoma, precursor T lymphoblastic lymphoma, T lymphoblastic; and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), lymphoid hematopoietic tumors, acute lymphoblastic leukemia, diffuse large B cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, cutaneous T cell lymphoma (CTLC) (also known as mycosis fungoides or Sézary syndrome) and lymphoplasmacytic lymphoma (LPL) with Waldenström macroglobulinemia; myeloma such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also known as indolent myeloma), solitary plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell leukemia; myeloid hematopoietic tumors, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nervous system, including astrocytoma, schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular carcinoma of the thyroid and teratocarcinoma, lymphoid hematopoietic tumors, such as T cell and B cell tumors, including but not limited to T cell disorders such as T prolymphocytic leukemia (T-PLL), including small cell and cerebriform cell types of T prolymphocytic leukemia (T-PLL); large granular lymphocyte leukemia (LGL), preferably T cell type of large granular lymphocyte leukemia (LGL); a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T cell lymphoma; head or neck cancer, kidney cancer, rectal cancer, thyroid cancer; acute myeloid lymphoma, and any combination of said cancers. The methods described herein can also be used to treat metastatic cancer.;

[0211] In certain embodiments, the composition is administered together with any additional anti-cancer agent. In other embodiments, the anti-cancer agent is any anti-cancer agent known in the art. In other embodiments, the anti-cancer agent is a third antibody. In some embodiments, the third antibody is any antibody disclosed herein.

[0212] In other embodiments, the composition is administered intravenously. In some embodiments, the composition is reconstituted prior to administration. In other embodiments, the composition is diluted prior to administration. In certain embodiments, the composition is administered in an absolute dose. In other embodiments, the composition is administered in a weight-based dose.

[0213] In some embodiments, the composition is administered at least about once a week, at least about twice a week, at least about every two weeks, at least about every three weeks, or at least about once a month. In some embodiments, the treatment lasts for at least about 4 weeks, at least about 8 weeks, at least about 12 weeks, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 18 months, at least about 2 years or greater than 2 years.

[0214] In some embodiments, the present invention relates to a method of modulating an immune response, comprising administering any composition disclosed herein.

[0215] In certain embodiments, the compositions of the invention (e.g., administration of an anti-PD-1 antibody or administration of an anti-PD-1 antibody and another anti-cancer therapy) effectively increase the survival of a subject. For example, the survival of a subject is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months or at least about 1 year or more compared to additional subjects treated with only another therapy (e.g., standard of care) or only one of the two members of the composition (e.g., anti-PD-1 antibody alone). In some embodiments, the survival is increased by at least about 2 months. In certain embodiments, the therapies of the invention effectively increase the progression-free survival of a subject. For example, the progression-free survival of a subject is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months or at least about 1 year compared to untreated subjects or subjects treated with only another therapy (e.g., standard of care for the treatment) or only one of the two members of the composition (e.g., anti-PD-1 or PD-L1 antibody alone). In some embodiments, the progression-free survival is increased by at least about 2 months. In certain embodiments, the therapies of the invention effectively increase the response rate of a group of subjects. For example, the response rate of a group of subjects is increased by at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or at least about 100% compared to another group of subjects treated with only another therapy (e.g., standard of care) or only one of the two members of the composition (e.g., anti-PD-1 antibody alone), i.e., single therapy.

[0216] Dose of the compositions disclosed herein

[0217] In some embodiments, the composition is administered at an absolute dose, regardless of the weight of the patient. For example, the anti-PD-1 antibody in combination with a second antibody can be administered at the following absolute doses: 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 50, 75, 80, 200, 240, 300, 360, 400, 480, 500, 750 or 1500 mg or any other dose disclosed herein, without regard to the weight of the patient. In some embodiments, the composition is administered at a weight-based dose at any dose disclosed herein. In some embodiments, the amounts of the first antibody and the second antibody administered to the patient in a single dose are the same as X amount and Y amount, respectively.

[0218] In certain embodiments of the combination treatment methods of the present invention, a therapeutically effective dose of an anti-PD-1 antibody or an antigen-binding portion thereof comprises 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg or about 300 mg. In some embodiments, a therapeutically effective dose of an anti-PD-1 antibody or an antigen-binding portion thereof comprises about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg or about 500 mg. In some embodiments, the dose of the anti-PD-1 antibody in the composition is from about 60 mg to about 300 mg, from about 60 mg to about 100 mg, from about 100 mg to about 200 mg, or from about 200 mg to about 300 mg. In some embodiments, the dose of the anti-PD-1 antibody in the composition is from about 300 mg to about 500 mg, from about 300 mg to about 450 mg, from about 300 mg to about 400 mg, from about 300 mg to about 350 mg, from about 350 mg to about 500 mg, from about 400 mg to about 500 mg, or from about 450 mg to about 500 mg. In some embodiments, the amount of the anti-PD-1 antibody in the composition is at least about 80 mg, about 160 mg or about 240 mg. In certain embodiments, the amount of the anti-PD-1 antibody in the composition is at least about 360 mg or 480 mg. In some embodiments, the dose of the anti-PD-1 antibody in the composition is at least about 240 mg or at least about 80 mg. In one embodiment, the amount of the anti-PD-1 antibody in the composition is about 360 mg. In another embodiment, the amount of the anti-PD-1 antibody in the composition is about 480 mg. In some embodiments, the dose of the anti-PD-1 antibody in the composition is at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg or at least about 5 mg / kg. In some embodiments, the dose of the anti-PD-1 antibody in the composition is from about 0.5 mg / kg to about 5 mg / kg, from about 0.5 mg / kg to about 5 mg / kg, from about 0.5 mg / kg to about 3 mg / kg or from about 0.5 mg / kg to about 2 mg / kg. In some embodiments, the dose of the anti-PD-1 antibody in the composition is at least about 1 mg / kg. Calculate the corresponding dose of the second antibody using the desired ratio.

[0219] In some embodiments, the anti-PD-1 antibody is administered at a sub-therapeutic dose, which is a dose of a therapeutic agent that is significantly lower than the dose typically or FDA-approved when administered as a single therapy for treating cancer. The amount of the second antibody in the composition is calculated based on the desired ratio. A dose of nivolumab that is less than the typical 3 mg / kg but not less than 0.001 mg / kg is a sub-therapeutic dose. The sub-therapeutic dose of the anti-PD-1 antibody used in the methods herein is greater than 0.001 mg / kg and less than 3 mg / kg. In some embodiments, the sub-therapeutic dose is about 0.001 mg / kg - about 1 mg / kg, about 0.01 mg / kg - about 1 mg / kg, about 0.1 mg / kg - about 1 mg / kg or about 0.001 mg / kg - about 0.1 mg / kg body weight. In some embodiments, the sub-therapeutic dose is at least about 0.001 mg / kg, at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg or at least about 1.0 mg / kg body weight. Receptor occupancy data for 15 subjects receiving doses of nivolumab from 0.3 mg / kg to 10 mg / kg indicated that PD-1 occupancy was dose-independent within this dose range. The mean occupancy across all doses was 85% (range 70% to 97%), and the mean steady-state occupancy was 72% (range 59% to 81%). In some embodiments, a 0.3 mg / kg dose may allow sufficient exposure to result in maximal biological activity.

[0220] In some embodiments, the composition is administered by intravenous infusion about once a week, about once every 2 weeks, about once every 3 weeks, or about once a month. In certain embodiments, the composition is administered about once every 3 weeks. In one embodiment, 360 mg of the anti-PD-1 antibody or antigen-binding fragment is administered once every 3 weeks. In another embodiment, 480 mg of the anti-PD-1 antibody or antigen-binding fragment is administered about once every 4 weeks. In some embodiments, the infusion is carried out for at least about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours, about 4 hours or about 5 hours.

[0221] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be absolute or variable to obtain an amount of the active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition, and mode of administration without causing undue toxicity to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention used, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts. The compositions of the present invention can be administered by one or more routes of administration using one or more methods well known in the art. As will be understood by those skilled in the art, the route of administration and / or mode will vary depending on the desired results.

[0222] Kit

[0223] Also within the scope of the present invention are kits that contain the anti-PD-1 antibody / second antibody composition and instructions for therapeutic use. Kits generally include a label and instructions for use indicating the intended use of the contents of the kit. The term label includes any written or recorded material provided on, with, or otherwise attached to the kit. Accordingly, the present disclosure provides kits that contain: (a) a suitable dose of the composition disclosed herein and (b) instructions for using the composition by any of the methods disclosed herein.

[0224] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited in this application are expressly incorporated herein by reference. Detailed Description

[0225] The present invention also relates to the following embodiments:

[0226] 1. A pharmaceutical composition comprising an amount X of a first antibody or an antigen-binding fragment thereof, which comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, and an amount Y of a second antibody or an antigen-binding fragment thereof, wherein the ratio of the amount X to the amount Y is from about 50:1 to about 1:50.

[0227] 2. The composition of embodiment 1, wherein the ratio of X to Y is about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 5:1, about 3:1, about 1:1, about 1:3, about 1:5, about 1:10, about 1:20, about 1:30, about 1:40, or about 1:50.

[0228] 3. The composition of embodiment 1 or 2, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.

[0229] 4. The composition of embodiment 3, wherein the anti-PD-1 antibody is nivolumab.

[0230] 5. The composition of any one of embodiments 1 to 4, wherein the amount of X of the first antibody or its antigen-binding fragment is at least about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg or about 300 mg.

[0231] 6. The composition of embodiment 5, wherein the amount of X of the first antibody is at least about 80 mg, about 160 mg or about 240 mg.

[0232] 7. The composition of any one of embodiments 1 to 6, wherein the amount of X of the first antibody or its antigen-binding fragment is about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg or about 300 mg.

[0233] 8. The composition of embodiment 7, wherein the amount of X of the first antibody or its antigen-binding fragment is about 80 mg or about 240 mg.

[0234] 9. The composition of any one of embodiments 1 - 8, wherein the second antibody or its antigen-binding fragment is an anti-CTLA4 antibody.

[0235] 10. The composition of embodiment 9, wherein the ratio of X to Y is about 3:1, about 1:1 or about 1:3.

[0236] 11. The composition of embodiment 9, wherein (i) the amount of X is about 240 mg and the amount of Y is about 80 mg, (ii) the amount of X is about 80 mg and the amount of Y is about 80 mg; (iii) the amount of X is about 160 mg and the amount of Y is about 160 mg; (iv) the amount of X is about 240 mg and the amount of Y is about 240 mg; or (v) the amount of X is about 80 mg and the amount of Y is about 240 mg.

[0237] 12. The composition of embodiment 10 or 11, wherein the anti-CTLA4 antibody is tremelimumab or ipilimumab.

[0238] 13. The composition of any one of embodiments 1 - 8, wherein the second antibody is an anti-LAG3 antibody.

[0239] 14. The composition of embodiment 13, wherein the ratio of X to Y is about 12:1, about 3:1 or about 1:1.

[0240] 15. The composition of embodiment 13 or 14, wherein the anti-LAG3 antibody is 25F7.

[0241] 16. The composition of any one of embodiments 1-8, wherein the second antibody is an anti-CD137 antibody.

[0242] 17. The composition of embodiment 16, wherein the ratio of X to Y is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 50:1, about 40:1, about 30:1, about 20:1, about 10:1, about 5:1, about 4:1 or about 2:1.

