EA042223B1 - PHARMACEUTICAL COMPOSITION FOR TREATMENT OF INFECTIOUS DISEASE OR CANCER - Google Patents

Description

Throughout this document, various publications are indicated in brackets by author's name and date, or by patent number or patent publication number. The entire contents of these publications are incorporated herein by reference for a more complete description of the prior art known to those skilled in the art at the date of the description and filing of the application for the present invention. In addition, the citation of a reference herein should not be construed as an admission that such reference is prior to the present invention.

The technical field to which the invention belongs

The present invention relates to pharmaceutical compositions that contain a combination of an anti-immune checkpoint antibody and a second antibody, in a fixed dose dosage form.

Background Art Information

Human cancers undergo numerous genetic and epigenetic changes that generate neoantigens potentially recognized by the immune system (Sjoblom et al. (2006) Science 314:268-74). The adaptive immune system, composed of T- and B-lymphocytes, has a powerful anti-cancer potential with broad capabilities and excellent specificity in response to various tumor antigens. In addition, the immune system exhibits significant flexibility and a memory component. Successful exploitation of all these characteristics of the adaptive immune system will make immunotherapy unique among all cancer treatments.

Recently, several immune pathway inhibitors have begun to provide new immunotherapeutic approaches to cancer treatment, including the development of an antibody (Ab), ipilimumab (YERVOY®), that binds to and inhibits cytotoxic T lymphocyte antigen 4 (CTLA-4), to treatment of patients with advanced melanoma, and the development of antibodies such as nivolumab and pembrolizumab (formerly lambrolizumab; USAN Council Statement (2013) Pembrolizumab: Statement on a nonproprietary name adopted by the USAN Council (ZZ-165), November 27, 2013), which bind specifically to the programmed cell death receptor-1 (PD-1) and block the inhibitory PD-1/PD-1 ligand pathway.

Anti-immune checkpoint antibodies can be administered in combination with other antibodies. However, administration of two antibodies can be cumbersome due to the difference in dosages and dosing intervals between the two antibodies, thereby leading to multiple intravenous injections at different time points. In addition, the stability profiles of the two antibodies can differ significantly. Due to the unique nature of each antibody, such as variations in Fc glycosylation, partial processing of heavy chain C-terminal Lys, Fc methionine oxidation, hinge cleavage, and glycation of Lys residues, each antibody has different physicochemical and/or thermodynamic properties, such as different degradation profiles. exposed to heat, freezing, light, pH extremes, agitation, shear stress, certain metals and organic solvents. Thus, although a single dosage form containing two antibodies will increase convenience, the unique nature of each antibody makes it difficult to identify.

Brief description of the invention

The present invention relates to a pharmaceutical composition for the treatment of an infectious disease or cancer in a subject in need thereof, wherein the pharmaceutical composition contains nivolumab and ipilimumab in a ratio of 1:3 in Tris-citrate buffer containing 4.62 mg/ml ipilimumab, 1.54 mg/ml nivolumab, 1.15% mannitol, 96.15 mM NaCl, 93.85 μM DTPA and 0.012% PS80, and having a pH of 5.6 to 6.6.

In some embodiments, the Tris-citrate buffer contains sodium citrate dihydrate and Tris hydrochloride (Tris-HCl).

In some embodiments, the Tris-citrate buffer contains 18.5 mM Tris-HCl.

In some embodiments, the tris-citrate buffer contains 1.5 mM sodium citrate dihydrate.

In some embodiments, the pH of the composition is 6.0, 6.2, 6.5, or 6.6.

In some embodiments, the pH is 6.2. In some embodiments, the pH is 6.1.

The present invention also relates to a pharmaceutical composition for the treatment of an infectious disease or cancer, wherein the pharmaceutical composition contains nivolumab and ipilimumab in a ratio of 1:3, containing ipilimumab 4.62 mg/ml, nivolumab 1.54 mg/ml, Trishydrochloride 18.46 mM ( Tris-HCl), 1.54 mM sodium citrate dihydrate, 96.15 mM NaCl, 1.15% (w/v) mannitol, 93.85 μM pentetic acid, 0.012% w/v PS80, and has a pH of about 6.0.

In some embodiments, the implementation of the composition:

a) is stable at 5°C for at least 1 week;

b) is stable at 40°C for at least 1 week; or

c) is stable at 25°C for at least 1 week.

- 1 042223

In some embodiments, the implementation of the composition:

a) exhibits an acid peak change that is less than 10% after storage for 6 months or 3 months at 5°C;

b) exhibits an acid peak change that is less than 15% after 3 months storage at 25°C;

c) exhibits an acid peak change that is less than 15% after storage for 3 months at 40°C;

d) exhibits a high molecular weight peak change that is less than 5% after 3 months storage at 4°C;

e) exhibits a high molecular weight peak change that is less than 5% after storage for 2 months or 3 months at 25°C;

f) exhibits a high molecular weight peak change that is less than 5% after storage for 2 months or 3 months at 40°C;

g) exhibits a major peak change in capillary isoelectric focusing (cIEF) analysis that is less than 5% after 1 month storage at 4°C;

h) exhibits a major peak change in capillary isoelectric focusing (cIEF) analysis that is less than 5% after 1 month storage at 25°C;

i) exhibits a major peak change in capillary isoelectric focusing (cIEF) analysis that is less than 5% after 1 month storage at 40°C;

j) exhibits a low molecular weight peak change that is less than 5% after 2 months storage at 40°C;

k) exhibits a low molecular weight peak change that is less than 5% after 2 months storage at 25°C; or

l) shows a low molecular weight peak change that is less than 5% after 2 months storage at 4°C.

In some embodiments, the cancer is selected from melanoma, 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, cancer ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer 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 lympho-regional leukemia, chronic lymphocytic leukemia, solid childhood tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancers and any combination of these types of cancer.

The pharmaceutical composition according to the invention may also contain one or more additional components selected from the group consisting of a bulking agent, a stabilizing agent, a chelating agent, a surfactant, a buffering agent, an ionic surfactant, and any combination thereof.

The composition may furthermore contain a bulking agent. In certain embodiments, the bulking agent is selected from the group consisting of NaCl, mannitol, glycine, alanine, and any combination thereof.

In certain embodiments, the composition contains a stabilizing agent. The stabilizing agent is selected from the group consisting of sucrose, trehalose, raffinose, arginine; or any combination of them.

In other embodiments, the composition contains a chelating agent. The chelating agent is selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid, nitrilotriacetic acid, and any combination thereof.

In certain embodiments, the surfactant is selected from the group consisting of polysorbate 80 (PS80), polysorbate 20 (PS20), and any combination thereof.

In some embodiments, the composition contains PS80 (% w/v) at a concentration of 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 PS80 concentration is about 0.01%, about 0.012%, about 0.013%, about 0.02%, about 0.23%, about 0.04%, or about 0.05%.

- 2 042223

In some embodiments, the composition contains sucrose (% w/v) at a concentration of 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 least about 15%. In some embodiments, the sucrose concentration is about 6 to 8.5%.

The composition according to the invention after formulation can remain stable and stored at a temperature of 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 remains stable and can be stored at a temperature of 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. In other embodiments, the composition remains stable and can be stored 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.

The pharmaceutical composition according to the invention may exhibit minimal change in the acid peak under stressful conditions, for example after storage at a certain temperature for an extended period of time. In one embodiment, the composition exhibits an acid peak change 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 for about 6 months or about 3 months at about 5°C. In certain embodiments, the composition exhibits an acid peak change 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 for about 3 months at about 25°C. In other embodiments, the composition exhibits an acid peak change 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 for about 3 months at about 40°C.

The composition according to the invention may also show a minimal change in the high molecular weight peak after storage for an extended period of time, several weeks, months or years. In one embodiment, the composition exhibits a high molecular weight peak change 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 for 3 months at about 4°C. In another embodiment, the composition exhibits a high molecular weight peak change 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 for about 2 months or about 3 months at about 25°C. In other embodiments, the composition exhibits a high molecular weight peak change 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 for about 2 months or about 3 months at about 40°C.

In addition, in certain embodiments, the implementation of the composition may show a minimum change in the main peak according to the analysis by the method of capillary isoelectric focusing (cIEF). In one embodiment, the composition shows a major peak change 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 for about 1 month at about 4°C. In some embodiments, the composition exhibits a major peak change as measured by 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% after storage for about 1 month at about 25°C. In some embodiments, the implementation of the composition shows a change in the main peak according to the analysis

- 3 042223 method of capillary isoelectric focusing (cIEF), 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 for about 1 month at about 40°C.

In some embodiments, the composition exhibits minimal change in the low molecular weight peak. In one embodiment, the composition exhibits a low molecular weight peak change that is 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 for about 2 months at about 40°C. In certain embodiments, the composition exhibits a low molecular weight peak change that is 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 for about 2 months at about 25°C. In certain embodiments, the composition exhibits a low molecular weight peak change that is 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 for about 2 months at about 4°C.

In some embodiments, the composition is diluted prior to use. In some embodiments, the composition is diluted with 0.9% sodium chloride injection, USP or 5% dextrose injection, USP, before use. In some embodiments, the composition is diluted to obtain the desired concentration of the first and second antibodies.

In some embodiments, the disease or condition is an infectious disease. In some embodiments, the disease is cancer. In some embodiments, the cancer is melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer. , ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, 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 myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid childhood tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, carcinoma renal pelvis, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal cord tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers and any combination of these types of cancer.

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 constant dose. In some embodiments, the amount of the first antibody and the amount of the second antibody administered to the patient as a single dose are identical to the amount of X and the amount of Y, respectively. In some embodiments, the composition is administered at a dose based on body weight. In some embodiments, the amount of first antibody administered to a 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 first antibody administered to a patient is at least about 1 mg/kg.

In some embodiments, the composition is administered at least once a week, at least twice a week, at least every two weeks, at least every three weeks, or at least once a month. In some embodiments, administration is continued 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 more than 2 years. In some embodiments, another anticancer agent is also used to treat the patient.

Brief description of the figures

In FIG. 1 shows compositions of dosage forms containing pharmaceutical substances (DS) and drug preparations (DP) based on an anti-CTLA4 antibody (ie ipilimumab) and an anti-PD-1 antibody (ie nivolumab).

In FIG. 2A shows Size Exclusion Chromatography (SEC) results of a combination dosage form containing a 1:1 fixed dose ratio combination (FDRC = combination with

- 4 042223 fixed dose ratio) anti-PD-l antibodies (eg nivolumab) and anti-CTLA-4 antibodies (eg ipilimumab). In FIG. 2B shows the results of capillary isoelectric focusing (cIEF) analyzes of a combined dosage form containing a 1:1 fixed dose ratio combination (FDRC = fixed dose ratio combination) of an anti-PD-1 antibody (eg, nivolumab) and an anti-CTLA-4 antibodies (eg ipilimumab). EC refers to a mixture of tris and citrate buffers. Results for nivolumab DP and ipilimumab DP are presented as controls; and the combination of an anti-PD-1 antibody and an anti-CTLA4 antibody (eg, nivolumab and ipilimumab) is shown as EC FDRC in FIG. 2A, B. FIG. 2A shows the actual change in high molecular weight (HMW) peak size (%) in dosage forms stored for 3 months at 40°C compared to day-0 controls. The adjusted pH at 25° C., and the concentrations of polysorbate 80 (PS80), NaCl, and mannitol for each prototype dosage form are shown below the x-axis in FIG. 2A. In FIG. 2B shows the actual change in acid peak size (%) of nivolumab and ipilimumab in dosage forms stored for 6 months at 5°C compared to day-0 controls. The adjusted pH at 25°C and the concentrations of NaCl and mannitol for each dosage form are shown below the x-axis in FIG. 2B. The experimental points are labeled N (nivolumab), C (combination of nivolumab and ipilimumab), and I (ipilimumab) in FIG. 2A, B.