[0243] 18. The composition of embodiment 16 or 17, wherein the anti-CD137 antibody is urelumab.

[0244] 19. The composition of any one of embodiments 1-8, wherein the second antibody is an anti-KIR antibody.

[0245] 20. The composition of embodiment 19, wherein the ratio of X to Y is about 30:1, about 10:1, about 3:1, about 1:1 or about 1:2.

[0246] 21. The composition of embodiment 19 or 20, wherein the anti-KIR antibody is 1-7F9 or lirilumab.

[0247] 22. The composition of any one of embodiments 1-8, wherein the second antibody is selected from anti-TGFβ antibody, anti-IL-10 antibody, anti-B7-H4 antibody, anti-Fas ligand antibody, anti-CXCR4 antibody, anti-mesothelin antibody, anti-CD27 antibody, anti-CD27 antibody and any combination thereof.

[0248] 23. The composition of any one of embodiments 1-8, wherein the second antibody is an anti-GITR antibody.

[0249] 24. The composition of embodiment 23, wherein the anti-GITR antibody is MK4166 or TRX518.

[0250] 25. The composition of any one of embodiments 1 to 8 or 22 to 24, wherein the ratio of X:Y is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1 or about 10:1.

[0251] 26. The composition of any one of embodiments 1 to 25, wherein the composition is formulated in a Tris-Cl, histidine, citrate, or Tris-citrate buffer.

[0252] 27. The composition of embodiment 26, wherein the composition is formulated in a Tris-Cl buffer, and the concentration of Tris-Cl is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 50 mM.

[0253] 28. The composition of embodiment 27, wherein the concentration of Tris-Cl is about 20 mM.

[0254] 29. The composition of embodiment 26, wherein the composition is formulated in a citrate buffer, and the concentration of citrate is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 50 mM.

[0255] 30. The composition of embodiment 29, wherein the citrate concentration is about 10 mM or about 20 mM.

[0256] 31. The composition of embodiment 26, wherein the composition is formulated in a histidine buffer, and the concentration of histidine is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 50 mM.

[0257] 32. The composition of embodiment 31, wherein the histidine concentration is about 20 mM.

[0258] 33. The composition of embodiment 26, wherein the composition is formulated in a Tris-citrate buffer, the concentration of Tris-Cl is at least about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 50 mM, and the concentration of citrate is at least about 2 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or about 50 mM.

[0259] 34. The composition of embodiment 33, wherein the concentration of Tris-Cl is about 13.3 mM and the concentration of citrate is about 6.7 mM.

[0260] The composition of any one of embodiments 1-34, wherein the pH of the composition is at least about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0261] 36. The composition of embodiment 35, wherein the pH of the composition is at least about 6.0, about 6.2, about 6.5, about 6.6 or about 7.0.

[0262] 37. The composition of any one of embodiments 1-36, wherein the composition comprises one or more additional components selected from: bulking agents, stabilizers, chelating agents, surfactants, buffering agents, and any combination thereof.

[0263] 38. The composition of embodiment 37, wherein the bulking agent is selected from NaCl, mannitol, glycine, alanine, and any combination thereof.

[0264] 39. The composition of embodiment 37 or 38, wherein the stabilizer is selected from sucrose, trehalose, raffinose, arginine; or any combination thereof.

[0265] 40. The composition of any one of embodiments 37-39, wherein the chelating agent is selected from diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid, nitrilotriacetic acid, and any combination thereof.

[0266] 41. The composition of any one of embodiments 37 to 40, wherein the surfactant is selected from polysorbate 80 (PS80), polysorbate 20 (PS20), and any combination thereof.

[0267] 42. The composition of embodiment 37, wherein the composition comprises NaCl at the following concentrations: at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 60 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 90 mM, at least about 100 mM, at least about 110 mM, at least about 120 mM, at least about 130 mM, at least about 140 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, at least about 225 mM, at least about 250 mM, at least about 275 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 450 mM or at least about 450 mM.

[0268] 43. The composition of embodiment 42, wherein the concentration of NaCl is about 100 mM, about 96.15 mM, about 83.3 mM, about 78.57 mM or about 50 mM.

[0269] 44. The composition of embodiment 37, wherein the composition comprises mannitol USP at the following concentrations (% w / v): at least about 0.25%, at least about 0.5%, at least about 0.75%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 7.5% or at least about 10%.

[0270] 45. The composition of embodiment 44, wherein the concentration of mannitol is about 1%, about 1.15%, about 1.67%, about 1.86% or about 3%.

[0271] 46. The composition of embodiment 37, wherein the composition comprises DTPA USP at the following concentrations: at least about 5 μM, at least about 10 μM, at least about 15 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 40 μM, at least about 50 μM, at least about 60 μM, at least about 70 μM, at least about 75 μM, at least about 80 μM, at least about 90 μM, at least about 100 μM, at least about 110 μM, at least about 120 μM, at least about 130 μM, at least about 140 μM, at least about 150 μM, at least about 175 μM or at least about 200 μM.

[0272] 47. The composition of embodiment 46, wherein the concentration of DTPA is about 20 μM, about 50 μM, about 65.71 μM, about 73.3 μM, about 93.85 μM or 100 μM.

[0273] 48. The composition of embodiment 37, wherein the composition comprises PS80 at the following concentrations (% w / v): at least about 0.005%, at least about 0.01%, at least about 0.015%, at least about 0.02%, at least about 0.03%, at least about 0.04%, at least about 0.05%, at least about 0.06%, at least about 0.07%, at least about 0.08%, at least about 0.09%, or at least about 0.1%.

[0274] 49. The composition of embodiment 48, wherein the concentration of PS80 is about 0.01%, about 0.012%, about 0.013%, about 0.02%, about 0.23%, about 0.04%, or about 0.05%.

[0275] 50. The composition of embodiment 37, wherein the composition comprises sucrose at the following concentrations (% w / v): at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.5%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5%, at least about 10%, at least about 12%, or at least about 15%.

[0276] 51. The composition of embodiment 50, wherein the concentration of sucrose is about 6% or about 8.5%.

[0277] 52. A pharmaceutical composition, which, in a buffer of about 6.2 pH, comprising about 13.3 mM Tris, about 6.7 mM citrate, about 1.67% mannitol, about 83.3 mM NaCl, about 73.3 μM DTPA, and about 0.013% PS80, comprises nivolumab and ipilimumab in a 1:1 ratio.

[0278] 53. A pharmaceutical composition, which, in a Tris-citrate buffer of about 6.6 pH, comprising about 1.15% mannitol, about 96.15 mM NaCl, about 93.85 μM DTPA, and about 0.012% PS80, comprises nivolumab and ipilimumab in a 3:1 ratio.

[0279] 54. A pharmaceutical composition, which, in a Tris-citrate buffer of about 6.0 pH, comprising about 1.86% mannitol, about 78.57 mM NaCl, about 65.71 μM DTPA, and about 0.023% PS80, comprises nivolumab and ipilimumab in a 1:3 ratio.

[0280] 55. A pharmaceutical composition, which, at about pH 6, contains about 50 mM NaCl, about 50 μM DTPA, about 6% sucrose and about 0.05% PS80 in a 20 mM histidine buffer, and contains nivolumab and ipilimumab in a ratio of 3:1.

[0281] 56. A pharmaceutical composition, which, at about pH 7, contains about 50 mM NaCl, about 50 μM DTPA, about 6% sucrose and about 0.05% PS80 in an about 20 mM histidine buffer, and contains nivolumab and ipilimumab in a ratio of 3:1.

[0282] 57. A pharmaceutical composition, which, at about pH 6, contains about 50 μM DTPA, about 8.5% sucrose and about 0.05% PS80 in an about 20 mM histidine buffer, and contains nivolumab and ipilimumab in a ratio of 3:1.

[0283] 58. A pharmaceutical composition, which, at about pH 6, contains about 50 mM NaCl, about 50 μM DTPA, about 6% sucrose and about 0.05% PS80 in an about 20 mM citrate buffer, and contains nivolumab and ipilimumab in a ratio of 3:1.

[0284] 59. A pharmaceutical composition, which, at about pH 6, contains about 50 mM NaCl, about 20 μM DTPA, about 3% mannitol and about 0.04% PS80 in an about 20 mM citrate buffer, and contains nivolumab and ipilimumab in a ratio of 3:1.

[0285] 60. A pharmaceutical composition, which, at about pH 6, contains about 50 mM NaCl, about 100 μM DTPA, about 3% mannitol and about 0.02% PS80 in an about 20 mM citrate buffer, and contains nivolumab and ipilimumab in a ratio of 1:1.

[0286] 61. A pharmaceutical composition, which, at about pH 6.5, contains about 50 mM NaCl, about 100 μM DTPA, about 3% mannitol and about 0.02% PS80 in an about 20 mM citrate buffer, and contains nivolumab and ipilimumab in a ratio of 1:1.

[0287] 62. A pharmaceutical composition, which, at about pH 6.5, contains about 100 mM NaCl, about 100 μM DTPA, about 1.0% mannitol and about 0.02% PS80 in an about 20 mM citrate buffer, and contains nivolumab and ipilimumab in a ratio of 1:1.

[0288] 63. A pharmaceutical composition, which contains nivolumab and ipilimumab in a 1:1 ratio in a citrate buffer of about 20 mM containing about 50 mM NaCl, about 100 μM DTPA, about 6% sucrose and about 0.02% PS80 at about pH 6.0.

[0289] 64. A pharmaceutical composition, which contains nivolumab and ipilimumab in a 1:3 ratio, and contains about 4.62 mg / ml nivolumab, about 1.54 mg / ml ipilimumab, about 18.5 mM Tris hydrochloride, about 1.5 mM sodium citrate dihydrate, about 96.2 mM NaCl, about 1.2% mannitol, about 93.9 μM pentetic acid, about 0.012% of PS80 at about pH 6.0.

[0290] 65. A pharmaceutical composition, which contains nivolumab and ipilimumab in a 1:3 ratio, and contains about 4.61 mg / ml nivolumab, about 1.54 mg / ml ipilimumab, about 18.46 mM Tris hydrochloride, about 1.54 mM sodium citrate dihydrate, about 96.15 mM NaCl, about 1.15% mannitol, about 93.85 μM pentetic acid, about 0.012% PS80 at about pH 6.3.

[0291] 66. The composition according to any one of embodiments 1-65, wherein the composition is stable at about 5 °C for at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 2 years or at least about 5 years.

[0292] 67. The composition according to any one of embodiments 1-66, wherein the composition is stable at about 40 °C for at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 2 years or at least about 5 years.

[0293] 68. The composition according to any one of embodiments 1-67, wherein the composition is stable at about 25 °C for at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 2 years or at least about 5 years.

[0294] 69. The composition according to any one of embodiments 1 to 68, which shows a change in the acidic peak of less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1% after storage at about 5 °C for about 6 months or about 3 months.

[0295] The composition of any one of embodiments 1 to 68, which after storage at about 25 °C for about 3 months, exhibits a change in the acidic peak of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%.

[0296] 71. The composition of any one of embodiments 1 to 68, which after storage at about 40 °C for about 3 months, exhibits a change in the acidic peak of less than about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%.

[0297] 72. The composition of any one of embodiments 1 to 72, which after storage at about 4 °C for about 3 months, exhibits a change in the high molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

[0298] 73. The composition of any one of embodiments 1 to 71, which after storage at about 25 °C for about 2 months or about 3 months, exhibits a change in the high molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

[0299] 74. The composition of any one of embodiments 1 to 71, which after storage at about 40 °C for about 2 months or about 3 months, exhibits a change in the high molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

[0300] 75. The composition of any one of embodiments 1 to 74, which after storage at about 4 °C for about 1 month, exhibits a change in the main peak of capillary isoelectric focusing (cIEF) analysis of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

[0301] 76. The composition according to any one of embodiments 1 to 74, which, after storage at about 25 °C for about 1 month, exhibits a change in the main peak of capillary isoelectric focusing (cIEF) analysis of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

[0302] 77. The composition according to any one of embodiments 1 to 74, which, after storage at about 40 °C for about 1 month, exhibits a change in the main peak of capillary isoelectric focusing (cIEF) analysis of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2% or about 0.1%.

[0303] 78. The composition according to any one of embodiments 1 - 77, which, after storage at about 40 °C for about 2 months, exhibits a change in the low molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

[0304] 79. The composition according to any one of embodiments 1 - 77, which, after storage at about 25 °C for about 2 months, exhibits a change in the low molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2% or about 0.1%.

[0305] 80. The composition according to any one of embodiments 1 - 77, which, after storage at about 4 °C for about 2 months, exhibits a change in the low molecular weight peak of less than about 5%, about 4%, about 3%, about 2%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

[0306] 81. The composition according to any one of embodiments 1 - 80, wherein the composition is diluted before use.

[0307] 82. The composition of embodiment 81, wherein the composition is diluted with 0.9% Sodium Chloride Injection USP or 5% Dextrose Injection USP before use.

[0308] 83. The composition of embodiment 61 or 82, wherein the composition is diluted to obtain the desired concentrations of the first antibody and the second antibody.

[0309] 84. A kit comprising the composition of any one of embodiments 1 - 83.

[0310] 85. A method for preparing the composition of any one of embodiments 1 - 83.

[0311] 86. The method of embodiment 85, wherein a formulation comprising an anti - PD - 1 antibody drug product is mixed with a formulation comprising a second antibody drug product to obtain the desired ratio in the final drug product without buffer change.

[0312] 87. The method of embodiment 85, wherein a formulation comprising an anti - PD - 1 antibody drug substance and a formulation comprising a second antibody drug substance are subjected to buffer exchange and / or concentration and then mixed to obtain the desired ratio in the final drug product.

[0313] 88. A method of modulating an immune response in a patient in need thereof, comprising administering to the patient the composition of any one of embodiments 1 - 83.

[0314] 89. A method of co - administering two antibodies to a patient in need thereof, comprising administering to the patient the composition of any one of embodiments 1 - 83, wherein the antibodies are capable of treating at least one disease or disorder.

[0315] 90. A method of treating a disease or disorder, comprising administering to the patient the composition of any one of embodiments 1 - 83.

[0316] 91. The method of embodiment 89 or 90, wherein the disease or disorder is an infectious disease.

[0317] 92. The method of embodiment 89 or 90, wherein the disease is cancer.

[0318] 93. The method of embodiment 92, wherein the cancer is melanoma cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer; chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia; childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma; environmentally induced cancers, including cancers induced by asbestos; and any combination thereof.

[0319] 94. The method of embodiment 92 or 93, wherein the composition is administered intravenously.

[0320] 95. The method of any one of embodiments 88 to 94, wherein the composition is diluted before administration.

[0321] 96. The method of any one of embodiments 84 to 95, wherein the composition is administered in an absolute dose.

[0322] 97. The method of embodiment 96, wherein the amounts of the first antibody and the second antibody administered to the patient in a single dose are the same as X amount and Y amount, respectively.

[0323] 98. The method of any one of embodiments 88 to 95, wherein the composition is administered in a weight-based dose.

[0324] 99. The composition of embodiment 98, wherein the amount of the first antibody administered to the patient is at least about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg or about 5 mg / kg.

[0325] 100. The composition of embodiment 98, wherein the X amount of the first antibody administered to the patient is at least about 1 mg / kg.

[0326] 101. The method of any one of embodiments 88 - 100, wherein the composition is administered at least about weekly, at least about twice a week, at least about every two weeks, at least about every three weeks, or at least about monthly.

[0327] The method of any one of embodiments 88 - 101, wherein the administration lasts for at least about 8 weeks, at least about 12 weeks, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 18 months, at least about 2 years, or greater than 2 years.

[0328] The method of any one of embodiments 88 - 102, wherein the patient is also treated with another anti - cancer agent.

[0329] Examples

[0330] Several feasibility studies were conducted to evaluate the stability of ipilimumab and nivolumab in a single fixed dose ratio combination (FDRC) formulation. Figure 1 Formulations of ipilimumab and nivolumab in drug substance (DS) or drug product (DP) of ipilimumab and nivolumab are shown and used as controls when indicated in the following examples.

[0331] Example 1

[0332] A feasibility study was conducted to evaluate the stability of ipilimumab and nivolumab in a single fixed dose ratio combination (FDRC) formulation resulting from mixing separate formulations of ipilimumab and nivolumab ( Figure 1 ) to obtain a final ratio of ipilimumab to nivolumab of 1:1.

[0333] Ipilimumab (BMS - 734016) DP contains 5 mg / mL of ipilimumab in 20 mM Tris - HCl, 100 mM NaCl, 1.0% (w / v) mannitol, 100 μM diethylenetriaminepentaacetic acid (DTPA), and 0.01% polysorbate 80 (PS80), with a pH of 7.0, and is available in 40 mL in 50 mL vials and 10 mL in 10 mL vials ( Figure 1 ). Nivolumab (BMS - 936558) DP contains 10 mg / mL of nivolumab in 20 mM citrate buffer (sodium citrate dihydrate), 50 mM NaCl, 3.0% (w / v) mannitol, 20 μM DTPA, and 0.02% PS80, with a pH of 6.0, and is available in 10 mL in 10 mL vials ( Figure 1 ).

[0334] To achieve a 1:1 ratio of ipilimumab to nivolumab, 80 mL of ipilimumab DP (2 vials) was mixed with 40 mL of nivolumab DP (4 vials) to obtain a combination product containing 3.3 mg / mL of ipilimumab and 3.3 mg / mL of nivolumab. As shown in Table 1, the resulting FDRC formulation contained 13.3 mM Tris-HCl, 6.7 mM citrate, 83.3 mM NaCl, 1.67% (w / v) mannitol, 73.3 μM DTPA, and 0.013% w / v PS80, with a pH of 6.2.

[0335] Table 1: Combined EC FDRC (1:1) Formulation

[0336]

[0337] The FDRC (1:1) formulation was filtered and aliquoted into 10 cc glass vials (5 mL per vial), stoppered, and sealed. The vials were then stored at 5 °C or 40 °C. Samples were analyzed at 0 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, and 6 months. The day 0 sample was used as a control.

[0338] Sample Analysis - Methods

[0339] At each time point, the sample vials were analyzed by visual appearance, pH at room temperature, HIAC, size exclusion chromatography, and imaging capillary isoelectric focusing (cIEF). HIAC (Royco) is an instrument based on light obscuration particle counting technology.

[0340] Using EMPOWERTM 2 software, with a 2497 dual-wavelength UV detector, on 2695 HPLC, with Guard SW XL a guard column of G3000SW XL , size exclusion chromatography (SEC) was performed by analytical size exclusion HPLC (SE-HPLC). Using 0.1 M NaH 2 PO 4 , 0.1 M Na 2 SO 4The system was equilibrated with 85% buffer and 15% acetonitrile (ACN) (mobile phase). Samples were analyzed without dilution, unless the concentration was greater than 125 mg / mL. If the sample concentration was greater than 125 mg / mL, the sample was diluted to 50 mg / mL with the appropriate buffer. Samples were transferred to HPLC vials before analysis and stored in an analytical HPLC system at a temperature of 5°C ± 3°C. A total of 100 μg of sample was injected for analysis and run isocratically using the mobile phase at a column temperature of 22°C. The flow rate was 1.0 mL / min, the run time for each sample was 20 minutes, and the detection wavelength was 280 nm.

[0341] Imaging capillary isoelectric focusing (cIEF) was performed using a Protein SIMPLETM iCE3 instrument with an Alcott sampler. The concentration of the sample to be analyzed was 25 mg / mL, with 2 M urea and 0.35% methylcellulose (MC). Separation was carried out using a 50 mm capillary with an inner diameter of 100 microns. The electrolyte solution was 80 mM H 3 PO 4 in 0.1% MC, and the cathode electrolyte solution was 100 mM NaOH in 0.1% MC. The carrier ampholyte was 1% 5 - 8 and 3% 8 - 10.5. The focusing time was 13 minutes, the focusing voltage started at 1.5 kV (300 V / cm) for the first minute, and then 3 kV (600 V / cm) for the remaining 12 minutes. Detection was carried out at 280 nm.

[0342] Sample Analysis - Results

[0343] After storage at 40°C for 3 months, SEC ( Figure 2A ) was performed on nivolumab DP and ipilimumab DP controls and a 1:1 ratio fixed - dose combination (EC FDRC(1:1)) formulation. After storage at 40°C for 3 months, the HMW peak size of the nivolumab DP control increased by approximately 1.6%, and the HMW peak size of the ipilimumab DP control increased by approximately 0.25% ( Figure 2A ). After storage at 40°C for 3 months, the HMW peak size of the EC FDRC(1:1) formulation increased by approximately 0.7% ( Figure 2A ). After storage at 40°C for 6 months, the HMW peak size of the EC FDRC(1:1) formulation increased from 0.555% to a final HMW peak size of 2.82%, an increase of approximately 2.265% (Table 2). After storage at 5°C for 6 months, the HMW peak size of the EC FDRC(1:1) formulation decreased from 0.555% to a final HMW peak size of 0.525% (Table 2).

[0344] Table 2: Results of capillary isoelectric focusing and size - exclusion chromatography for the combined EC FDRC(1:1)

[0345]

[0346] After storage at 5 °C for 6 months, cIEF ( Figure 2B ) was performed on nivolumab DP and ipilimumab DP control and EC FDRC (1:1) formulations. After storage at 5 °C for 6 months, the acidic peak size of the nivolumab DP control increased by approximately 1.3%, and the acidic peak size of the ipilimumab DP increased by approximately 3% ( Figure 2B ). For the EC FDRC (1:1) formulation, the acidic peak size of nivolumab increased by approximately 3.56%, from 35.09% at day 0 (initial) to 38.65% at 6 months, while the acidic peak size of ipilimumab increased by approximately 4.16%, from 34% at day 0 (initial) to 38.16% at 6 months (Table 2 and Figure 2B ).

[0347] This study can be used to support a mixed buffer system over a wide concentration range, namely the Tris-citrate buffer composition.

[0348] Example 2

[0349] A feasibility study was conducted to evaluate the stability of ipilimumab / nivolumab FDRC generated by mixing individual formulations of ipilimumab and nivolumab to final ratios of 3:1, 1:1, and 1:3 (Table 3). FDRC formulations were generated by mixing 5 mg / mL ipilimumab DS and 20 mg / mL nivolumab DS to achieve protein ratios of 3:1, 1:1, and 1:3 (Table 3). Each combined solution was further mixed with a stir bar at room temperature for 30 minutes, transferred to vials, and stored for stability over time. The vials were stored at 5 °C, 25 °C, and 40 °C for up to 12 months.