In FIG. 3A,B show the results of SEC analyzes of dosage forms containing a fixed dose combination of 1:3, 1:1, or 3:1 of an anti-PD-1 antibody (eg, nivolumab) and an anti-CTLA-4 antibody (eg, ipilimumab). ). Results for nivolumab DP and ipilimumab DP are presented as controls; and the combination of an anti-PD-1 antibody and an anti-CTLA4 antibody (eg, nivolumab and ipilimumab) is shown as EC (FIG. 3A, B). In FIG. 3A shows HMW peak size (%) at day-0 (Baseline), HMW peak size after 2 months storage at 40°C, and change in HMW peak size between day-0 control and formulation after 2 months storage at 40°C. for each sample and prototype dosage form (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) ). In FIG. 3B shows LMW peak size (%) at day-0 (Baseline), LMW peak size after 2 months storage at 40°C, LMW peak size change between day-0 control and each formulation after 2 months storage at 40 °C, and change in LMW peak size between control at day-0 and each dosage form after storage for 3 months at 25°C. Adjusted pH at 25° C., ipilimumab concentration, nivolumab concentration, PS80 and NaCl for each dosage form are shown below the x-axis (Fig. 3A, B).

In FIG. 4A-C show the results of a cIEF analysis of dosage forms containing a fixed dose combination of 1:3, 1:1, or 3:1 of an anti-PD-1 antibody (eg, nivolumab) and an anti-CTLA-4 antibody (eg, ipilimumab). ) after storage for 3 months at 25°C (Fig. 4A), 3 months at 5°C (Fig. 4B) and 1 month at 25°C (Fig. 4C). The results for nivolumab DP and ipilimumab DP are shown as controls (FIGS. 4A-C). Actual differences in acid peak sizes (%) of nivolumab (N) and ipilimumab (I) at selected time points relative to controls on day-0 are shown for controls and each prototype formulation (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)) (FIGS. 4A-C). The theoretical pH at 25° C., buffer type, and ipilimumab to nivolumab ratio for each dosage form are shown below the x-axis in FIG. 4A, B; and NaCl concentration, theoretical pH at 25°C, and ipilimumab to nivolumab ratio for each dosage form are shown below the x-axis in FIG. 4C. The theoretical pH value is equivalent to the pH value in the stability study (Fig. 4A-C).

In FIG. 5A, B show the results of SEC (FIG. 5A) and cIEF (FIG. 5B) analyzes of novel Design-of-Experiment (DoE) dosage forms containing a 3:1 fixed dose combination of anti- PD-1 antibodies (eg nivolumab) and anti-CTLA antibodies (eg ipilimumab). The results for nivolumab DP and ipilimumab DP are shown as controls (FIGS. 5A-B). In FIG. 5A shows HMW peak size (%) at day-0 (Baseline), HMW peak size after 3 months storage at 40° C., change in HMW peak size between day-0 control and formulation after 3 months storage at 40° C. C, and HMW peak size change between day-0 control and formulation after 3 months storage at 25°C for formulation controls and each prototype formulation (Combo New, Combo 4, Combo 5, Combo 6 and Combo 8 ). In FIG. 5B shows the actual differences in acid peak sizes (%) of nivolumab (N) and ipilimumab (I) versus controls at day 0 for each prototype formulation after 3 months storage at 25°C. Ratio of ipilimumab to nivolumab; concentrations of NaCl, mannitol and sucrose; theoretical pH values at 25°C and the type of buffer for each dosage form are shown under the x-axes (Fig. 5A, B).

In FIG. 6A, B show the results of SEC analyzes of platform combined (PC) formulations containing a 1:1 fixed dose ratio combination; 1:3 or 3:1 anti-PD-1 antibodies (eg, nivolumab) and anti-CTLA-4 antibodies (eg, ipilimumab), after storage for 3 months at 40°C (Figure 6A) and 3 months at 5°C (Fig. 6B). results

- 5 042223 for nivolumab DP and ipilimumab DP are shown as controls (FIGS. 6A, B). In FIG. 6A shows the actual change in HMW peak size (%) between controls at day-0 and each control and prototype dosage form (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 for 3 months at 40°C. In FIG. 6B shows HMW peak size (%) on Day 0 (Baseline) and HMW peak size after 3 months storage at 5°C for each dosage form. The type of buffer and the ratio of ipilimumab to nivolumab for each dosage form are shown below the x-axes (FIGS. 6A, B).

In FIG. 7A, B show the results of cIEF assays of pooled platform (PC) formulations containing a 1:1 fixed dose combination; 1:3 or 3:1 anti-PD1 antibodies (eg, nivolumab) and anti-CTLA-4 antibodies (eg, ipilimumab), after storage for 3 months at 25°C (Figure 7A) and 3 months at 5°C C (Fig. 7B). The results for nivlumab DP and ipilimumab DP are shown as controls (Fig. 7A, B). In FIG. 7A shows actual differences in acid peak sizes (%) of nivolumab (N) and ipilimumab (I) versus controls at day-0 for each control and prototype formulation (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 for 3 months at 25°C . In FIG. 7B shows actual differences in acid peak sizes (%) of nivolumab (N) and ipilimumab (I) versus controls at day-0 for each dosage form after 3 months storage at 5°C. The buffer type and ratio of ipilimumab to nivolumab for each dosage form is shown below the x-axes (FIGS. 7A, B).

In FIG. 8 shows the results of SEC analyzes of dosage forms containing FDRC (1:1) based on nivolumab DP after storage for 1 month at 40°C. Results for nivolumab DP and ipilimumab DP are shown as controls (FIG. 8). The actual change in HMW peak size (%) between day-0 controls and formulations after 1 month storage at 40° C. is shown for each control and prototype formulation (A, B, C, and D) (FIG. 8). The ipilimumab to nivolumab ratio, buffer type, and theoretical adjusted pH for each dosage form are shown below the x-axis (FIG. 8).

In FIG. 9 shows the results of cIEF analyzes of formulations containing FDRC (1:1) based on nivolumab DP after storage for 3 months at 25°C. Results for nivolumab DP and ipilimumab DP are shown as controls. In FIG. 9 shows the actual differences in acid peak sizes (%) of nivolumab (N) and ipilimumab (I) versus controls at day-0 for each control and prototype formulation (A, B, C, and D) after 3 months storage at 25 °C. The ipilimumab to nivolumab ratio, buffer type, and theoretical adjusted pH for each dosage form are shown below the x-axis.

In FIG. 10 shows the degradation rate of ipilimumab, estimated by change in acid peak area, in FDRC and commercial formulation at 25°C/60% RH (relative humidity). The composition of FDRC is shown in table. 7.

In FIG. 11 shows the degradation rate of nivolumab, estimated from the change in acid peak area, in FDRC and commercial formulation at 25°C/60% RH (relative humidity). The composition of FDRC is shown in table. 7.

In FIG. 12 shows the acid peak profile at 25°C for ipilimumab and nivolumab in the study at various pH values.

In FIG. 13 shows the high molecular weight SEC profile of DP prototypes in a stability study. The HMW DP FDRC profile remained unchanged after 6 months of storage at 2-8°C and 25°C.

In FIG. 14 shows the SEC profile of DP FDRC monomer after 6 months storage at 2-8°C and 25°C.

In FIG. 15 shows the acid peak profile of ipilimumab. The evaluation showed that deamidation was dependent on pH at an elevated temperature of 25° C., as indicated by an increase in the acid peak profile in the higher temperature range at pH 7.0.

In FIG. 16 shows the acid peak profile of nivolumab. The evaluation showed that deamidation was dependent on pH at an elevated temperature of 25° C., as indicated by an increase in the acid peak profile in the higher temperature range at pH 7.0.

In FIG. 17 shows the main peak profile of ipilimumab. The evaluation showed that deamidation was dependent on pH at an elevated temperature of 25° C., as indicated by an increase in the acid peak profile in the higher temperature range at pH 7.0.

In FIG. 18 shows the main peak profile of nivolumab. The evaluation showed that deamidation was dependent on pH at an elevated temperature of 25° C., as indicated by an increase in the acid peak profile in the higher temperature range at pH 7.0.

In FIG. 19 shows the effect of pH on the cIEF profile.

In FIG. 20 shows the iCIEF profile over the pH range 5.4-6.6.

Detailed description of the invention

The present invention relates to pharmaceutical compositions containing anti-PD-1 an

- 6 042223 titel and second antibody. In some embodiments, the implementation of the composition is a dosage form containing a combination with a fixed dose combination. Advantages of such a single dosage form formulation containing a fixed dose combination may include increased adherence to treatment by reducing treatment time (for a given formulation, e.g., intravenous administration) or reduction in the number of administrations (e.g., multiple intravenous (i.v.) injections), and the possibility of obtaining a combined profile for both drugs. However, such a single fixed dose combination formulation may cause undesirable interactions between the two antibodies, thereby reducing the total amount of active ingredient, as well as limiting the therapist's ability to dose.

Terms

To facilitate understanding of the present invention, it is first necessary to define some terms. As used in this invention, except as expressly provided herein, each of the following terms will have the meaning given below. Additional definitions are contained in the description.

As used herein, the term and/or should be considered as a specific description of each of the two specified features or components, with or without the other. Thus, the term and/or used in an expression such as A and/or B herein is understood to include A and B, A or B, A (only) and B (only). Similarly, the term and/or used in an expression such as A, B and/or C is understood to cover each of the following: 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 (only); B (only); and C (only).

It is intended that where aspects are described herein in terms of the term comprising, other similar aspects described in terms of consisting of and/or consisting primarily of are also contemplated.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as generally understood by a person skilled in the art to which this invention pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide one of skill in the art with a general vocabulary for many of the terms used in this invention.

Units of measure, prefixes and symbols are designated in their commonly used form according to the International System of Units (SI) (Systeme International de Unites, SI). Numeric ranges include numbers that define the range. The capital letters presented here are not intended to limit the various aspects of the invention that may be made with reference to the description as a whole. Thus, the terms defined below are more fully defined by reference to the description as a whole.

Administration refers to the physical administration to a subject of a composition containing a therapeutic agent using various delivery methods and systems known to those skilled in the art. Preferred routes of administration of the dosage forms described herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, parenteral administration means routes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intradermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the dosage form is administered by a non-parenteral route, preferably orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, for example, intranasally, vaginally, rectally, sublingually, or topically. The administration may also be performed, for example, once, multiple times, and/or over one or more extended periods.

An adverse event (AE) as used herein is any adverse and usually unintended or undesirable sign (including laboratory abnormality), symptom or disease associated with the use of a therapeutic treatment. For example, an adverse event may be associated with the activation of the immune system or the proliferation of cells of the immune system (eg, T cells) in response to treatment. Therapeutic treatment may cause one or more associated adverse events, and each adverse event may have the same or different levels of severity. Reference to methods capable of modifying adverse events means a treatment regimen that reduces the occurrence and/or severity of one or more adverse events associated with the use of another treatment regimen.

- 7 042223

An antibody (Ab) will include, without limitation, an immunoglobulin glycoprotein that specifically binds to an antigen and contains at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen-binding portion thereof. Each heavy (H) chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three constant domains, CH1, CH2 and C _H3 . Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a single constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), scattered between regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells), and the first component (C1q) of the classical complement system.

The immunoglobulin may be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3 and IgG4. An isotype refers to the class or subclass of an antibody (eg, IgM or IgG1) that is encoded by heavy chain constant region genes. By way of example, the term antibody includes natural and non-natural antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and 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 indicated and unless the context dictates otherwise, the term antibody also includes an antigen-binding fragment or antigen-binding portion of any of the above immunoglobulins, and includes a monovalent and divalent fragment or portion, and a single chain antibody.