[0350] Table 3: Combinations of EC FDRC (3:1; 1:1; 1:3) – Formulations of ipilimumab DP and nivolumab DP

[0351]

[0352] Prototype EC: pH 6.6, with a 3:1 ratio of ipilimumab to nivolumab, containing 4.62 mg / mL ipilimumab, 1.54 mg / mL nivolumab, 1.15% w / v mannitol, 96.15 mM NaCl, 93.85 μM DTPA, and 0.012% w / v PS80, pH 6.6. Prototype EC: pH 6.0, with a 1:3 ratio of ipilimumab to nivolumab, containing 2.86 mg / mL ipilimumab, 8.57 mg / mL nivolumab, 1.86% w / v mannitol, 78.57 mM NaCl, 65.71 μM DTPA, and 0.023% w / v PS80, pH 6.0. Prototype EC: pH 6.2, with a 1:1 ratio of ipilimumab to nivolumab, containing 4.00 mg / mL ipilimumab, 4.00 mg / mL nivolumab, 1.67% w / v mannitol, 83.33 mM NaCl, 73.33 μM DTPA, and 0.013% w / v PS80, pH 6.2.

[0353] SEC analysis

[0354] Generally, a slight increase in HMW and LMW was observed in all 3 prototypes ( Figure 3A -B). After 2 months of storage at 40 °C, SEC was performed on nivolumab DP control, ipilimumab DP control, and EC FDRC formulations EC:pH 6.0 (1:3), EC:pH 6.2 (1:1), and EC:pH 6.6 (3:1) Figure 3A and 3B ). The ipilimumab control formulation had an initial HMW peak size of approximately 0.4% on day 0, which increased by approximately 0.1% after 2 months at 40 °C, reaching a final HMW peak size just over 0.5% ( Figure 3A ). The nivolumab control formulation had an initial HMW peak size of approximately 0.8% on day 0, which increased by approximately 0.7% after 2 months at 40 °C, reaching a final HMW peak size of over 1.5% ( Figure 3A ). The EC:pH 6.0 FDRC formulation (1:3) had an initial HMW peak size of approximately 0.6% on day 0, which increased by approximately 0.7% after 2 months at 40 °C, reaching a final HMW peak size of approximately 1.3% ( Figure 3A ). The EC:pH 6.2 FDRC formulation (1:1) had an initial HMW peak size of approximately 0.5% on day 0, which increased by approximately 0.5% after 2 months at 40 °C, reaching a final HMW peak size of approximately 1.0% ( Figure 3A ). The EC:pH 6.6 FDRC formulation (3:1) had an initial HMW peak size of approximately 0.5% on day 0, which increased by approximately 0.3% after 2 months at 40 °C, reaching a final HMW peak size of approximately 0.8% (Figure 3A )。

[0355] The low molecular weight (LMW) peak sizes of various formulations were also measured at day 0, after 2 months at 40 °C, and after 3 months at 25 °C ( Figure 3B ). The ipilimumab control formulation had an initial LMW peak size of approximately 0.2% at day 0, which increased by approximately 0.65% after 2 months at 40 °C, reaching a final LMW peak size of approximately 0.85% ( Figure 3B ). After storage at 25 °C for 3 months, the LMW peak size of the ipilimumab control formulation increased by approximately 0.1% ( Figure 3B ). The nivolumab control formulation had an initial LMW peak size of approximately 0.2% at day 0, which increased by approximately 0.6% after 2 months at 40 °C, reaching a final LMW peak size of approximately 0.8% ( Figure 3B ). After storage at 25 °C for 3 months, the LMW peak size of the nivolumab control formulation increased by less than 0.1% ( Figure 3B ). The EC:pH 6.0 FDRC formulation (1:3) had an initial LMW peak size of approximately 0.15% at day 0, which increased by approximately 0.8% after 2 months at 40 °C, reaching a final LMW peak size of approximately 0.95% ( Figure 3B ). After storage at 25 °C for 3 months, the LMW peak size of the EC:pH 6.0 (1:3) FDRC formulation increased by approximately 0.2% ( Figure 3B ). The EC:pH 6.2 FDRC formulation (1:1) had an initial LMW peak size of approximately 0.15% at day 0, which increased by approximately 1.2% after 2 months at 40 °C, reaching a final LMW peak size of approximately 1.35% ( Figure 3B ). After storage at 25 °C for 3 months, the LMW peak size of the EC:pH 6.2 (1:1) FDRC formulation increased by approximately 0.3% ( Figure 3B ). The EC:pH 6.6 FDRC formulation (3:1) had an initial LMW peak size of approximately 0.15% at day 0, which increased by approximately 1.5% after 2 months at 40 °C, reaching a final LMW peak size of approximately 1.65% ( Figure 3B ). After storage at 25 °C for 3 months, the LMW peak size of the EC:pH 6.6 (3:1) FDRC formulation increased by approximately 0.1%.

[0356] cIEF analysis

[0357] After storage at 25 °C for 3 months ( Figure 4A ), after storage at 5 °C for 3 months ( Figure 4B ), and after storage at 25 °C for 1 month ( Figure 4C) cIEF was performed on nivolumab DP control, ipilimumab DP control, and EC FDRC formulations EC:pH 6.0 (1:3), EC:pH 6.2 (1:1), and EC:pH 6.6 (3:1). After storage at 25 °C for 3 months, the acidic peak size of nivolumab DP control decreased by approximately 0.05%, and the acidic peak size of ipilimumab DP control increased by approximately 5.59% ( Figure 4A ). After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the FDRC formulation EC:pH 6.0 (1:3) increased by approximately 5% and approximately 5.7%, respectively ( Figure 4A ). After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the FDRC formulation EC:pH 6.2 (1:1) increased by approximately 6.8% and approximately 6.3%, respectively ( Figure 4A ). After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the FDRC formulation EC:pH 6.6 (3:1) increased by approximately 4% and approximately 7.8%, respectively ( Figure 4A ). Among the three FDRC formulations, the acidic peak size of ipilimumab increased by approximately 5.7% - 7.8%, or approximately 2.2% per month on average; the acidic peak size of nivolumab increased by approximately 4% - 6.8%, or less than 2% (approximately 1.76%) per month on average ( Figure 4A ).

[0358] Figure 4B shows the actual changes in the acidic peak sizes of the samples stored at 5 °C for 3 months relative to the initial (day 0) control as analyzed by cIEF. After storage at 5 °C for 3 months, the acidic peak size of nivolumab DP control decreased by approximately 5.1%, and the acidic peak size of ipilimumab DP control decreased by approximately 1% ( Figure 4B ). After storage at 5 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the FDRC formulation EC:pH 6.0 (1:3) increased by approximately 0.1% and decreased by approximately 1.5%, respectively ( Figure 4B ). After storage at 5 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the FDRC formulation EC:pH 6.2 (1:1) increased by approximately 2.1% and approximately 0.5%, respectively ( Figure 4B ). After storage at 5 °C for 3 months, the FDRC formulation EC:pH 6.6 (3:1) showed no change in the acidic peak size of ipilimumab and a decrease of less than 0.1% in the acidic peak size of nivolumab ( Figure 4B ).

[0359] Figure 4CShows the actual change in the acidic peak size of the samples stored at 25 °C for 1 month relative to the initial (day 0) control. After storage at 25 °C for 1 month, the acidic peak size of the nivolumab DP control increased by approximately 1.05%, and the acidic peak size of the ipilimumab DP control increased by approximately 1.16%( Figure 4C ). After storage at 25 °C for 1 month, the acidic peak sizes of nivolumab and ipilimumab in the FDRC formulation EC:pH6.0 (1:3) increased by approximately 2.8% and 1%, respectively( Figure 4C ). After storage at 25 °C for 1 month, the acidic peak sizes of nivolumab and ipilimumab in the FDRC formulation EC:pH6.2 (1:1) increased by approximately 3.1% and 1.6%, respectively( Figure 4C ). After storage at 25 °C for 1 month, the acidic peak size of nivolumab in the FDRC formulation EC:pH6.6 (3:1) did not change, and the acidic peak size of ipilimumab increased by approximately 2.8%( Figure 4C ).

[0360] Example 3

[0361] A design of experiments (DoE) study was conducted to identify new candidate ipilimumab / nivolumab formulations. Prototype ipilimumab / nivolumab FDRC (3:1) formulations were prepared in selected histidine or citrate formulations, as shown in Table 4. All DoE FDRC prototypes were prepared at a final concentration of 10 mg / mL of ipilimumab / nivolumab at a ratio of 3:1 (Table 4). The FDRC prototype "Combo 4" contained 20 mM citrate, 50 mM NaCl, 50 μM DTPA, 6% w / v sucrose, and 0.05% w / v PS80, with a theoretical pH of 6. The FDRC prototype "Combo 5" contained 20 mM histidine, 50 mM NaCl, 50 μM DTPA, 6% w / v sucrose, and 0.05% w / v PS80, with a theoretical pH of 6.0. The FDRC prototype "Combo 6" contained 20 mM histidine, 50 mM NaCl, 50 μM DTPA, 6% w / v sucrose, and 0.05% w / v PS80, with a theoretical pH of 7. The FDRC prototype "Combo new" contained 20 mM histidine, 50 μM DTPA, 8.5% w / v sucrose, and 0.05% w / v PS80, with a theoretical pH of 6. The FDRC prototype "Combo8" was similar to the current nivolumab DP formulation and contained 20 mM citrate, 50 mM NaCl, 20 μM DTPA, 3% w / v mannitol, and 0.04% w / v PS80, with a theoretical pH of 6.

[0362] Table 4: DoE FDRC (3:1) - New formulations

[0363]

[0364] The DoE FDRC formulation is produced from the following “Combo new” preparation. “Combo new” is prepared by first subjecting ipilimumab DS and nivolumab DS (ELN 96488-024 and -025) to ultrafiltration / diafiltration. Specifically, a disposable UFDF cassette is used for nivolumab DS and ipilimumab DS. Using the diafiltration / concentration mode, approximately 250 mL of unformulated DS of nivolumab (~21 mg / mL) is used for UF / DF. The transmembrane pressure (TMP) is set at 15 psi, and a flow rate of 0.3 liters per minute is set for the feed pump. Diafiltration is completed after using 3 liters of buffer. The sample in the container is further concentrated based on scale weight reduction and collected in 250 PETG bottles. The concentration of nivolumab after UFDF is 30.6 mg / mL. Using the diafiltration / concentration mode, approximately 500 mL of unformulated DS of ipilimumab (~5.2 mg / mL) is used for UF / DF. The concentration of ipilimumab in the final product is 16.2 mg / mL as determined by A280 measurement.

[0365] Next, 20 mL of ipilimumab DS in a histidine-sucrose-based buffer and 7.5 mL of nivolumab DS in a histidine-sucrose-based buffer are added to a D-Tube Dialyzer device and dialyzed against the “Combo new” buffer as shown in Table 4 for 24 hours (3x buffer change) in a cold room with sufficient volume. Then the protein concentrations of ipilimumab and nivolumab are measured by HIAC. Then additional ipilimumab DS and / or nivolumab DS and appropriate buffer are added to bring the final concentration of ipilimumab to 7.5 mg / mL and the final concentration of nivolumab to 2.5 mg / mL (3:1). The remaining prototypes Combo 4, Combo 5, Combo 6, and Combo 8 are prepared in the same manner as “Combo new” with modifications to the specific concentrations shown in Table 4.

[0366] Then the combined DP formulation is filtered and aseptically filled into 10 cc vials (SAP#1215125, lot #2L68780), stoppered (SAP#1239068, lot #0H49862), and crimped. Some vials are retained for day 0 control analysis. The remaining vials are placed on stability stations at 5 °C, 25 °C, and 40 °C until vials are pulled for analysis at specific time points.

[0367] SEC analysis

[0368] After storage at 40 °C for 3 months, nivolumab DP control, ipilimumab DP control, and DoE FDRC (3:1) formulations “Combo new”, Combo 4, Combo 5, Combo 6, and Combo 8 were subjected to SEC( Figure 5A ). The ipilimumab control formulation had an initial HMW peak size of approximately 0.4% on day 0, which increased by approximately 0.2% after 3 months at 40 °C to a final HMW peak size of approximately 0.6%( Figure 5A ). The nivolumab control formulation had an initial HMW peak size of approximately 0.7% on day 0, which increased by approximately 1.6% after 3 months at 40 °C to a final HMW peak size of approximately 2.4%( Figure 5A ). The “Combo new” FDRC formulation had an initial HMW peak size of approximately 0.4% on day 0, which increased by approximately 0.1% after 3 months at 40 °C to a final HMW peak size just over 0.5%( Figure 5A ). The Combo 4 FDRC formulation had an initial HMW peak size of approximately 0.6% on day 0, which increased by approximately 0.7% after 3 months at 40 °C to a final HMW peak size of approximately 1.3%( Figure 5A ). The Combo 5 FDRC formulation had an initial HMW peak size just below 0.5% on day 0, which increased by approximately 0.3% after 3 months at 40 °C to a final HMW peak size less than 0.8%( Figure 5A ). The Combo 6 FDRC formulation had an initial HMW peak size of approximately 0.5% on day 0, which increased by approximately 0.3% after 3 months at 40 °C to a final HMW peak size of approximately 0.8%( Figure 5A ). The Combo 8 FDRC formulation had an initial HMW peak size of approximately 0.5% on day 0, which increased by approximately 1.0% after 3 months at 40 °C to a final HMW peak size of approximately 1.5%( Figure 5A ).

[0369] After storage at 25 °C for 3 months, the same formulations were analyzed by SEC( Figure 5A ). After storage at 25 °C for 3 months, the HMW peak sizes of the ipilimumab control formulation and the nivolumab control formulation each increased by 0.1% or less( Figure 5A ). After storage at 25 °C for 3 months, the HMW peak sizes of the “Combo new” and Combo 8 FDRC formulations each increased by 0.1% or less, and the HMW peak sizes of the Combo 4, Combo 5, and Combo 6 FDRC formulations each decreased by approximately 0.1% or less( Figure 5A ).

[0370] cIEF analysis

[0371] After storage at 25 °C for 3 months, cIEF was performed on nivolumab DP control, ipilimumab DP control, and DoE FDRC (3:1) formulations “Combo new”, Combo 4, Combo 5, Combo6, and Combo 8 ( Figure 5B ). After storage at 25 °C for 3 months, the acidic peak size of the ipilimumab control increased by approximately 5.59%, and the acidic peak size of the nivolumab DP control decreased by approximately 0.05% ( Figure 5B ). After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the “Combo new” FDRC formulation increased by approximately 0.64% and 5.98%, respectively ( Figure 5B ). After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the Combo 4 FDRC formulation increased by approximately 5.32% and 6.97%, respectively ( Figure 5B ). After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the Combo 5 FDRC formulation increased by approximately 0.12% and 5.34%, respectively ( Figure 5B ). After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the Combo 6 FDRC formulation increased by approximately 7.01% and 12.19%, respectively ( Figure 5B ). After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the Combo 8 FDRC formulation each increased by approximately 7.17% ( Figure 5B ).

[0372] Example 4

[0373] A feasibility study was conducted to evaluate the stability of ipilimumab / nivolumab FDRC at different ratios of ipilimumab to nivolumab, using a modified form of the DoE FDRC(3:1) "Combo new" formulation characterized in Example 3 as the base formulation. Ipilimumab / nivolumab FDRC platform combination (PC) formulations were prepared at ratios of ipilimumab to nivolumab of 3:1, 1:3, and 1:1, as shown in Table 5. All formulations were prepared in histidine buffer at a final concentration of 50 μM DTPA, 0.05% w / v PS80, and 8.0% w / v sucrose (Table 5). FDRC PC prototype 4 ("PC:pH 5.5-1:3") had a ratio of 1:3 and a pH of 5.5; FDRC PC prototype 5 ("PC:pH 6.0-1:3") had a ratio of 1:3 and a pH of 6.0; FDRC PC prototype 6 ("PC:pH 6.5-1:3") had a ratio of 1:3 and a pH of 6.5; FDRC PC prototype 7 ("PC:pH 6.0-1:1") had a ratio of 1:1 and a pH of 6.0; and FDRC PC prototype 8 ("PC:pH 6.0-3:1") had a ratio of 3:1 and a pH of 6.0 (Table 5).

[0374] Table 5: Ipilimumab / Nivolumab FDRC Platform Combination Formulations

[0375]

[0376]

[0377] SEC analysis

[0378] After storage at 40 °C for 3 months, SEC ( Figure 6A ) was performed on nivolumab DP control, ipilimumab DP control, and platform combination (PC) FDRC formulations PC:pH 6.0-1:1, PC:pH 5.5-1:3, PC:pH 6.0-1:3, PC:pH 6.5-1:3, and PC:pH 6.0-3:1. After storage at 40 °C for 3 months, the HMW peak sizes of the nivolumab and ipilimumab control formulations increased by approximately 1.7% and 0.25% respectively ( Figure 6A ). After storage at 40 °C for 3 months, the HMW peak size of the PC:pH6.0-1:1 FDRC formulation increased by approximately 0.5% ( Figure 6A ). After storage at 40 °C for 3 months, the HMW peak size of the PC:pH 5.5-1:3 FDRC formulation increased by approximately 1.25% ( Figure 6A ). After storage at 40 °C for 3 months, the HMW peak size of the PC:pH 6.0-1:3 FDRC formulation increased by approximately 0.75% ( Figure 6A)。After storage at 40 °C for 3 months, the HMW peak size of the PC:pH 6.5-1:3 FDRC formulation increased by approximately 0.1%( Figure 6A )。After storage at 40 °C for 3 months, the HMW peak size of the PC:pH 6.0-3:1 FDRC formulation increased by approximately 0.25%( Figure 6A )。

[0379] After storage at 5 °C for 3 months, the same formulations were analyzed by SEC( Figure 6B )。The nivolumab control formulation had an initial HMW peak size of approximately 0.70% on day 0, which increased to a final HMW peak size of approximately 0.71% after 3 months at 5 °C( Figure 6B )。The ipilimumab control formulation had an initial HMW peak size of approximately 0.4% on day 0, which did not change after 3 months at 5 °C( Figure 6B )。The PC:pH 6.0-1:1 FDRC formulation had an initial HMW peak size of approximately 0.44% on day 0, which increased to a final HMW peak size of approximately 0.45% after 3 months at 5 °C( Figure 6B )。The PC:pH 5.5-1:3 FDRC formulation had an initial HMW peak size of approximately 0.47% on day 0, which increased to a final HMW peak size of approximately 0.48% after 3 months at 5 °C( Figure 6B )。The PC:pH 6.0-1:3 FDRC formulation had an initial HMW peak size of approximately 0.51% on day 0, which did not change after 3 months at 5 °C( Figure 6B )。The PC:pH 6.5-1:3 FDRC formulation had an initial HMW peak size of approximately 0.56% on day 0, which increased to a final HMW peak size of approximately 0.58% after 3 months at 5 °C( Figure 6B )。The PC:pH 6.0-3:1 FDRC formulation had an initial HMW peak size of approximately 0.37% on day 0, which increased to a final HMW peak size of approximately 0.39% after 3 months at 5 °C( Figure 6B )。

[0380] cIEF analysis

[0381] After storage at 25 °C for 3 months( Figure 7A ) and after storage at 5 °C for 3 months( Figure 7B ), cIEF was performed on nivolumab DP control, ipilimumab DP control, and platform combination (PC) FDRC formulations PC:pH 6.0-1:1, PC:pH 5.5-1:3, PC:pH 6.0-1:3, PC:pH 6.5-1:3, and PC:pH 6.0-3:1.

[0382] After storage at 25 °C for 3 months, the size of the acidic peak of nivolumab control decreased by approximately 0.05%, and the size of the acidic peak of ipilimumab control increased by approximately 5.59%( Figure 7A ). After storage at 25 °C for 3 months, the size of the acidic peaks of nivolumab and ipilimumab in the PC:pH 6.0-1:1 FDRC formulation increased by approximately 2.6% and 7%, respectively( Figure 7A ). After storage at 25 °C for 3 months, the size of the acidic peaks of nivolumab and ipilimumab in the PC:pH5.5-1:3 FDRC formulation increased by approximately 2.1% and 5.9%, respectively( Figure 7A ). After storage at 25 °C for 3 months, the size of the acidic peaks of nivolumab and ipilimumab in the PC:pH 6.0-1:3 FDRC formulation increased by approximately 3.7% and 6.8%, respectively( Figure 7A ). After storage at 25 °C for 3 months, the size of the acidic peaks of nivolumab and ipilimumab in the PC:pH 6.5-1:3 FDRC formulation increased by approximately 5.9% and 6.3%, respectively( Figure 7A ). After storage at 25 °C for 3 months, the size of the acidic peaks of nivolumab and ipilimumab in the PC:pH 6.0-3:1 FDRC formulation increased by approximately 1.3% and 6.2%, respectively( Figure 7A ). After storage at 25 °C for 3 months, the size of the acidic peak of ipilimumab in all PC FDRC formulations increased by approximately 5.9 - 7.0%, or approximately 2.0% per month on average( Figure 7A ). The size of the acidic peak of nivolumab in the FC FDRC formulation increased by approximately 1.3 - 5.9%, or a maximum of approximately 2% per month( Figure 7A ).