The term monoclonal antibody (mAb) is used to describe a non-natural preparation of antibody molecules of a single molecular composition, i. antibody molecules whose primary sequences are substantially identical and which exhibit the same binding specificity and affinity for a particular epitope. A monoclonal antibody (mAb) is an example of an isolated antibody. Monoclonal antibodies (mAbs) can be generated using hybridoma, recombinant, transgenic, or other methods known to those skilled in the art.

A human antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, then the constant region is also derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by in vitro random or site-specific mutagenesis or somatic mutation in vivo). However, the term human antibody as used herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms human antibodies and fully human antibodies are used interchangeably.

A humanized antibody refers to an antibody in which some, most or all of the amino acids outside the CDR regions of the non-human antibody have been replaced with the corresponding amino acids derived from human immunoglobulins. In one embodiment of the humanized form of the antibody, some, most, or all of the amino acids outside the CDRs are changed to amino acids derived from human immunoglobulins, while some, most, or all of the amino acids in one or more of the CDRs are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are acceptable as long as they do not deprive the antibody of its ability to bind to a given antigen. The humanized antibody retains antigenic specificity similar to the original antibody.

A chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

An anti-antigen antibody refers to an antibody that binds specifically to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, and an anti-CTLA-4 antibody specifically binds to CTLA-4.

The antigen-binding portion of an antibody (also referred to as the antigen-binding fragment)

- 8 042223 refers to one or more antibody fragments that retain the ability to specifically bind to an antigen bound by a full-length antibody.

Cancer refers to a broad group of different diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer or cancerous tissue may include a tumor.

CD137, CD-137, tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 41BB, and induced lymphocyte activation (ILA) all belong to the same member of the tumor necrosis factor receptor family. One activity of CD137 is associated with costimulatory 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 homologues of hCD137, and analogs sharing at least one epitope with hCD137. The amino acid sequence of hCD137 can be found under GenBank accession number NP_001552.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an immunoinhibitory receptor belonging to the CD28 family. CTLA-4 is exclusively expressed on T cells in vivo and binds to two ligands, CD80 and CD86 (also referred to as B7-1 and B7-2, respectively). The term CTLA-4 as used herein includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologues of hCTLA-4, and analogues that share at least one epitope with hCTLA-4. The complete hCTLA-4 sequence can be found under GenBank accession no. AAB59385.

Disease refers to any disturbance of structure or function in an organism, such as a person, that is not a direct result of physical injury. An infectious disease is a disease that is caused by an organism such as a bacterium, fungus, parasite, virus, or other pathogen.

Dosing interval as used herein refers to the amount of time between administration of multiple doses to a subject of a dosage form described herein. The dosing interval can thus be specified as ranges.

As used herein, the term dosing frequency refers to the frequency of administration of doses of the dosage form described herein at a given time. Dosing frequency may be indicated in terms of the number of doses at a given time, such as once a week or once every two weeks.

The use of the term fixed dose in relation to a composition according to the invention means that two or more different antibodies in a single composition are present in specific (fixed) ratios relative to each other. In some embodiments, the fixed dose is based on the weight (eg, mg) of antibodies. In certain embodiments, the fixed dose is based on the concentration (eg, mg/mL) of antibodies. 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 so 2:1 mg of the first antibody to mg of the second antibody. For example, a 3:1 ratio of the first antibody and the second antibody may mean that the vial may 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.

As used herein, the term constant dose in relation to a composition of the invention means the dose that is administered to a patient regardless of the weight or body surface area (BSA) of the patient. Therefore, a constant dose is not given as a dose in mg/kg, but is given as an absolute amount of the agent (eg, anti-CTLA4 antibodies and/or anti-PD-1 antibodies). For example, a 60 kg patient and a 100 kg patient will receive the same dose of composition (e.g., 240 mg of anti-PD-1 antibody and 80 mg of anti-CTLA4 antibody presented in a single bottle containing a fixed dose combination dosage form, i.e. 240 mg anti-PD-1 antibody and 80 mg anti-CTLA4 antibody (or in two fixed dose combination vials, i.e. 120 mg anti-PD-1 antibody and 40 mg anti- CTLA4 antibodies, etc.)).

As used herein, dose based on body weight means that the dose administered to a patient is calculated based on the patient's body weight. For example, when a patient with a body weight of 60 kg requires 3 mg/kg of anti-PD-1 antibody in combination with 1 mg/kg of anti-CTLA4 antibody, the corresponding amounts of anti-PD-1 antibody can be calculated (i.e. 180 mg) and anti-CTLA4 antibodies (ie 60 mg) from a 3:1 fixed dose combination formulation of anti-PD-1 antibody and anti-CTLA4 antibody.

As used herein, the term reference composition refers to a composition containing either the first antibody or the second antibody, but not both antibodies. The reference composition may contain

- 9 042223 the same components as the composition containing the first antibody and the second antibody, except that only one antibody is present. In other embodiments, the reference composition is a commercially available corresponding composition, such as OPDIVO® or KEYRUDA® for an anti-PD-1 antibody, or YERVOY® for an anti-CTLA-4 antibody.

The terms GITR, member of the tumor necrosis factor receptor superfamily 18, activation-induced TNFR family receptor, or glucocorticoid-inducible TNFR-related protein 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 241 amino acid type I transmembrane protein characterized by three cysteine-rich pseudo-repeats in the extracellular domain and specifically protects T-cell receptor-induced apoptosis but does not protect cells from other apoptotic signals including triggering through Fas, dexamethasone treatment or UV irradiation (Nocentini, G, et al. (1997) Proc. Natl. Acad. Sci, USA 94:6216622). As used herein, the term GITR includes human GITR (hGITR), variants, isoforms, and species homologues of hGITR, and analogues sharing at least one epitope with hGITR. Three isoforms of hGITR have been identified, all of which contain the same extracellular domain except for its C-terminal portion. Option 1 (accession number NP_004186) consists of 241 amino acids and represents the longest transcript. It contains an additional coding segment, resulting in a frameshift compared to variant 2. The resulting protein (isoform 1) contains a different and shorter C-terminus compared to isoform 2. Option 2 (accession number NP_683699) encodes the longest protein (isoform 2), consisting of 255 amino acids, and is soluble. Option 3 (accession number NP_683700) contains an additional coding segment, resulting in a frameshift compared to variant 2. The resulting protein (isoform 3) contains a different and shorter C-terminus compared to isoform 2, and consists of 234 amino acids.

The term immunotherapy refers to the treatment of a subject afflicted or at risk of infection, or suffering from a relapse of a disease, by a method, including inducing, enhancing, suppressing or otherwise modifying an immune response.

The term LAG3, LAG-3, or lymphocyte activation gene 3 refers to the lymphocyte activation gene 3. As used herein, the term LAG-3 includes human LAG-3 (hLAG-3), hLAG-3 variants, isoforms, and species homologues, and analogues having at least one common epitope with hLAG-3. The term human LAG-3 refers to a human LAG-3 sequence, such as the complete human LAG-3 amino acid sequence, having Genbank access code NP_02277. The term mouse LAG-3 refers to a mouse LAG-3 sequence, such as the complete mouse LAG-3 amino acid sequence of Genbank access code NP_032505. Also, LAG-3 is known in the art as, for example, CD223. The sequence of human LAG-3 may differ from human LAG-3 in Genbank accession number NP_002277 by containing, for example, conserved mutations or mutations in non-conserved regions, and LAG-3 has essentially the same biological function as human LAG-3 in Genbank under 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 specifically binds an antibody of the present invention, or the biological function of human LAG-3 is to bind to class II MHC molecules.

The term lyophilisate, as used in connection with a dosage form according to the invention, means a dosage form that is prepared using freeze-drying techniques known in the art. The solvent (eg water) is removed by freezing followed by sublimation under vacuum and desorption of residual water at elevated temperature. In the pharmaceutical field, the lyophilisate typically has a residual moisture content of about 0.1-5% (w/w) and is presented as a powder or a physically stable porous mass. The lyophilizate is characterized by rapid dissolution after the addition of the reducing medium.

The term Ig-like killer cell receptor, cytotoxicity suppression receptor or KTR refers to a protein or polypeptide encoded by a gene that is a member of the KIR gene family or a cDNA derived from such a gene. For a detailed description of the KIR gene family, including KTR gene nomenclature and KTR gene products, and Genbank accession numbers for exemplary KTRs, see M. Carrington and P. Norman The KIR Gene Cluster, available from the NCBI Bookshelf website ( available at ncbi.nlm.nih.gov/books). As used herein, the term KIR includes human KIR (hKIR), variants, isoforms, and species homologues of hKIR, and analogues sharing at least one epitope with hKIR. Human KIR gene and cDNA sequences, as well as their protein products, are available from public databases, including GenBank. Non-limiting exemplary human KIRs in GenBank have the following access numbers: KIR2DL1: Genbank access number U24076, NM_014218, AAR16197, or L41267; KIR2DL2: Genbank access number U24075 or L76669; KIR2DL3: Genbank access number U24074 or L41268; KIR2DL4: Genbank access number X97229; KIR2DS1: Genbank access number X89892; KIR2DS2: Genbank access number L76667; KIR2DS3: Genbank access number NM_012312 or L76670 (splice option); KIR3DL1: Genbank access number L41269; and KIR2DS4: number

- 10 042223 access Genbank AAR26325. KIR may contain from 1 to 3 extracellular domains, and may have a long (ie, more than 40 amino acids) or short (ie, less than 40 amino acids) cytoplasmic tail. As described earlier, these features define the KIR nomenclature. In addition, a description of KIR is contained in international publication WO/2014/055648, the entire content of which is incorporated herein by reference.

Programmed death-1 (PD-1) protein is an immunoinhibitory receptor belonging to the CD28 family. PD-1 is predominantly expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term PD-1 includes human PD-1 (hPD-1), variants, isoforms, and species homologues of hPD-1, and analogues that share at least one epitope 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 here.

Programmed death protein ligand-1 (PD-L1) is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that inhibits T cell activation and cytokine secretion when bound to PD-1. As used herein, the term PD-L1 includes human PD-L1 (hPD-L1), variants, isoforms, and species homologues of hPD-L1, and analogues that share at least one epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

The term reconstituted dosage form as used herein means a dosage form that has been lyophilized and re-dissolved by the addition of a diluent. The diluent may be, for example, 0.9% sodium chloride injection, USP or 5% dextrose injection, USP.

The 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.

A therapeutically effective amount or therapeutically effective dose of a drug or therapeutic agent is any amount of a drug that, when used alone or in combination with another therapeutic agent, protects the subject from the occurrence of a disease or promotes regression of the disease, as evidenced by a reduction in the severity of the symptoms of the disease, an increase in the frequency or the duration of periods of absence of symptoms of the disease, or the prevention of deterioration or disability due to the defeat of the disease. The ability of a therapeutic agent to induce disease regression can be assessed using various methods known to one of skill in the art, for example, in human patients during clinical trials, in animal model systems predictive of efficacy in humans, or by analyzing the activity of the agent in assays in vitro.

As used herein, a subtherapeutic dose means a dose of a therapeutic compound (eg, antibody) that is lower than the usual or typical dose of a therapeutic compound when administered alone for the treatment of a hyperproliferative disease (eg, cancer).

Treatment or therapy of a subject refers to any type of intervention or process administered to a subject, or administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or biochemical indicator associated with the disease.

The use of an alternative (for example, or) is to be understood to mean one, both, or any combination of these alternatives. The indefinite articles a or an must be understood as one or more of any of the specified or listed components.