[0383] After storage at 5 °C for 3 months, the size of the acidic peak of nivolumab control decreased by approximately 5.2%, and the size of the acidic peak of ipilimumab control decreased by approximately 1%( Figure 7B ). After storage at 5 °C for 3 months, the size of the acidic peaks of nivolumab and ipilimumab in the PC:pH 6.0-1:1 FDRC formulation decreased by approximately 2%, and increased by approximately 2.2%, respectively( Figure 7B ). After storage at 5 °C for 3 months, the size of the acidic peaks of nivolumab and ipilimumab in the PC:pH5.5-1:3 FDRC formulation decreased by approximately 1.1% and approximately 0.3%, respectively( Figure 7B ). After storage at 5 °C for 3 months, the size of the acidic peaks of nivolumab and ipilimumab in the PC:pH 6.0-1:3 FDRC formulation each decreased by approximately 0.2%( Figure 7B ). After storage at 5 °C for 3 months, the size of the acidic peaks of nivolumab and ipilimumab in the PC:pH 6.5-1:3 FDRC formulation increased by approximately 0.5%, and decreased by approximately 3.1%, respectively( Figure 7B)。After storage at 5 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the PC:pH 6.0-3:1 FDRC formulation increased by approximately 0.1% and decreased by approximately 0.2% respectively ( Figure 7B )。In summary, the acidic peak sizes of ipilimumab and nivolumab in the 1:3 formulation at pH 5.5-6.5 showed essentially no change after storage at 5 °C for 3 months, and there were no discernible changes in ipilimumab and nivolumab at the three different ratios ( Figure 7B )。

[0384] Example 5

[0385] A feasibility study was conducted to evaluate the stability of ipilimumab / nivolumab (1:1) FDRC in several nivolumab DP-based formulations, as shown in Table 6. These formulations were designed by modifying the nivolumab DP formulation ( Figure 1 )。Using a centrifugal filter device with a molecular weight cut-off of 50 kDa, a total of 24 vials of ipilimumab DP and nivolumab DP were buffer-exchanged from their original DP buffer formulations to a buffer formulation at pH 6.0 containing 20 mM citric acid and 50 mM NaCl (Prototype A). Prototypes B-D were prepared in the same manner to meet the specifications shown in Table 6. Prototype A contained 7.5 mg / mL ipilimumab, 7.5 mg / mL nivolumab, 20 mM citrate, 50 mM NaCl, 3.0% w / v mannitol, 100 μM diethylenetriaminepentaacetic acid (DTPA), and 0.02% PS80 at pH 6.0. Prototype A was the same as nivolumab DP, except that Prototype A had 100 μM diethylenetriaminepentaacetic acid, while nivolumab DP had 20 μM diethylenetriaminepentaacetic acid. Prototype B contained 7.5 mg / mL ipilimumab, 7.5 mg / mL nivolumab, 20 mM citrate, 50 mM NaCl, 3.0% w / v mannitol, 100 μM diethylenetriaminepentaacetic acid (DTPA), and 0.02% PS80 at pH 6.5. Prototype C contained 7.5 mg / mL ipilimumab, 7.5 mg / mL nivolumab, 20 mM citrate, 100 mM NaCl, 1.0% w / v mannitol, 100 μM diethylenetriaminepentaacetic acid (DTPA), and 0.02% PS80 at pH 6.5. Prototype D contained 7.5 mg / mL ipilimumab, 7.5 mg / mL nivolumab, 20 mM citrate, 50 mM NaCl, 6% w / v sucrose, 100 μM diethylenetriaminepentaacetic acid (DTPA), and 0.02% PS80 at pH 6.0.

[0386] Table 6: Nivolumab DP-based FDRC (1:1) formulations

[0387]

[0388]

[0389] *Note: Prototype A is similar to the nivolumab DP formulation, except for the concentration of diethylenetriaminepentaacetic acid (DTPA) which is the same as that in the ipilimumab DP formulation (see Figure 1 ).

[0390] FDRC prototypes A, B, C, and D were filtered through a 0.2 micron device and filled into 10 cc vials (1 or 2 mL per vial), stoppered, and sealed. Then they were placed on a stability station for up to 12 months and analyzed for stability by appearance, pH, SEC, HIAC, and cIEF.

[0391] SEC analysis

[0392] After storage at 40 °C for 1 month, SEC ( Figure 8 ) was performed on nivolumab DP control, ipilimumab DP control, and FDRC (1:1) prototypes A, B, C, and D based on nivolumab DP. After storage at 40 °C for 1 month, the HMW peak sizes of the nivolumab and ipilimumab control formulations increased by approximately 0.38% and 0.02% respectively ( Figure 8 ). After storage at 40 °C for 1 month, the HMW peak size of the FDRC prototype A formulation increased by approximately 0.36% ( Figure 8 ). After storage at 40 °C for 1 month, the HMW peak size of the FDRC prototype B formulation increased by approximately 0.41% ( Figure 8 ). After storage at 40 °C for 1 month, the HMW peak size of FDRC prototype C increased by approximately 0.37% ( Figure 8 ). After storage at 40 °C for 1 month, the HMW peak size of FDRC prototype D increased by approximately 0.24% ( Figure 8 ). The nivolumab control formulation and FDRC prototype A and B formulations each contain 3% w / v mannitol, while the ipilimumab control formulation and FDRC prototype C formulation have 1% mannitol, and the FDRC prototype D formulation has no mannitol (see Table 6).

[0393] cIEF analysis

[0394] After storage at 25 °C for 3 months, cIEF ( Figure 9 ) was performed on nivolumab DP control, ipilimumab DP control, and FDRC (1:1) prototypes A, B, C, and D formulations. After storage at 25 °C for 3 months, the acidic peak size of the nivolumab control increased by approximately 7.5%, and the acidic peak size of the ipilimumab control increased by approximately 8.8% ( Figure 9 ). After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the FDRC prototype A formulation each increased by approximately 9.4% ( Figure 9) After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the FDRC prototype B formulation increased by approximately 8.2% and 13.8%, respectively ( Figure 9 ) After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the FDRC prototype C formulation increased by approximately 8.7% and 10.2%, respectively ( Figure 9 ) After storage at 25 °C for 3 months, the acidic peak sizes of nivolumab and ipilimumab in the FDRC prototype D formulation increased by approximately 10.1% and 9%, respectively ( Figure 9 ) The effect of NaCl on the acidic peak changes can be observed by comparing the ipilimumab control formulation and the FDRC prototype C formulation each with 100 mM NaCl with the nivolumab control formulation and the FDRC prototypes A, B, and D formulations each with 50 mM NaCl ( Figure 9 ; Table 6).

[0395] Example 6

[0396] A fixed-dose ratio combination (FDRC) drug product of nivolumab and ipilimumab was developed in a 1:3 ratio. The ipilimumab / nivolumab FDRC was prepared from the commercial drug substances of ipilimumab and nivolumab. See Figure 1 . The ipilimumab drug substance is an aqueous solution at pH 7.0 containing 5 mg / mL ipilimumab in 20 mM Tris hydrochloride, 100 mM sodium chloride, 1.0% (w / v) mannitol, 100 μM pentetic acid, 0.01% (w / v) polysorbate 80. The nivolumab drug substance is an aqueous solution at pH 6.0 containing 20 mg / mL nivolumab in 20 mM sodium citrate, 50 mM sodium chloride, 3.0% (w / v) mannitol, 20 μM pentetic acid, 0.04% (w / v) polysorbate 80. Both the ipilimumab and nivolumab drug substances are stored at 2 °C - 8 °C.

[0397] The ipilimumab / nivolumab FDRC (3:1) drug product was formulated by combining the ipilimumab and nivolumab drug substances in a 3 to 1 ipilimumab to nivolumab protein ratio. Development stability data up to 6 months indicated that the FDRC drug product was stable when stored at 2 °C to 8 °C (the intended storage conditions). The FDRC drug product is a sterile, non-pyrogenic, single-use, preservative-free isotonic aqueous solution for IV administration. The FDRC drug product can be administered undiluted at a total protein concentration of 6.2 mg / mL, or further diluted to the desired concentration with 0.9% Sodium Chloride Injection USP or 5% Dextrose Injection USP. The FDRC is packaged in type I flint glass tubes or molded vials and stoppered with a membrane-coated butyl rubber stopper. The composition of the FDRC is provided in Table 7.

[0398] Table 7. Comparison of the compositions of the FDRC, ipilimumab, and nivolumab drug products

[0399]

[0400]

[0401] 1 Also known as diethylenetriaminepentaacetic acid (DTPA)

[0402] 2 Dilute solutions of hydrochloric acid and sodium hydroxide can be used for pH adjustment during the manufacture of ipilimumab and nivolumab DS. The solution pH is not adjusted during DP manufacture.

[0403] The stability of the FDRC DP samples prepared as shown in Table 7 was monitored under the expected (5 °C), accelerated (25 °C), and stress (40 °C) storage conditions.

[0404] The major degradation pathways identified for ipilimumab and nivolumab were aggregation (HMW species detected by SE-HPLC), fragmentation (LMW species detected by SE-HPLC), and deamidation (acidic charge variants detected by CEX or iCIEF), as shown in Examples 1-5. These changes were monitored by SE-HPLC and iCIEF in the FDRC development stability studies. Additionally, in these studies, particulate matter and binding activity were monitored at specified time points by HIAC and ELISA binding, respectively.

[0405] The results obtained from the studies showed that after 6 months of storage at 2 °C to 8 °C, in the FDRC DP, the levels of combined HMW species, combined LMW species, acidic charge variants of nivolumab, acidic charge variants of ipilimumab, and particulate matter remained essentially unchanged.

[0406] The 6-month study conducted under accelerated conditions at 25 °C showed that the formation rates of HMW species were comparable among the FDRC DP, nivolumab DP, and ipilimumab DP. The formation rate of LMW species in the FDRC DP was 0.15% per month, comparable to the formation rate of 0.18% per month in the ipilimumab DP, as FDRC is mainly composed of ipilimumab. The formation rate of acidic variants of nivolumab in the FDRC DP was 1.98% per month, comparable to the formation rate of 1.76% per month in the nivolumab DP. It was considered that the formation rates of acidic variants of ipilimumab were comparable in the FDRC and ipilimumab DP, being 2.4% and 1.9% per month, respectively. The particulate matter levels remained essentially unchanged.

[0407] Studies conducted by storing at 40 °C for 3 months showed that under this condition, similar but greater changes were observed in the FDRC DP.

[0408] The use of time study data demonstrated the stability, compatibility, and equivalence of the administration solutions prepared from the FDRC DP and from the combination of nivolumab and ipilimumab DP in IV bags.

[0409] In summary, the comparable formation rates of critical quality attributes (CQAs) such as HMW substances, LMW substances, and acidic variants under stress and accelerated conditions and the negligible changes in these CQAs under the recommended storage conditions indicate the potential of developing the FDRC DP with the commercialized DS of nivolumab and ipilimumab. Each of the studies listed above is shown in more detail as follows:

[0410] Aggregation and fragmentation detected by SE-HPLC

[0411] The degree of aggregation (HMW substances) and fragmentation (LMW substances) of the FDRC was examined by SE-HPLC. The HMW substances, monomers, and LMW substances of ipilimumab co-eluted with the HMW substances, monomers, and LMW substances of nivolumab, respectively. The results listed in Table 8 are reported as the area percentages of the combined monomers, combined HMW substances, and combined LMW substances of ipilimumab and nivolumab. The level of the combined HMW substances was initially tested at 0.5% and remained essentially unchanged (range 0.5 to 0.6%) after storage at 5 °C and 25 °C for 6 months, and increased to 1.0% after storage at 40 °C for 3 months. The level of the combined LMW substances was initially tested at 0.1%, remained essentially unchanged (range 0.1 to 0.2%) after storage at 5 °C for 6 months, increased to 1.0% after storage at 25 °C for 6 months, and increased to 2.4% after storage at 40 °C for 3 months.

[0412] Table 8. Combined monomers, HMW substances, and LMW substances in FDRC detected by SE-HPLC

[0413]

[0414] NT = Not tested

[0415] These results were compared with commercial DP formulations of ipilimumab (5 mg / mL) and nivolumab (10 mg / mL) controls, which were placed on the station with FDRC DP at 5 °C, 25 °C, and 40 °C and analyzed in a similar manner using this modified SEC-HPLC method, as shown in Tables 9 and 10, respectively. Based on the available data, it was shown that at the recommended storage temperature of 2 - 8 °C, ipilimumab, nivolumab, and FDRC were not prone to form HMW substances; and at 25 °C and 40 °C, the monthly formation rates of HMW substances among ipilimumab, nivolumab, and FDRC were comparable, as shown in Table 11. More importantly, the formation rates of HMW and LMW substances in FDRC were equivalent to those of HMW&LMW substances in ipilimumab, because FDRC mainly consists of ipilimumab and the total protein concentration in FDRC, i.e., 6.2 mg / mL, is very close to the ipilimumab DP concentration of 5 mg / mL. The comparable formation rates of CQAs such as HMW and LMW substances under stress and accelerated conditions and the negligible changes in these CQAs under the recommended storage conditions indicate the potential for developing FDRC DP.

[0416] Table 9. Monomers, HMW substances, and LMW substances in ipilimumab detected by SE-HPLC

[0417]

[0418]

[0419] Table 10. Monomers, HMW substances, and LMW substances in nivolumab detected by SE-HPLC

[0420]

[0421] Table 11. Monthly formation rates of monomers, HMW substances, and LMW substances in ipilimumab, nivolumab, and FDRC detected by SEHPLC

[0422]

[0423] Charge variants detected by iCIEF

[0424] The charge variant profile of FDRC was determined by iCIEF analysis. The ipilimumab and nivolumab peaks were separated in the chromatographic profile. The relative amounts of the acidic, main, and basic peak areas of ipilimumab are listed in Table 12, and the relative amounts of the acidic, main, and basic peak areas of nivolumab are listed in Table 13. The acidic, main, and basic peak areas of ipilimumab and nivolumab remained essentially unchanged after storage at 5 °C for 6 months. For ipilimumab and nivolumab, changes in the charge profile were observed at 25 °C and 40 °C. Degradation was significant within a short time at 40 °C, so it was not used for comparison and was considered too severe for the evaluation of DP stability.

[0425] Table 12. Charge profile of ipilimumab in FDRC detected by iCIEF

[0426]

[0427] Table 13. Charge profile of nivolumab in FDRC detected by iCIEF

[0428]

[0429] In addition, as discussed previously, the FDRC composition evaluated above consists of tris.HCl and sodium citrate dihydrate and is thus prone to pH changes with temperature due to the amine buffer Tris-HCl. Therefore, the charge profile changes between FDRC and nivolumab were carried out at 25 °C, consistent with the sample preparation temperature and storage temperature.

[0430] Comparison (Table 14) of the acidic charge profile of ipilimumab in FDRC shown in Table 12 with the ipilimumab control DP (commercial composition) in Tris-HCl buffer at pH 7 (4 °C) at 25 °C conditions showed that the formation of acidic peaks was comparable, at 2.4% and 1.93% per month, respectively, as Figure 10 shown. This relatively small difference under stress conditions was considered insignificant for the stability of the FDRC drug product under the recommended storage conditions (2 - 8 °C), as shown in Table 12.

[0431] Previously, changes in the ipilimumab charge profile have been monitored by CEX, and thus data are being collected to identify the comparability of the ipilimumab charge profile under various conditions. However, the potential for deamidation (the main degradation pathway of ipilimumab) may be reduced because deamidation kinetics are generally slower at lower pH.

[0432] Table 14. Charge profile of ipilimumab detected by iCIEF at 25 °C

[0433]

[0434] The comparison of the nivolumab acidic charge profile in the FDRC shown in Table 15 with three nivolumab long-term stability batches (LTSB) at 25 °C indicates that the formation of acidic peaks is comparable, at 1.97% and 1.75% per month, respectively, as Figure 11 shown.

[0435] Charge profile of nivolumab (GMP batch) at 25 °C detected by iCIEF

[0436]

[0437] Particulate matter detected by HIAC

[0438] Samples stored at 5 °C and 25 °C for up to 6 months were examined using an optical resistance particle counting procedure (HIAC) to determine the size and number of particles based on the size in the FDRC DP. As shown in Table 16, the particulate matter values for ≥2 µm, ≥5 µm, ≥10 µm, and ≥25 µm are variable but fully fall within the acceptance criteria outlined in USP <787>.

[0439] Table 16. Particulate matter detected by HIAC

[0440]

[0441] Binding activity determined by ELISA assay

[0442] The specific binding of ipilimumab to the human CTLA-4 receptor and nivolumab to the human PD-1 receptor was detected using ELISA assays. The binding activities of ipilimumab and nivolumab in the FDRC samples were calculated relative to the ipilimumab and nivolumab reference standards, respectively. The binding activities of the FDRC samples stored at 25 °C for 2 months were within the recommended acceptance criteria (70% - 130%) (Table 17).

[0443] Table 17. Binding activity determined by ELISA assay

[0444]

[0445] Trypsin peptide mapping assay

[0446] Trypsin peptide mapping assays were performed to measure deamidation and oxidation. Samples were reduced, alkylated and digested with trypsin. Trypsin peptides were separated on a C-18 column and detected by a UV detector at 215 and 280 nm, followed by detection with a mass spectrometer (LTQ-Orbitrap-Elite). Relative quantification was achieved by comparing the peak areas of intact peptides and modified peptides in the selected ion chromatograms. The assay results are shown in Tables 18 and 19.

[0447] Table 18. Trypsin Peptide Mapping - Deamidation

[0448]

[0449]

[0450] 3 Nivolumab H 4 = Heavy chain trypsin peptide #4 of nivolumab

[0451] 4 Ipilimumab H 5 = Heavy chain trypsin peptide #5 of ipilimumab

[0452] 5 Ipilimumab H 37 / Nivolumab H 36 Deamidation 1 = Heavy chain trypsin peptide #37 (Asn#) of ipilimumab and heavy chain trypsin peptide #36 (Asn#) of nivolumab

[0453] 6 Ipilimumab H 37 / Nivolumab H 36 Deamidation 2 = Heavy chain trypsin peptide #37 (Asn#) of ipilimumab and heavy chain trypsin peptide #36 (Asn#) of nivolumab

[0454] 7 Ipilimumab H 37 / Nivolumab H 36 Deamidation 3 = Heavy chain trypsin peptide #37 (Asn#) of ipilimumab and heavy chain trypsin peptide #36 (Asn#) of nivolumab

[0455] 8 Ipilimumab H 37 / Nivolumab H 36 Deamidation 4 = Heavy chain trypsin peptide #37 (Asn#) of ipilimumab and heavy chain trypsin peptide #36 (Asn#) of nivolumab

[0456] Table 19. Trypsin Peptide Mapping - Oxidation

[0457]

[0458] 9 Ipilimumab H 21 / Nivolumab H 22 = Trypsin peptide #21 (His / Met#) of the heavy chain of ipilimumab and trypsin peptide #22 (His / Met#) of the heavy chain of nivolumab

[0459] 10 Nivolumab H 4 = Trypsin peptide #4 of the heavy chain of nivolumab

[0460] 11 Ipilimumab H 3 = Trypsin peptide #3 of the heavy chain of ipilimumab

[0461] pH analysis of the sample

[0462] Measure the pH of FDCR DP as shown in Table 20.

[0463] Table 20. pH values of DP solutions

[0464]

[0465]

[0466] Use-time stability of FDRC drug product

[0467] Studies were conducted to demonstrate the stability and compatibility of FDRC DP together with 0.9% Sodium Chloride Injection USP (NS), IV bags, IV infusion sets, and in-line filters. After storage at 5 °C for 2 months, the FDRC DP samples were diluted in NS in IV bags, stored at 25 °C for 4 hours, and then stored at 5 °C for 20 hours. The solution in the IV bag was then infused through the IV set and in-line filter. Samples were collected and analyzed by HIAC, microflow imaging (MFI), SE-HPLC, CE-SDS, iCIEF, and reversed-phase ultra-high performance liquid chromatography (RP-UPLC).

[0468] The study results are shown in Tables 21 - 23. The data show that after the compatibility study, there were little or no changes between the initial values for particulate matter (by HIAC), aggregation (by SE-HPLC), fragmentation (by (SE-HPLC), purity (by CE-SDS), charge variant profile (by iCIEF), and the ipilimumab / nivolumab protein ratio (by RP-UPLC).

[0469] Results showed that FDRC DP can be diluted with 0.9% Sodium Chloride Injection USP to a concentration range of 1.5 / 0.5 to 4.2 / 1.4 mg / mL ipilimumab / nivolumab for IV infusion. The diluted solution in the IV bag can be stored at 5°C for up to 24 hours, and up to 4 hours of the 24 hours can be stored at room temperature (25°C).

[0470] Table 21. Use-time stability and comparability of FDRC drug product

[0471]

[0472]

[0473] 12 Samples collected from the IV bag at zero time after dilution of FDRC DP in the IV bag

[0474] 13 Samples collected after storage for 24 hours and infusion through an IV set and in-line filter

[0475] Table 22. Use-time stability and comparability of FDRC drug product

[0476]

[0477] Table 23. Use-time stability and comparability of FDRC drug product

[0478]

[0479] The use-time stability of co-administered ipilimumab and nivolumab drug products was studied to demonstrate the stability and compatibility of the co-administered DP together with 0.9% Sodium Chloride Injection USP (NS), IV bag, IV infusion set, and in-line filter. The ipilimumab and nivolumab single-therapy DP vials were diluted in NS in an IV bag, stored at 25°C for 4 hours, and then stored at 5°C for 20 hours. Then the solution in the IV bag was infused through an IV set and in-line filter. Samples were collected and analyzed by HIAC, microflow imaging (MFI), SE-HPLC, CE-SDS, iCIEF, and reversed-phase ultra-high-performance liquid chromatography (RP-UPLC).

[0480] The research results are shown in Tables 24 - 26. The data showed that after the compatibility study, there was little or no change from the initial values for particulate matter (by HIAC), aggregation (by SE - HPLC), fragmentation (by SE - HPLC), purity (by CE - SDS), charge variant profile (by iCIEF), and the ipilimumab / nivolumab protein ratio (by RP - UPLC). The results indicated that the co - administered DP can be diluted with 0.9% Sodium Chloride Injection USP to a concentration range of 1.5 / 0.5 to 4.2 / 1.4 mg / mL ipilimumab / nivolumab for IV infusion. The diluted solution in the IV bag can be stored at 5°C for up to 24 hours, and up to 4 hours of the 24 - hour period can be at room temperature (25°C).

[0481] Table 24. Use - time stability and comparability of co - administered ipilimumab and nivolumab drug products

[0482]

[0483]

[0484] 14 Samples collected from the IV bag at zero time after dilution of the co - administered DP in the IV bag

[0485] 15 Samples collected after storage for 24 hours and infusion through an IV set and an in - line filter

[0486] Table 25. Use - time stability and comparability of co - administered ipilimumab and nivolumab drug products (continued 2)

[0487]

[0488] Table 26. Use - time stability and comparability of co - administered ipilimumab and nivolumab drug products (continued 3)

[0489]

[0490] Example 7

[0491] For the nivolumab - ipilimumab 1:3 fixed - dose ratio combination, the process performance qualification (PPQ) limits for pH and polysorbate 80 in the nivolumab - ipilimumab 1:3 fixed - dose ratio combination (FDRC) were determined. The quantitative composition of the FDRC drug product is shown in Table 27.