The terms approximately or consisting essentially of refer to a quantity or composition that is within an acceptable error range for a particular quantity or composition as determined by one skilled in the art, which will depend in part on how that quantity or composition is measured or determined, i.e. e. limitations of a particular system of measurement. For example, approximately or consisting essentially of can mean within 1 standard deviation or more than 1 standard deviation according to practice in this field. Alternatively, approximately or consisting essentially of can mean a range of up to 10% or 20% (ie, ±10% or ±20%). For example, about 3 mg may include any number in the range of 2.7 mg to 3.3 mg (for 10%) or 2.4 mg to 3.6 mg (for 20%). In addition, in particular in relation to biological systems or processes, the terms can mean up to a value or up to 5 times the value of a value. When using specific values or compositions in this specification and claims, unless otherwise indicated, a value approximately or consisting essentially of is expected to be within the acceptable error range for that particular value or composition.

As used herein, once approximately every week, once approximately every two weeks, or other similar dosing intervals are approximate numbers. Once approximately every week may include every seven days ± one day, i.e. from every six days to every seven days. Once every two weeks may include every fourteen days ± three days, i.e. from every eleven days to every seventeen days. Similar approximations apply, for example, once about every three weeks, once about every four weeks, once about every five weeks, once about every six weeks, and once about every twelve weeks. In some embodiments, a dosing interval of once about every six weeks or once about every twelve weeks means that the first dose may be given on any day in the first week and then the next dose may be given on any day in the sixth or twelfth week. , respectively. In other embodiments, a dosing interval of once approximately every six weeks or once approximately 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 of the sixth or the twelfth week (i.e. Monday) respectively.

As described herein, any concentration range, percentage range, ratio range, or range of integers should be understood to include the value of any integer within the specified range and, if necessary, its fractions (for example, one tenth or one hundredth of an integer), unless otherwise indicated.

Various aspects of the invention are described in more detail in the following subsections.

Anti-PD-I and anti-PD-L1 antibodies

The composition according to the invention includes a first antibody and a second antibody in a ratio of 1:100 to 100:1. In one aspect, 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 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 for PD-1, Programmed Death Ligand-1 (PD-L1) and Programmed Death Ligand-2 (PDL2), have been identified that are expressed on antigen-presenting cells, and also in many types of cancer and has been shown to inhibit T-cell activation and cytokine secretion when bound to PD-1. Inhibition of the PD-1/PD-L1 interaction mediates high antitumor activity in preclinical models.

Human monoclonal antibodies (HuMAbs) that specifically bind to PD-1 with high affinity are described in US Pat. Nos. 8,008,449 and 8,779,105. PCT WO 2012/145493. Each of the anti-PD-1 HuMAbs described in US Pat. No. 8,008,449 exhibits one or more of the following properties: (a) binds to human PD-1 with a K _D of 1 x 10' ⁷ M or less as determined by surface plasmon resonance using the Biacore biosensor system; (b) does not substantially bind to human CD28, CTLA-4 or ICOS; (c) increases T cell proliferation in a mixed culture lymphocyte response (MLR) assay; (d) increases interferon-gamma production in the MLR assay; (e) increases secretion of interleukin-2 (IL-2) in the MLR assay; (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and/or PD-L2 to PD-1; (h) stimulates antigen-specific memory responses; (i) stimulates antibody responses; and (j) inhibits the growth of tumor cells in vivo. Anti-PD-I antibodies useful in the present invention will include mAbs that specifically bind to human PD-1 and exhibit at least one, preferably at least five of the above properties.

In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab (also known as OPDIVO®; previously designated 5C4, BMS-936558, MDX-1106, or ONO4538) is a fully human IgG4 (S228P) antibody against the PD-1 immune checkpoint inhibitor that selectively prevents interaction with PD ligands. -1 (PD-L1 and PD-L2), thereby blocking the suppression of antitumor functions of T cells (US patent 8008449; 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.

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 KEYTRUDA®, lambrolizumab and MK-3475) is a humanized IgG4 monoclonal antibody directed against the human cell surface PD-1 receptor (programmed death-1 or

- 12 042223 programmed cell death-1). Pembrolizumab is described, for example, in US Pat. Nos. 8,354,509 and

8900587; see also https://www.cancer.gov/drugdictionary?cdrid=695789 (accessed 14 Dec

2014). The FDA has approved the use of pembrolizumab for the treatment of relapsed or refractory melanoma.

In other embodiments, the anti-PD-1 antibody or fragment thereof cross-competes with MEDI0608. In still 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 (formerly AMP-514), which is a monoclonal antibody. A description of MEDI0608 is found, for example, in US Pat.

In certain embodiments, the first antibody is an anti-PD-1 antagonist. One example of an anti-PD-1 antagonist is AMP-224, which is an Fc-B7-DC fusion protein. AMP-224 is described in publication 2013/0017199 or at https://www.cancer.gov/publications/dictionaries/cancer-drug?cdrid=700595 (accessed July 9, 2015).

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 to the same epitope as BGB-A317. In certain embodiments, the anti-PD-1 antibody contains 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 publication 2015/0079109.

Ahtu-PD-I antibodies useful in the disclosed compositions also include isolated antibodies that specifically bind to human PD-1 and cross-compete for binding to human PD-1 with nivolumab (see, for example, US Pat. Nos. 8,008,449 and 8,779,105; WO 2013/173223). The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same region of an antigen epitope and sterically make it difficult for other cross-competing antibodies to bind to that particular epitope region. These cross-competing antibodies are expected to have functional properties highly similar to those of nivolumab due to their binding to the same region of the PD-1 epitope. Cross-competing antibodies can be easily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays such as Biacore assay, ELISA assays or flow cytometry (see, for example, WO 2013/173223).

In certain embodiments, antibodies that cross-compete for binding to human PD-1, or bind to the same region of the human PD-1 epitope as nivolumab, are monoclonal antibodies (mAb). For administration to human subjects, these cross-competing antibodies may be chimeric antibodies or humanized or human antibodies. Such chimeric, humanized or human antibodies can be made and isolated using methods well known in the art.

Ahtu-PD-1 antibodies useful in the compositions of the disclosed invention also include the antigen-binding portions of the above antibodies. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Examples of binding fragments encompassed by the term antigen-binding portion of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, V _H , C _L and C _H1 domains; (ii) an F(ab')2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fdfragment consisting of V _H - and C _H1 domains; and (iv) an Fv fragment consisting of the VL and VH domains of a single antibody arm.

Ahtu-PD-1 antibodies suitable for use in the disclosed compositions are antibodies that bind to PD-1 with high specificity and affinity, block PD-L1 and/or PD-L2 binding, and inhibit the immunosuppressive effect of the PD-singal pathway. 1. In any of the compositions or in any of the methods disclosed herein, an anti-PD-1 antibody comprises an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits functional properties similar to those of full antibodies in terms of inhibition of ligand binding and activation immune system. In certain embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, cross-competes with nivolumab for binding to human PD-1. In other embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is a chimeric, humanized, or human monoclonal antibody, or 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.

In certain embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, comprises a heavy chain constant region that is of the human IgG1 or IgG4 isotype. In certain other embodiments, the heavy chain constant region sequence

- 13 042223

The IgG4 anti-PD-l antibody, or antigen-binding portion thereof, contains the S228P mutation, which replaces a serine residue in the hinge region with a proline residue normally found at the corresponding position in antibodies of the IgG1 isotype. This mutation, which is present in nivolumab, prevents replacement of the Fab arm with endogenous IgG4 antibodies while maintaining low affinity for activation of Fc receptors associated with wild-type IgG4 antibodies (Wang et al., 2014). In still other embodiments, the antibody comprises a light chain constant region that is a human kappa or lambda constant region. In other embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is a mAb or antigen-binding portion thereof. In certain embodiments of any of the therapeutic methods described herein involving the administration of an anti-PD-1 antibody, said 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 human antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in US Pat. No. 8,008,449. In still other embodiments, the anti-PD-1 antibody is MEDI0608 (formerly AMP-514), AMP224, or pidilizumab (CT-011).

In certain embodiments, the first antibody for the disclosed composition is an anti-PD-L1 antibody. Since anti-PD-1 and anti-PD-L1 antibodies target the same signaling pathway and have been shown in clinical studies to exhibit similar levels of efficacy in various types of cancer, the anti-PD-L1 antibody can be replaced by an anti -PD-1 antibody in any of the therapeutic methods or in any of the compositions disclosed here. In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (formerly referred to as 12A4 or MDX-1105) (see, for example, US Pat. 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; US Pat. No. 8,217,149), MEDI4736 (Khleif , 2013, In: Proceedings from the European Cancer Congress 2013; September 27-October 1, 2013; Amsterdam, The Netherlands. Abstract 802) or MSB0010718C (also referred to as avelumab; see US 2014/0341917). In certain embodiments, antibodies that cross-compete for binding to human PD-L1 or bind to the same region of the human PD-L1 epitope as the above anti-PD-L1 antibodies are monoclonal antibodies (mAb). For administration to human subjects, these cross-competing antibodies may be chimeric antibodies, or may be humanized or human antibodies. Such chimeric, humanized or human monoclonal antibodies (mAbs) may be prepared and isolated using methods well known in the art.

Ahtu-CTLA-4 antibodies

The Ahtu-CTLA-4 antibodies used for the present invention bind to human CTLA-4 in such a way as to interfere with the interaction of CTLA-4 with the human B7 receptor. Since the interaction of CTLA-4 with B7 transduces a signal, resulting in the inactivation of T cells bearing the CTLA-4 receptor, disruption of the interaction effectively induces, enhances, or prolongs the activation of such T cells, thereby inducing, enhancing, or prolonging the immune response.

Human monoclonal antibodies (HuMAbs) that specifically bind to CTLA-4 with high affinity are described in US Pat. Nos. 6,984,720 and 7,605,238. 7034121. Antu-CTLA-4 human monoclonal antibodies (HuMAb) described in US Pat. Nos. 6,984,720 and 7,605,238 have been shown to have one or more of the following properties: an association constant (K _a ) of at least about 10 ⁷ M ^-1 or about 109 M ^-1 , or from about 10 ¹⁰ M ^-1 to 10 ¹¹ M ^-1 or higher, determined using Biacore analysis; (b) an association kinetic constant (ka) of at least about 10 ³ , about 10 ⁴ , or about 105 m ^-1 m ^-1 ; (c) a kinetic dissociation constant (kd) of at least about 10 ³ , about 10 ⁴ , or about 105 m ^-1 s ^-1 ; and (d) inhibits CTLA-4 binding to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies useful in the present invention include monoclonal antibodies (mAbs) that specifically bind to human CTLA-4 and have at least one, at least two, or at least three of the above properties. An exemplary clinical anti-CTLA-4 antibody is the human mAb 10D1 (currently known as ipilimumab and marketed under the trade name YERVOY®) as described in US Pat. No. 6,984,720.

An exemplary clinical anti-CTLA-4 antibody is the human monoclonal antibody (mAb) 10D1 (currently known as ipilimumab and marketed under the tradename YERVOY®) as described in US Pat. No. 6,984,720. Ipilimumab is an anti-CTLA-4 an antibody for use in the methods disclosed herein. Ipilimumab is a fully human IgG1 monoclonal antibody that blocks CTLA-4 binding to its B7 ligands, thereby stimulating T cell activation and improving overall survival (OS) in patients with advanced melanoma.

Another anti-CTLL-4 antibody useful for the methods of the present invention is tremelimumab (also known as CP-675,206). Tremelimumab is a human monoclonal anti-CTLL-4 IgG2 antibody.