[0492] Table 27. Quantitative composition of the FDRC drug product

[0493]

[0494] q.s. = sufficient quantity

[0495] pH limit value

[0496] 16 Also known as diethylenetriaminepentaacetic acid

[0497] 17 Dilute solutions of hydrochloric acid and sodium hydroxide can be used for pH adjustment during the manufacture of nivolumab and ipilimumab DS. The solution pH is not adjusted during FDRCDP manufacture.

[0498] Commercially available ipilimumab DS and nivolumab DS have pH acceptance criteria of 6.6 - 7.6 (4°C) and 5.5 - 6.5, respectively. Since FDRC DP is manufactured without any further manipulation of the introduced DS, studies were conducted to understand the possible range of pH in FDRC DP due to the variability of the introduced DS.

[0499] Table 28. Variability of FDRC DP pH due to the variability of the introduced DS

[0500]

[0501] A solution was prepared to simulate the FDRC DP formulation by adding 42 mL of 20 mM Tris - HCl and 3.5 mL of 20 mM sodium citrate buffer. The results of this evaluation (Table 28) indicate that the pH range of FDRC DP can be within the range of 5.7 - 7.0, with a target pH of 6.2 - 6.3 under ambient conditions. This property is well - controlled in the introduced nivolumab and ipilimumab DS, and thus, FDRC DP is unlikely to experience pH extremes of 5.7 or 7.0. Additionally, based on the current understanding of the CQAs of ipilimumab and nivolumab, the risk of FDRC DP quality attributes is expected to be higher in the higher pH range. Based on this understanding, two additional studies were initiated to evaluate the effect of pH on DP quality attributes and to understand the effect of the variability of various excipients (including pH) from the introduced DS on DP quality attributes.

[0502] The evaluation of the data from the pH range study focused on the quality attributes of FDRC affected by pH changes, such as the charge profile monitored by capillary isoelectric focusing (icIEF) and the high molecular weight aggregates monitored by size exclusion chromatography (SEC). Under the storage conditions recommended at 2-8°C or up to 3 months at 25°C, within the pH range (environment) of 5.4-6.6, the SEC profile of FDRCDP had no discernible changes. Only under accelerated conditions (40°C) was quantifiable change observed, where the pH range evaluated showed no effect on the SEC profile (Table 29).

[0503] Table 29. Monomer, HMW and LMW species due to pH variability (%)

[0504]

[0505]

[0506] As shown in Table 30, the charge profiles of ipilimumab and nivolumab did not show any significant differences beyond the analytical error after 6 months at the recommended storage temperature of 2-8°C. The charge profiles of ipilimumab and nivolumab were primarily evaluated at a storage temperature of 25°C because the differences were more discernible, unlike at 40°C where the profiles changed significantly and the molecules clearly dissociated. Figure 12 The acidic profiles of ipilimumab and nivolumab in FDRC DP compared to their respective controls at pH 6.0 are shown.

[0507] Table 30. Acidic and main peak profiles due to pH variability after 6 months at 5°C (cIEF)

[0508]

[0509] Furthermore, pH robustness studies initiated with the variables shown in Table 31, where the pH range evaluated varied between 5.8-7.0, have yielded similar SEC and cIEF profile observations.

[0510] Table 31. DP durability study design

[0511]

[0512]

[0513] The HMW profile of FDRC DP remained unchanged after storage at 2-8°C and 25°C for 6 months, as Figure 13 As shown (monomer overview see Figure 14) indicates that there is no pH effect even in the presence of other variables such as the concentrations of sodium chloride, mannitol, and PS80. Evaluation of the acidic peak and main peak profiles of ipilimumab and nivolumab in FDRC DP ( Figure 15 - 18 ) clearly shows the pH-dependence of deamidation at the accelerated temperature of 25 °C, as indicated by the increase in the acidic peak profile in the higher temperature range at pH 7.0. However, this effect at the accelerated temperature did not translate into a quantifiable difference at the recommended storage temperature of 2 - 8 °C.

[0514] The effect of the cIEF peak profile and pH value is as Figure 19 shown, the iCIEF profile: the pH range of 5.4 - 6.6 is as Figure 20 shown.

[0515] Polysorbate 80 limit: NLT 60 μg / mL

[0516] The concentration of polysorbate 80 in FDRC DP is mainly limited by the ratio of ipilimumab and nivolumab DS used to prepare FDRC DP by mixing, where the target concentration of PS80 in FDRC DP is 120 μg / mL, and the nominal concentrations of ipilimumab and nivolumab DS are 100 μg / mL and 400 μg / mL, respectively. For nivolumab and ipilimumab DS, there are no release acceptance criteria for PS80; however, in-process limits of 275 - 525 μg / mL and 60 - 140 μg / mL are respectively applicable for the manufacture of nivolumab DS and ipilimumab DS.

[0517] Preliminary analysis of DP attributes affected by PS80 variability such as SEC HMW (%) showed no discernible changes ( Figure 13 ), and particles measured by HIAC in the range of 10 - 25 microns met the current USP acceptance criteria. Additionally, the FDRC DP manufacturing method is designed such that no filter flushing is required before filling the DP vials, due to the presence of an intermediate tank (35 - 40 L) downstream of redundant sterile filters, which is filled before starting the filling operation.

[0518] Furthermore, during the DP optimization study, a range of PS80 concentrations from 120 μg / ml to 1000 μg / ml was evaluated in FDRC DP under the worst-case agitation condition of shaking on a horizontal shaker at 300 rpm for up to 72 hours. These studies showed no visible particles after 72 hours upon visual appearance analysis, and the SEC profiles of all prototypes had no discernible differences from the initial time point. Based on this, it was decided to add concentrated PS80 concentrate at the DP production site without changing the target concentration of FDRC DP.

[0519] However, to understand the potential risk of particle generation or HMW substance formation due to the significant decrease in PS80 levels upon dilution with an infusion solution such as saline, a study was conducted in which an FDRC DP solution with a target concentration of 120 μg / ml of PS80 was diluted 20-fold (6 μg / mL) with normal saline, and the resulting solution was evaluated for up to 24 hours by visual appearance, particles measured by HIAC, and SEC HMW (%). This study showed that a concentration of PS80 reduced to 6 μg / ml in an infusion solution prepared from FDRC DP did not cause any changes in the visual appearance, HMW profile, or HIAC characteristics of the solution, which strengthens the rationale for maintaining the target concentration at 120 μg / ml. In addition, during clinical and commercial administration, it is expected that the FDRC DP will be diluted ~3X with an infusion solution, resulting in a PS80 concentration of 40 μg / ml. The proposed NLT PS80 concentration of 60 μg / ml still results in a final infusion solution concentration of 20 μg / ml, which exceeds the evaluated concentration of 6 μg / ml in the dilution study discussed above.

[0520] This application claims the benefit of U.S. Provisional Application No. 62 / 303,855, filed March 4, 2016, U.S. Provisional Application No. 62 / 269,000, filed December 17, 2015, U.S. Provisional Application No. 62 / 265,268, filed December 9, 2015, and U.S. Provisional Application No. 62 / 149,325, filed April 17, 2015, the entire contents of which are incorporated herein by reference. Sequence Listing <110> BRISTOL-MYERS SQUIBB COMPANY SADINENI, VIKRAM QUAN, YONG KASERER, WALLACE <120> Composition Comprising a Combination of an Anti-PD-1 Antibody and an Additional Antibody <130> 3338.026PC04 / ELE / C-K / E-H <150> US 62 / 149,325 <151> 2015-04-17 <150> US 62 / 303,855 <151> 2016-03-04 <150> US 62 / 269,000 <151> 2015-12-17 <150> US 62 / 265,268 <151> 2015-12-09 <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH chain <400> 1 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ser Met Val Arg Gly Asp Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 <210> 2 <211> 107 <212> PRT <213> Artificial sequence <220> <223> VL chain <400> 2 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 123 <212> PRT <213> Artificial sequence <220> <223> VH chain <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Phe His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Ala Gly Ser Asn Lys Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Gln Leu Asp Tyr Tyr Tyr Tyr Tyr Val Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL chain <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> VH chain <400> 5 Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly 20 25 30 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 35 40 45 Val Ile Trp Tyr Ala Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Arg Ile Ala Val Ala Phe Tyr Tyr Ser Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> VL chain <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain <400> 7 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ser Met Val Arg Gly Asp Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 130 135 140 Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn 195 200 205 Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg 210 215 220 Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg 290 295 300 Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly <210> 8 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light Chain <400> 8 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 9 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain <400> 9 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Ser Met Val Arg Gly Asp Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235 240 Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly 450

Claims

1. A pharmaceutical composition comprising a buffer and nivolumab and ipilimumab in a 1:1 ratio, said buffer comprising 13.3 mM Tris-HCl, 6.7 mM citrate, 1.67% w / v mannitol, 83.3 mM NaCl, 73.3 μM DTPA, and 0.013% PS80, with a pH of 6.2; wherein the concentration of nivolumab is 3.3 mg / mL or 4.0 mg / mL, and wherein the concentration of ipilimumab is 3.3 mg / mL or 4.0 mg / mL.

2. The pharmaceutical composition of claim 1, wherein the composition: (i) is stable for 1 week to 6 months at 5°C; (ii) is stable for 1 week to 1 month at 40°C; or (ii) is stable for 1 week to 2 months at 25°C.

3. The pharmaceutical composition of claim 1 or 2, said pharmaceutical composition comprising 3.3 mg / mL nivolumab and 3.3 mg / mL ipilimumab.

4. The pharmaceutical composition of claim 1 or 2, said pharmaceutical composition comprising 4.0 mg / mL nivolumab and 4.0 mg / mL ipilimumab.

5. A kit comprising the pharmaceutical composition of any one of claims 1-4 and instructions for administering said pharmaceutical composition to a subject in need thereof, wherein said subject has melanoma.

6. Use of the pharmaceutical composition of any one of claims 1-4 or the kit of claim 5 in the manufacture of a medicament for treating a disease or disorder in a human patient in need thereof, wherein said human patient has melanoma.

7. Use of a composition in the manufacture of a medicament for treating a disease or disorder in a human patient in need thereof, wherein said human patient has melanoma, said composition comprising a buffer and nivolumab and ipilimumab in a 1:1 ratio, said buffer comprising 13.3 mM Tris-HCl, 6.7 mM citrate, 83.3 mM NaCl, 1.67% w / v mannitol, 73.3 μM DTPA, and 0.013% PS80, with a pH of 6.2; wherein the concentration of nivolumab is 3.3 mg / mL, and the concentration of ipilimumab is 3.3 mg / mL.

8. Use of a composition in the manufacture of a medicament for treating a disease or disorder in a human patient in need thereof, wherein said human patient has melanoma, said composition comprising a buffer and nivolumab and ipilimumab in a 1:1 ratio, said buffer comprising 1.67% w / v mannitol, 83.3 mM NaCl, 73.3 μM DTPA, and 0.013% PS80, with a pH of 6.2; wherein the concentration of nivolumab is 4.0 mg / mL, and the concentration of ipilimumab is 4.0 mg / mL.

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