Tremelimumab is described in WO/2012/122444, US Patent Application Publication 2012/263677 or WO 2007/113648 A2.

Anti-CTLA-4 antibodies useful in the disclosed composition 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 CTLA-4 epitope region. 4 people as ipilimumab or tremelimumab. In certain embodiments, antibodies that cross-compete for binding to human CTLA-4, or bind to the same human CTLA-4 epitope region as ipilimumab or tremelimumab, are antibodies containing the 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-CTLL-4 antibodies also include antigen-binding portions of the above antibodies, such as Fab, F(ab') ₂ , Fd or Fv fragments.

Anti-LLG-3 antibodies

The anti-LLG-3 antibodies of the present invention bind to human LAG-3. Antibodies that bind to LAG-3 are described in International Publication WO/2015/042246 and US Publications 2014/0093511 and 2011/0150892.

An exemplary anti-LLG-3 antibody useful for the present invention is 25F7 (described in US Publication 2011/0150892). An additional illustrative anti-LLG-3 antibody useful for the present invention is BMS-986016. In one embodiment, the anti-LLG-3 antibody useful for the composition cross-competes with 25F7 or BMS986016. In another embodiment, an anti-LLG-3 antibody useful for the composition binds to the same epitope as 25F7 or BMS-986016. In other embodiments, the anti-LLG-3 antibody contains six CDRs of 25F7 or BMS-986016.

Anti-CD137 antibodies

Anti-CD137 antibodies specifically bind to and activate CD137-expressing immune cells, stimulating an immune response, in particular a cytotoxic T-cell response against tumor cells. Antibodies that bind to CD137 are described in US Publication No. 2005/0095244 and US Pat.

In some embodiments, the anti-CD137 antibody is urelumab (BMS663513) described in US Pat. No. 7,288,638 (20H4.9-IgG4 [10C7 or BMS-663513]). In some embodiments, the anti-CD137 antibody is BMS-663031 (20H4.9-IgG1) as described in US Pat. No. 7,288,638. In some embodiments, the anti-CD137 antibody is 4E9 or BMS-554271 as described in US Pat. in some embodiments, the anti-CD137 antibody is the antibody described in US Pat. Nos. 7,214,493; 6303121; 6569997; 6905685 or 6355476. In some embodiments, the anti-CD137 antibody is 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1 as described in US Pat. No. 6,362,325. In some embodiments, the anti-CD137 antibody is the antibody described in US Published Patent 6,974,863 (such as 53A2). In some embodiments, the anti-CD137 antibody is an antibody disclosed in US Published Patent 6,210,669 (such as 1D8, 3B8, or 3E1). In some embodiments, the antibody is PF-05082566 (PF2566) developed by Pfizer. In other embodiments, an anti-CD137 antibody useful for the invention cross-competes with anti-CD137 antibodies disclosed herein. In some embodiments, an anti-CD137 antibody binds to the same epitope as an anti-CD137 antibody disclosed herein. In other embodiments, an anti-CD137 antibody useful for the invention comprises six CDRs of the anti-CD137 antibodies disclosed herein.

Anti-KIR antibodies

Antibodies that specifically bind to KIR block the interaction between killer-cell immunoglobulin-like receptors (KIRs) on NK cells with their ligands. Blocking these receptors promotes the activation of NK cells and, potentially, the destruction of tumor cells in the future. Examples of anti-KIR antibodies are contained in international publications WO 2014/055648, WO 2005/003168, WO 2005/009465, WO 2006/072625, WO 2006/072626, WO 2007/042573, WO 2008/084106, WO 2010/06 2012/071411 and WO 2012/160448.

One anti-KIR antibody useful for the present invention is lirilumab (also referred to as BMS-986015, IPH2102, or S241P variant 1-7F9), first described in international publication WO 2008/084106. Another anti-KIR antibody useful for the present invention is

- 15 042223 is 1-7F9 (also referred to as IPH2101) described in international publication WO 2006/003179. In one embodiment, an anti-KIR antibody useful in a composition of the present invention cross-competes for binding to lirimumab or I-7F9 to KIR. In another embodiment, the anti-KIR antibody binds to the same epitope as lirilumab or I-7F9. In other embodiments, the anti-KIR antibody comprises six CDRs of lirilumab or I-7F9.

Anti-GITR antibodies

Anti-GITR antibodies to be combined with a fixed dose anti-PD-1 antibody can be any anti-GITR antibody that specifically binds to the target, human GITR, and activates glucocorticoid-induced tumor necrosis factor receptor (GITR). GITR is a member of the TNF receptor superfamily that is expressed on the surface of many types of immune cells, including regulatory T cells, effector T cells, B cells, natural killer (NK) cells, and activated dendritic cells (anti-GITR agonist antibodies). In particular, GITR activation increases the proliferation and function of effector T cells, and also suppresses the suppression induced by activated regulatory cells. In addition, GITR stimulation promotes antitumor immunity by increasing the activity of other immune cells such as NK cells, antigen presenting cells, and B cells. Examples of anti-GITR antibodies are described in international publications WO 2015/031667, WO 2015/184099, WO 2015/026684, WO 11/028683 and WO 2006/105021, US Pat. , 2013/0183321 and 2014/0348841.

In one embodiment, the anti-GITR antibody useful for the present invention is TRX518 (described, for example, in Schaer et al. Curr Ort Immunol. (2012) Apr; 24(2): 217-224 and WO 2006/105021) . In another embodiment, an anti-GITR antibody useful in the present invention is MK4166 or MK1248 and the antibodies described in WO 11/028683 and US Pat. a VL chain containing the sequence of SEQ ID NO: 105, with the sequences of SEQ ID NO: taken from WO11/028683 or US patent 8709424). In certain embodiments, the anti-GITR antibody is an anti-GITR antibody as described in WO2015/031667, e.g., an antibody comprising VH CDRs 1-3 comprising the sequences of SEQ ID NOs: 31, 71, and 63 of WO 2015/031667, respectively, and VL CDR 1-3 containing the sequences of SEQ ID NOS: 5, 14 and 30 according to WO 2015/031667. In certain embodiments, the anti-GITR antibody is an anti-GITR antibody as described in WO 2015/184099, e.g., a Hum231#1 or Hum231#2 antibody, or CDRs thereof, or derivatives thereof (e.g., pab1967, pab1975, or pab1979) . In certain embodiments, the anti-GITR antibody is an anti-GITR antibody as disclosed in JP 2008278814, WO 09/009116, WO 2013/039954, US 20140072566, US 20140072565, US 20140065152 or WO 2015/026684-, or is INBRX- 110 (INHIBRx), LKZ-145 (Novartis) or MEDI-1873 (Medlmmune). In certain embodiments, the anti-GITR antibody is an anti-GITR antibody as described in PCT/US2015/033991 (eg, an antibody containing the 28F3, 18E10, or 19D3 variable regions). For example, an anti-GITR antibody can be an antibody containing the following VH and VL chains or their CDRs:

VH:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEG SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYG MDVWGQGTTVTVS (SEQ GO NO: 1), and VL:

AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIK (SEQ GO NO: 2); or

VH:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLEWVAVIWYAG SNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVM DVWGQGTTVTVSS (SEQ GO NO: 3), and VL:

- 16 042223

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGV

PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK (SEQ ID NO: 4); or

VH:

VQLVESGGGVVQPGRSLRLSC AASGFTF S S YGMHWVRQ APGKGLEW VAVIWYAGS NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMD VWGQGTTVTVSS (SEQ ID NO: 5), and

VL:

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGV

PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK (SEQ ID NO: 6)

In certain embodiments, an antibody comprising the aforementioned VH and VL light chain pair or CDRs thereof contains a heavy chain constant region of an IgG1 isotype, wild-type or mutated, eg, to lack effector functions. In one embodiment, the anti-GITR antibody comprises the following heavy and light chain amino acid sequences: heavy chain:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEG

SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYG MDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQIDNO: 7), и легкая цепь:

AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ Ш NO: 8), или тяжелая цепь:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEG

SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYG MDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSS IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

G(SEQIDNO: 9), and light chain:

AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVP

SRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ SH NO: 8).

In certain embodiments, an anti-GITR antibody for a composition of the present invention cross-competes with an anti-GITR antibody described herein, e.g., TRX518, MK4166, or an antibody comprising the amino acid sequence of a VH domain and a VL domain described herein. In some embodiments, an anti-GITR antibody for a composition of the present invention binds to the same epitope as an anti-GITR antibody described herein, e.g., TRX518, MK4166, or an antibody comprising the amino acid sequence of a VH domain and a VL domain described herein. In certain embodiments, the anti-GITR antibody comprises six CDRs of TRX518, MK4166, or antibodies comprising the amino acid sequence of a VH domain and a VL domain as described herein. Illustrative pharmaceutical com

- 17 042223 position contains an anti-PD-1 antibody, for example, nivolumab, MK-3475 (pembrolizumab) or atezolizumab and an anti-GITR agonist antibody, for example, TRX518, MK4166 or an antibody containing the amino acid sequence of the VH domain and the VL domain described here, wherein the ratio of the amount (e.g., concentration (e.g., mg/mL) or weight (e.g., mg)) of anti-PD-1 antibody to the amount of anti-GITR antibody (e.g., concentration (e.g., mg/mL) or weight (eg mg), respectively) 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) anti-PD-1 or anti-PD-L1 antibody to (2) anti-GITR antibody may 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. : refers to e.g. 1:1-20 refers to a ratio of 1 to a number selected from 1-20. The combination can be administered once a week, twice a week, once every three weeks, or once a month.

In certain embodiments, an anti-PD-1 antibody or an anti-PD-L1 antibody, such as nivolumab, pembrolizumab, or atezolizumab, is formulated with an anti-GITR antibody, wherein the anti-GITR antibody is present at a dose, such as a constant dose of 0 .1mg to 1000mg, such as 0.1100mg, 0.5-100mg, 1-100mg, 5-100mg, 10-100mg, 50-100mg, 0.1-300mg, 0, 5-300 mg, 1-300 mg, 5-300 mg, 10300 mg, 50-300 mg, 100-300 mg or 200-300 mg. Exemplary amounts of anti-GITR antibody that can be formulated together with an anti-PD-1 antibody or an 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 or about 1000 mg. In certain embodiments, an anti-PD-1 antibody or an anti-PD-L1 antibody is formulated with an anti-GITR antibody, wherein the dose of the anti-PD-1 antibody or anti-PD-L1 antibody is a dose (e.g., a constant dose) , equal to 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 mg or about 300 mg.

In exemplary embodiments, an anti-PD-1 antibody or an anti-PD-Ll antibody, e.g., nivolumab, pembrolizumab, or atezolizumab, is formulated together with an anti-GITR antibody, e.g., an antibody containing: (i) VH and VL domains having amino acid the sequences of SEQ ID NOs: 1 and 2, respectively, SEQ ID NOs: 3 and 4, respectively, or SEQ ID NOs: 5 and 6, respectively; or CDR1, CDR2, CDR3 of the VH domain, and CDR1, CDR2 and CDR3 of the VL domain of any of these pairs of variable regions; or (ii) heavy and light chains containing the amino acid sequences of SEQ ID NOs: 7 and 8, respectively, or SEQ ID NOs: 7 and 9, respectively, at the following fixed dose ratios: 80-300 mg of an anti-PD-1 antibody, or anti-PD-L1 antibodies to 1-1000 mg of anti-GITR antibodies; 80300 mg anti-PD-1 antibody or anti-PD-L1 antibody to 1-100 mg anti-GITR antibody; 80-300 mg of anti-PD1 antibody or anti-PD-L1 antibody to 10-100 mg of anti-GITR antibody; 80-300mg anti-PD-1 antibody or anti-PD-L1 antibody to 10-300mg anti-GITR antibody, or 80-300mg anti-PD-1 antibody or anti-PD-L1 antibody to 100-300mg anti-GITR antibodies. In exemplary embodiments, nivolumab is formulated with an anti-GITR antibody, with the dose of nivolumab being about 80 mg or about 240 mg. The fixed dose combination may be administered by intravenous infusion over, for example, about 30 minutes, about 30-60 minutes, about 60 minutes, or about 60-90 minutes about every 1, 2, 3 or about 4 weeks.

In certain embodiments, about 3 mg/kg of an anti-PD-1 antibody, e.g., nivolumab, can be administered together, e.g., as a fixed dose combination, with 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 an anti-GITR antibody, e.g. TRX518, MK4166, or an antibody containing heavy and light chains or variable regions, or CDRs described herein, for example, as an intravenous infusion over, for example, about 30 minutes, about 30-60 minutes, about 60 minutes or about 60-90 minutes approximately every 1, 2, 3 or 4 weeks. In certain embodiments, about 2 mg/kg of an anti-PD1 antibody, e.g., nivolumab or MK-3475, is administered together, e.g., in a fixed dose ratio combination, with 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 kg of an anti-GITR antibody, e.g. MK4166, or an antibody containing heavy and light chains or variable regions, or CDRs as described herein, e.g., as an intravenous infusion over e.g., about 30 minutes, about 30-60 minutes, or about 60 min approximately every 1, 2, 3 or 4 weeks. The amount of antibodies in mg/kg can be calculated to determine the mass (mg) or concentration (mg/ml) of antibodies required for a dosage form with a fixed dose ratio. In certain embodiments, the anti-PD-1 antibody and anti-GITR antibody may be presented as a lyophilized composition, such as in a dual chambered vial or syringe. The lyophilized composition may contain, for example, about 50 mg of an anti-PD-1 antibody or anti-PD-L1 antibody, such as nivolumab, MK3475, or atezolizumab, and about 5-250 mg, about 10-250 mg, about 30-100 mg , about 30-70 mg or about 50 mg of an anti-GITR antibody, eg TRX-518, MK4166, or an antibody containing heavy and light chains or variable regions or CDRs as described herein.

- 18 042223

Additional antibodies

In some embodiments, the implementation of the second antibody to be combined with the first antibody is an anti-TGFe antibody described in international publication WO 2009/073533. In some embodiments, the second antibody is an anti-IL-10 antibody described in WO 2009/073533. In some other embodiments, the implementation of the second antibody is an anti-B7-H4 antibody described in international publication WO 2009/073533. In certain embodiments, the second antibody is an anti-Fas ligand antibody as described in WO 2009/073533. In some embodiments, the second antibody is an anti-CXCR4 antibody as described in US Publication 2014/0322208 (eg, ulocuplumab (BMS-936564)). In some embodiments, the second antibody is an anti-mesothelin antibody described in US Pat. No. 8,399,623. In some embodiments, the second antibody is an anti-HER2 antibody, such as Herceptin (US Pat. e.g. WO/2001/000244). In some embodiments, the second antibody to be combined with the first antibody is an anti-CD27 antibody. In some embodiments, the anti-CD-27 antibody is varlilumab (also known as CDX-1127 and 1F5), which is a human IgG1 antibody that is a human CD27 agonist, as described, for example, in US Pat. No. 9,169,325. In some embodiments, the second antibody to be combined with the first antibody is an anti-CD73 antibody. In certain embodiments, the anti-CD73 antibody is CD73.4.IgG2C219S.IgG1.1f.

Dosage Forms, Pharmaceutical Compositions and Doses

In the dosage form according to the present invention, the first antibody and the second antibody are formulated into a single composition according to the present invention, for example, a pharmaceutical composition containing the first antibody and 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. An anti-PD-LI antibody may be used in place of an anti-PD-1 antibody in any of the compositions or method described herein.

As used herein, the pharmaceutically acceptable carrier includes any or all solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonicizing agents, and absorption delaying agents, and the like, which are physiologically compatible. In certain embodiments, the implementation of the carrier for the composition containing the antibody is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). The pharmaceutical composition according to 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.

In one embodiment, the composition containing the first antibody and the second antibody is presented as a single use vial. In another embodiment, the composition containing the first antibody and the second antibody is presented in the form of a vial for multiple use.

In other embodiments, the first antibody (eg, an anti-PD-1 antibody or an anti-PDL1 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 antiLAG3 antibody is 25F7. In some embodiments, the implementation of the second antibody is an anti-GITR antibody. In some embodiments, the anti-GITR antibody is MK4166, TRX518, an antibody containing the CDRs, variable chains, or heavy and light chains of anti-GITR antibodies as described in PCT/US2015/033991 (e.g., antibodies 28F3, 18E10, or 19D3), or any other anti-GITR antibody described here. In some embodiments, the implementation of 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-TGFe antibody, anti-IL-10 antibody, anti-B7-H4 antibody, anti-Fas ligand antibody, anti-CXCR4 antibody, anti-mesothelin antibody, anti-CD27 antibody, anti-CD73 antibody, or any combination thereof.

In some embodiments, the implementation of the first antibody and the second antibody are contained in the composition in a fixed dose (ie, a fixed ratio). In other embodiments, this

- 19 042223 fixed dose is at least about 1:200 to about 200:1, at least about 1:150 to about 150:1, at least about 1:100 to about 100:1, at least at least about 1:75 to about 75:1, at least about 1:50 to about 50:1, at least about 1:25 to about 25:1, at least about 1:10 to about 10:1, at least about 1:5 to about 5:1, at least about 1:4 to about 4:1, at least about 1:3 to about 3:1, at least from about 1:2 to about 2:1 mg of anti-PD-1 antibody (or anti-PD-L1 antibody) to mg of 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 antibodies (or anti-PD-L1 antibodies) to 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 first antibody to mg of second antibody.

In other embodiments, the composition comprises a first antibody and a second antibody in a specified ratio (e.g., 200:1 to 1:200, 100:1 to 1:100, 20-1:1 to 1:1-20, or any ratio described herein), wherein the composition has one or more properties selected from the group consisting of the following properties: (i) aggregation in the composition is comparable to aggregation in the reference composition (i.e., composition containing the first antibody or second antibody) after 6 months of storage at 2 to 8°C; (ii) fragmentation in the composition is comparable to aggregation in the reference composition (ie, composition containing the first antibody or second antibody) after 6 months of storage at 2 to 8°C; (iii) the deamidation of the first antibody or the second antibody in the composition is comparable to the deamidation of the antibody in the reference composition (ie, the composition containing the first antibody or second antibody) after 6 months of storage at 2 to 8°C; (iv) the level of particulate matter in the compositions is comparable to the level of particulate matter in the reference composition (ie, the composition containing the first antibody or the second antibody) after 6 months of storage at 2 to 8°C; and (v) any combination of them.

In still other embodiments, the composition comprises a first antibody and a second antibody in a specified ratio (e.g., 200:1 to 1:200, 100:1 to 1:100, 20-1:1 to 1:1-20, or any the ratio described here), while the composition has one or more properties selected from the group consisting of the following properties: (i) aggregation in the composition is comparable to aggregation in the reference composition (i.e., the composition containing the first antibody or second antibody) after 6 months of storage at 25°C; (ii) fragmentation in the composition is comparable to fragmentation in the reference composition (ie composition containing the first antibody or second antibody) after 6 months of storage at 25°C; (iii) the deamidation of the first antibody or the second antibody in the composition is comparable to the deamidation of the antibody in the reference composition (ie the composition containing the first antibody or the second antibody) after 6 months of storage at 25°C; (iv) the level of particulate matter in the compositions is comparable to the level of particulate matter in the reference composition (ie, composition containing the first antibody or second antibody) after 6 months of storage at 25°C; and (v) any combination of them.

In some embodiments, the implementation of the aggregation of the composition assessed by the content of high molecular weight (HMW) fractions in the composition, measured by size exclusion high performance liquid chromatography (SE-HPLC). In some embodiments, fragmentation of the composition is assessed by the level of low molecular weight (LMW) fractions in the composition, measured by SE-HPLC. In some embodiments, deamidation of a composition is assessed by the level of acid-charged variants in the composition as measured by cation exchange chromatography (CEX) or capillary isoelectric focusing with visual detection along the entire length of the capillary (iCIEF).

In some embodiments, the amount of anti-PD-1 antibody in the composition 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 of 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 anti-PD-1 antibody in the composition is about 60 mg to 300 mg, about 60 mg to 100 mg, about 100 mg to 200 mg, or about 200 mg to 300 mg. In some embodiments, the amount of anti-PD-1 antibody in the composition is about 300 mg to 500 mg, about 300 mg to 450 mg, about 300 mg to 400 mg, about 300 mg to 350 mg, about 350 mg up to 500 mg, from about 400 mg to 500 mg, or from about 450 mg to 500 mg. In some

- 20 042223 embodiments, the amount of anti-PD-l antibody in the composition is at least about 80 mg, about 160 mg, or about 240 mg. In some embodiments, the amount of anti-PD1 antibody in the composition is at least about 240 mg, or at least about 80 mg. In some embodiments, the amount of 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 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. kg or at least about 5 mg/kg. In some embodiments, the amount of anti-PD-1 antibody in the composition is from about 0.5 mg/kg to 5 mg/kg, from about 0.5 mg/kg to 5 mg/kg, from about 0.5 mg/kg up to 3 mg/kg, or from about 0.5 mg/kg to 2 mg/kg. In some embodiments, the amount of anti-PD1 antibody in the composition is at least about 1 mg/kg. In some embodiments, the anti-PD-1 antibody is nivolumab or pembrolizumab.

In some embodiments, the anti-PD-1 antibody is pembrolizumab and the amount of 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 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 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 anti-PD-1 antibody used to treat the disease or condition may be a dose based on body weight, such as at least about 0.5 mg/day. 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 anti-PD-1 antibody that can be used to treat a disease or condition is a dose based on body weight, 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 of manti-PD-I antibody to mg of anti-CTLA4 antibody. In some embodiments, the amount of anti-CTLA4 antibody in the composition 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 of anti-CTLA4 antibody in the composition is about 60 mg to 300 mg, about 60 mg to 100 mg, about 100 mg to 200 mg, or about 200 mg to 300 mg. In some embodiments, the amount of 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 anti-CTLA4 antibody in the composition is at least about 240 mg. In some embodiments, the amount of 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 anti-CTLA4 antibody in the composition is used as a dosage based on body weight, e.g., about 1 mg/kg to 10 mg/kg, about 1 mg/kg to 5 mg/kg, or about 2 mg /kg up to 5 mg/kg. In some embodiments, the amount of 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.

In some embodiments, the second antibody is an anti-KIR antibody and the fixed dose ratio is about 30:1, about 10:1, about 3:1, about 1:1, about 1:2, or about 3:10 mg of anti -PD-1 antibody to mg anti-KIR antibody.

In some embodiments, the second antibody is an anti-LAG3 antibody and the fixed dose ratio is about 80:3, about 80:1, about 12:1, about 3:1, or about 1:1 mg of anti-PD-1 antibody to 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 mg, 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 mg to 350 mg, about 60 mg to 300 mg, about 100 mg to 300 mg, or about 150 mg to 250 mg. In some embodiments, the amount of anti-LAG3 antibody in the composition is at least about 240 mg.

In some embodiments, the second antibody is an anti-CD137 antibody,

- 21 042223 and the fixed dose ratio 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 to 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 20 mg, about 1 mg to 15 mg, about 5 mg to 12 mg, or about 5 mg to 10 mg. In some embodiments, the amount of anti-CD137 antibody in the composition is at least about 8 mg.

In some embodiments, the second antibody is an anti-CD73 antibody and the fixed dose ratio 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 to mg anti-CD73 antibody. In some embodiments, the amount of anti-CD73 antibody in the composition is from about 100 mg to 200 mg, or from about 150 mg to 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.

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

In some embodiments, the anti-PD-1 antibody and the second antibody are combined using existing dosage forms of the two antibodies (e.g., 2 ml of anti-PD-1 antibody in citrate buffer are combined with 2 ml of anti-CTLA4 antibody in Tris buffer without changing buffers ).

In some embodiments, the composition contains one or more additional components selected from the group consisting of: a bulking agent, a stabilizing agent, a chelating agent, a surfactant, a buffering agent, and any combination thereof. In some embodiments, the buffering agent comprises citrate buffer, tris buffer, tris-Cl buffer, histidine buffer, tris acetate buffer (TAE), HEPES buffer, tris borate buffer (TBE), 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-picoline buffer, 2-picoline buffer, 4-picoline buffer, 3-based buffer ,5-lutidine, 3,4-lutidine buffer, 2,4-lutidine buffer, Aces buffer, diethyl malonate buffer, N-methylimidazole buffer, 1,2-dimethylimidazole buffer, TAPS- buffer, bis-tris buffer, L-arginine buffer, lactate th buffer, glycolate buffer.

In some embodiments, the anti-PD-1 antibody and the second antibody are formulated in a buffer that is based on the buffer conditions of one of two individual antibody dosage forms. In some embodiments, the buffer conditions used are anti-PD-1 antibody buffer conditions. In some embodiments, the anti-PD-1 antibody is nivolumab and the two antibodies are formulated in a nivolumab citrate buffer system. In some embodiments, the buffer is a citrate buffer.

In some embodiments, the anti-PD-1 antibody and the second antibody are formulated under buffer conditions that are different from the buffer conditions of each of the two antibodies. 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 50 mM, in some embodiments, from about 5 mM to 40 mM, from about 5 mM to 30 mM, from about 5 mM to 20 mM, from about 5 mM to 15 mM , from about 10 mM to 30 mM, or from about 15 mM to 25 mM. In some embodiments, the citrate concentration is about 10 mM. In some embodiments, the citrate concentration is about 20 mM.

- 22 042223

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 50 mM, from about 10 mM to 50 mM, from about 10 mM to 40 mM, from about 10 mM to 30 mM, or from about 15 mM to 25 mM. In some embodiments, the Tris-Cl concentration is about 20 mM.

In some embodiments, the buffer used is a histidine-based buffer. In some embodiments, the histidine concentration 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 from about 5 mM to 50 mM, from about 5 mM to 40 mM, from about 5 mM to 30 mM, from about 5 mM to 25 mM, or from about 10 mM to 15 mM. In some embodiments, the histidine concentration is about 20 mM.

In some embodiments, the buffer used is 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 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 Tris-Cl concentration is from about 5 mM to 20 mM, from about 5 mM to 15 mM, or from about 10 mM to 15 mM, and the citrate concentration is from about 1 mM to 15 mM, from about 1 mM to 10 mM or about 5 mM to 10 mM. In some embodiments, the Tris-Cl concentration is about 13.3 mM and the citrate concentration is about 6.7 mM.

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 about 5.0 to 8.0, about 5.5 to 6.5, about 6.0 to 7.0, about 6.5 to 7.5. In some embodiments, the pH of the composition is about 6.0. In some embodiments, the pH of the composition is about 7.0. In some embodiments, the pH of the composition is about 6.2. In some embodiments, the pH of the composition is about 6.5. In some embodiments, the pH of the composition is about 6.6. In some embodiments, the pH of the composition is about 5.5.

In some embodiments, the implementation of the composition according to the invention, in addition, contains a bulking agent. The bulking agent may be selected from the group consisting of NaCl, mannitol, glycine, alanine, and any combination thereof. In other embodiments, the implementation of the composition according to the invention contains a stabilizing agent. The stabilizing agent may be selected from the group consisting of sucrose, trehalose, raffinose, arginine; or any combination of them. In other embodiments, the implementation of the composition according to the invention contains a surfactant. The surfactant may be selected from the group consisting of polysorbate 80 (PS80), polysorbate 20 (PS20), and any combination thereof. In certain embodiments, the composition further comprises a chelating agent. The chelating agent may be selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid, nitrilotriacetic acid, and any combination thereof.

In one embodiment, the composition contains NaCl, mannitol, pentetic acid (DTPA), sucrose, PS80, and any combination thereof. In another embodiment, the composition contains NaCl at a concentration of at least about 5 mM, at least about 10 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 contains about 10 mM to 200 mM NaCl, about 25 mM to 150 mM NaCl, about 40 mM to 125 mM NaCl, about 25 mM to 75 mM NaCl, about 50 mM to 100 mM NaCl or about 75 mM to 125 mM NaCl. In some embodiments, the implementation of the composition contains about 100 mm NaCl. In certain embodiments, the composition contains about 50 mM NaCl. In other embodiments, the implementation of the composition contains about 83.3 mm NaCl. In yet other embodiments, the composition contains about 96.15 mM

NaCl. In a particular embodiment, the composition contains about 78.57 mM NaCl.

In certain embodiments, the composition comprises mannitol (% w/v), USP at a concentration of 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 contains about 0.5% to 5% mannitol, about 0.5% to 4% mannitol, about 0.5% to 1.5% mannitol, about 1% to 2% mannitol, or about 2.5% to 3.5% mannitol. In yet other embodiments, the composition contains about 1% mannitol. In yet other embodiments, the composition contains about 3.0% mannitol. In some embodiments, the composition contains about 1.67% mannitol. In certain embodiments, the composition contains about 1.15% mannitol. In a specific embodiment, the composition contains about 1.86% mannitol.

In other embodiments, the composition contains pentetic acid (DTPA), USP at a concentration of at least about 5 μM, at least about 10 μM, at least μM, μM, μM, μM, by at least at least at least at least about about about about μM, μM, μM, μM, at least about 25 at least about 50 at least about 75 at least about 100 μM, μM, μM, μM, at least at least less than

110 µM, at least about

120 μM, at least about 130 μM, at least at least

140 µM, at least about 150 µM, at least about 175 µM, or at least about about about about about about about

200 µM. In some embodiments, the composition contains about 10 µM to 200 µM DTPA, about 10 µM to 150 µM DTPA, about 10 µM to 100 µM DTPA, about 10 µM to 30 µM DTPA, about 50 µM to 100 µM DTPA or about 75 µM to 125 µM

DTPA. In other embodiments, the implementation of the composition contains DTPA at a concentration of about 100 μm. In certain embodiments, the implementation of the composition contains DTPA at a concentration of about 20 μm. In still other embodiments, the composition contains DTPA at a concentration of about 73.3 μM. In a specific embodiment, the composition contains DTPA at a concentration of about 50 μM. In specific embodiments, the composition contains DTPA at a concentration of about 93.85 μM. In certain embodiments, the composition contains DTPA at a concentration of about 65.71 μM.

In some embodiments, the composition comprises polysorbate 80, NF (PS80) (% w/v) at a concentration of 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% or at least about 0.1%. In other embodiments, the composition contains about 0.005% to 0.1% PS80, about 0.005% to 0.02% PS80, about 0.005% to 0.05% PS80, about 0.01% to 0.02% PS80, about 0.02% to 0.1% PS80 or about 0.01% to 0.03% PS80. In still other embodiments, the implementation of the composition contains PS80 at a concentration of about 0.01%. In yet other embodiments, the composition comprises PS80 at a concentration of about 0.04%. In some embodiments, the implementation of the composition contains PS80 at a concentration of about 0.04%. In some embodiments, the composition contains PS80 at a concentration of about 0.013%. In a specific embodiment, the composition contains PS80 at a concentration of about 0.05%. In some embodiments, the implementation of the composition contains PS80 at a concentration of about 0.02%. In other embodiments, the composition contains PS80 at a concentration of about 0.012%. In a specific embodiment, the composition contains PS80 at a concentration of about 0.23%.

In certain embodiments, the composition contains sucrose (% w/v) at a concentration of 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 least about 15% sucrose. In other embodiments, the composition contains about 1% to 10%, about 2% to 10%, about 5% to 10%, about 5% to 7%, or about 7.5% to 10% sucrose. In yet other embodiments, the composition contains about 6% sucrose. In yet other embodiments, the composition contains about 8.5% sucrose. In yet other embodiments, the composition contains about 8% sucrose.

In certain embodiments, the composition comprises nivolumab and ipilimumab in tricitrate buffer. In some embodiments, the composition contains nivolumab and ipilimumab in a 1:1 ratio in a buffer containing about 13.3 mM Tris (or 13.3 mM Tris ± 10%, 20%, 30%,

- 24 042223

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% ) at a pH of about 6.2. In some embodiments, the composition contains nivolumab and ipilimumab in a 3:1 ratio in tricitrate buffer containing 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%) at a pH of about 6.6. In some embodiments, the composition contains nivolumab and ipilimumab in a 1:3 ratio in Tris-citrate buffer containing 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%) at a pH of about 6.0.

In other embodiments, the composition comprises nivolumab and ipilimumab in a histidine buffer. In some embodiments, the composition contains nivolumab and ipilimumab in a 3:1 ratio in 20 Mm histidine buffer (or 20 mM histidine buffer ± 10%, 20%, 30%, 40%, or 50%) containing about 50 mM NaCl (or 50 mM NaCl ± 10%, 20%, 30%, 40% or 50%), about 50 µM DTPA (or 50 µM DTP A ± 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% ), at pH 6. In some embodiments, the composition comprises nivolumab and ipilimumab in a 3:1 ratio in about 20 mM histidine buffer containing 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%), at a pH of about 7. In some embodiments, the composition contains nivolumab and ipilimumab in a ratio of 3:1 to about 20 mM histidine buffer (or 20 mM histidine buffer ± 10%, 20%, 30%, 40% or 50%) containing 50 μM DTPA (or 50 μM DTPA ± 10%, 20%, 30%, 40% or 50% ), about 8.5% sucrose (or 8.5% sucrose ± 10%, 20%, 30%, 40% or 50%) and about 0.05% PS80 (or 0.05% PS80 ± 10%, 20 %, 30%, 40%, or 50%) at a pH of about 6. In some embodiments, the composition contains nivolumab and ipilimumab in a ratio of 1:1, 3:1, or 1:3 in histidine buffer (20 mM ± 10%, 20% , 30%, 40%, or 50%) containing 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%), at pH 5.5 , 6.0 or 6.5. In one embodiment, the composition contains nivolumab and ipilimumab in citrate buffer. In another embodiment, the composition contains 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%) containing 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% ), at a pH of about 6. In other embodiments, the composition contains 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%), containing 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%), at a pH of about 6. In still other embodiments, the implementation of the composition contains nivolumab and ipilimumab in a 1:1 ratio in approximately 20 mM citrate buffer (or 20 mM citrate buffer ± 10%, 20%, 30%, 40%, or 50%) containing approximately 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%), at 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%) containing 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%), at 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%) containing 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%), at a pH of about 6.5. In yet other embodiments, the composition comprises

- 25 042223 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%) containing 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%), at a pH of about 6.0.

In some embodiments, a composition containing nivolumab and ipilimumab in a 1:3 ratio contains about 4.62 mg/ml nivolumab, about 1.54 mg/ml ipilimumab, about 18.5 mM tris hydrochloride, about 1.5 mM dihydrate sodium citrate, about 96.2 mM NaCl, about 1.2% mannitol, about 93.9 μM pentetic acid, and about 0.012% PS80 at a pH of about 6.0.

In some embodiments, a composition containing nivolumab and ipilimumab in a 1:3 ratio contains about 4.61 mg/ml nivolumab, about 1.54 mg/ml ipilimumab, about 18.46 mM tris hydrochloride, about 1.54 mM dihydrate sodium citrate, about 96.15 mM NaCl, about 1.15% mannitol, about 93.85 μM pentetic acid, and about 0.012% PS80 at a pH of about 6.3.

In some embodiments, the pharmaceutical composition contains 30 mg of nivolumab and 90 mg of ipilimumab per vial. In other embodiments, the composition contains 40 mg of nivolumab and 120 mg of ipilimumab per vial.

In other embodiments, the implementation of the composition contains a third antibody. In some embodiments, the third antibody is any antibody described herein.

Composition stability

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.

In another embodiment, the composition exhibits an acid peak change (e.g., deamidation) that is 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at about 5°C. In other embodiments, the composition exhibits an acid peak change that is 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at a temperature of about 25°C. In some embodiments, the composition exhibits an acid peak change that is 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at a temperature of about 40°C. In some embodiments, the acid peak is measured using capillary isoelectric focusing (cIEF) analysis.

In some embodiments, deamidation of a composition of the present invention is comparable to deamidation of a reference composition (a composition containing a first antibody or a second antibody) if the composition exhibits an acid peak change (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 acid peak of the reference composition.

In certain embodiments, the composition exhibits a high molecular weight (HMW) peak change (e.g., aggregation) that is 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 for about 1 month, about 2 months, about 3 months, about 4 months , about 6 months or about 1 year at about 5°C. In some embodiments, the composition exhibits an HMW peak change that is 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at a temperature of about 25°C. In some embodiments, the composition exhibits an HMW peak change that is 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at a temperature of about 40°C. In some embodiments, the composition exhibits an HMW peak change that is less than about 5%, about 4%, about 3%, about 2%, about 1.5%,

- 26 042223 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 an HMW peak change that is 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at about 5°C, about 25°C or about 40°C. In some embodiments, the high molecular weight peak is measured by chromatography. In some embodiments, the chromatography is size exclusion chromatography.

In some embodiments, the aggregation (e.g., level of HMW fractions) of a composition of the present invention is comparable to that of a reference composition (a composition containing a first antibody or a second antibody) if the composition exhibits a HMW peak change 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 peak of the reference composition.

In some embodiments, the composition exhibits a major peak change 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at temperature around 5°C. In some embodiments, the composition exhibits a major peak change 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at temperature around 25°C. In some embodiments, the composition exhibits a major peak change 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at temperature around 40°C. In some embodiments, the composition exhibits a major peak change 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 capillary isoelectric focusing (cIEF) analysis.

In some embodiments, the composition exhibits a low molecular weight (LMW) peak change (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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at about 5°C. In some embodiments, the composition exhibits an LMW peak change 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at temperature around 25°C. In some embodiments, the composition exhibits an LMW peak change 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at temperature around 40°C. In some embodiments, the composition exhibits an LMW peak change 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 an LMW peak change 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 for about 1 month, about 2 months, about 3 months, about 4 months, about 6 months or about 1 year at about 5°C, about 25°C or about 40°C. In some embodiments, the low molecular weight peak is measured by chromatography. In some embodiments, the chromatography is size exclusion chromatography.

In some embodiments, fragmentation (e.g., level of LMW fractions) of a composition of the present invention is comparable to fragmentation of a reference composition (a composition containing a first antibody or a second antibody) if the composition containing

- 27 042223 the first and second antibodies exhibit an LMW peak change 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 peak of the reference composition.

Method for making the compositions disclosed herein

In one embodiment, the invention is directed to a method for making any of the compositions disclosed herein. In another embodiment, the anti-PD-1 antibody drug formulation is mixed with the second antibody drug formulation to obtain the desired ratio in the final drug without buffer changes. In other embodiments, the implementation of the final composition is contained in Tris-citrate buffer.

In some embodiments, a dosage form containing an anti-PD-1 antibody pharmaceutical substance and a dosage form containing another antibody pharmaceutical substance are subjected to buffer changes and/or concentration prior to mixing to obtain the desired ratio in the final drug product.

In other embodiments, the composition is diluted prior to use. In certain embodiments, the composition is diluted prior to use with 0.9% sodium chloride injection, USP, or 5% dextrose injection, USP. In other embodiments, the composition is diluted to form an infusion solution with the desired concentration of the first and second antibodies. In still other embodiments, the final concentration of the first and second antibodies is from about 1 mg/ml to 500 mg/ml, from about 1 mg/ml to 450 mg/ml, from about 1 mg/ml to 400 mg/ml, from about 1 mg/mL to 350 mg/mL, from about 1 mg/mL to 300 mg/mL, from about 1 mg/mL to 250 mg/mL, from about 1 mg/mL to 200 mg/mL, from about 1 mg /ml to 150 mg/ml, from about 1 mg/ml to 100 mg/ml, from about 1 mg/ml to 90 mg/ml, from about 1 mg/ml to 80 mg/ml, from about 1 mg/ml up to 70 mg/ml, from about 1 mg/ml to 60 mg/ml, from about 1 mg/ml to 50 mg/ml, from about 1 mg/ml to 40 mg/ml, from about 1 mg/ml to 30 mg/ml, about 1 mg/ml to 20 mg/ml, about 1 mg/ml to 15 mg/ml, about 1 mg/ml to 10 mg/ml, about 1 mg/ml to 9 mg/ml ml, about 1 mg/ml to 8 mg/ml, about 1 mg/ml to 7 mg/ml, about 1 mg/ml to 6 mg/ml, about 1 mg/ml to 5 mg/ml, about 1 mg/ml to about 4 mg/ml, about 1 mg/ml to about 3 mg/ml, about 1 mg/ml to about 2 mg/ml, about 0.5 mg/ml to 3 mg/ml, about 50 mg/ml to 400 mg/ml, or about 100 mg/ml to 300 mg/ml.

In certain embodiments, the diluted infusion solution is stored for no more than about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 h at room temperature after dilution. In some embodiments, the diluted infusion solution is stored in a refrigerator (at about 2-8°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 after dilution.

Methods according to the invention

The present invention provides methods for treating a subject afflicted with a disease or condition with any of the compositions disclosed herein. In one embodiment, the method is directed to administering a pharmaceutical composition comprising an X amount of a first antibody, which is an anti-PD-1 antibody or an anti-PD-L1 antibody, and a Y amount of a second antibody, wherein the amount of the first antibody and the amount of the second antibody are in a fixed ratio of about 100:1 to 1:100.

In some embodiments, the disease or condition is an infectious disease. In other embodiments, the disease or condition is cancer. In still other embodiments, the cancer is melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, cancer uterine, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel 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 ureter cancer, carcinomas in the renal pelvis, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal cord tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancers and any combination of these types of cancer. In yet other embodiments, the cancer is lung cancer, metastatic melanoma, glioblastoma, or renal cell carcinoma.

- 28 042223

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 (eg, renal cell carcinoma (RCC)), prostate cancer (eg, hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer , stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer (or carcinoma), gastrointestinal cancer, germ cell tumor, childhood sarcoma, sinonasal natural killer, melanoma (eg, metastatic malignant melanoma such as cutaneous or intraocular malignant melanoma), bone cancer , skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, sarcoma soft tissue, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvis carcinoma, nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, virus-associated cancers (eg, human papillomavirus (HPV)-associated tumor), and hematological malignancies, originating from either of the two major blood cell lines, i.e. myeloid cell lines (producing granulocytes, erythrocytes, platelets, macrophages and mast cells) or lymphoid cell lines (producing B-, T-, NK-cells and plasma cells), such as all types of leukemias, lymphomas and myelomas, for example, acute, chronic, lymphocytic and/or myelogenous leukemias such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), AML undifferentiated leukemia (M0), myeloid leukemia (M1) , myeloblastic leukemia (M2; cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 eosinophilic variant [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megacariblastic leukemia (M7), isolated granulocytic sarcoma and chloroma; lymphomas such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematologic malignancy, eg B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, lymphoma arising from lymphoid tissue mucosal-associated lymphoma (MALT), anaplastic (eg, Ki 1+) large cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, progenitor T-cell lymphoblastic lymphoma, T-lymphoblastic lymphoma; and lymphomas/leukemias (T-Lb1y/T-ALL) such as peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic lymphoid lineage tumors, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, progenitor B-cell lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Cesari's syndrome) and lymphoplasmacytoid lymphoma (LPL) with Waldenström's macroglobulinemia; myeloma such as IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (also called indolent myeloma), single plasmacytoma and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of the myeloid line, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nervous system, including astrocytoma, schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoskarma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosa, keratoacanthoma, seminoma, follicular thyroid cancer, and teratocarcinoma, hematopoietic tumors of the lymphoid lineage, 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 medulla cell type; large-grained lymphocytic leukemia (LGL), preferably of the T-cell type; a/d T-NHL hepato-splenic lymphoma; peripheral/postthymic 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, as well as any combination of these types of cancer. The methods described herein can also be used to treat metastatic cancer.

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

In other embodiments, the composition is administered intravenously. In some embodiments, the composition is reconstituted prior to administration. In yet other embodiments, the composition is diluted prior to administration. In a specific embodiment, the composition is administered at a constant dose. In other embodiments, the composition is administered at a dose based on body weight.

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 every month. In some embodiments, treatment is continued 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 more than 2 years.

In some embodiments, the invention is directed to a method for modulating an immune response, comprising administering any of the compositions disclosed herein.

In certain embodiments, a composition of the present invention (eg, administering an anti-PD-1 antibody, or administering an anti-PD-1 antibody and other anti-cancer therapy) is effective in increasing the subject's survival time. For example, the subject's survival time 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 a year or more compared to another subject being treated with only another type of therapy (eg, standard therapy) or only one of the two components of the composition (eg, only anti-PD-1 antibody). In some embodiments, the duration of survival is increased by at least about 2 months. In certain embodiments, the implementation of therapy according to the present invention effectively increases the duration of survival of the subject without disease progression. For example, a subject's progression-free survival 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 for 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 an untreated subject or a subject treated with another therapy alone (e.g., standard therapy) or with only one of the two components of the formulation (e.g., anti-PD-1 alone or anti-PD-L1 antibody) . In some embodiments, progression-free survival is increased by at least about 2 months. In certain embodiments, the implementation of therapy according to the present invention effectively increases the response rate in a group of subjects. For example, the response rate in a group of subjects increased by at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least 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 treated subjects only another therapy (eg, standard therapy) or only one of the two components of the composition (eg, only anti-PD-1 antibody), ie. monotherapy.

Doses of the compositions disclosed herein

In some embodiments, the composition is administered at a constant dose regardless of the patient's body weight. For example, an anti-PD-1 antibody with a second antibody can be administered at a constant dose of 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, regardless of the patient's body weight. In some embodiments, the composition is administered at a dose based on body weight at any dose disclosed herein. In some embodiments, the amount of the first antibody and the amount of the second antibody administered to the patient as a single dose are identical to the amount of X and the amount of Y, respectively.

In certain embodiments of the combination therapy methods of the present invention, a therapeutically effective dose of an anti-PD-1 antibody or antigen-binding portion thereof is 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about

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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 antigen-binding portion thereof is 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 dosage of the anti-PD-1 antibody in the composition is about 60 mg to 300 mg, about 60 mg to 100 mg, about 100 mg to 200 mg, or about 200 mg to 300 mg. In some embodiments, the dosage of the anti-PD-1 antibody in the composition is about 300 mg to 500 mg, about 300 mg to 450 mg, about 300 mg to 400 mg, about 300 mg to 350 mg, about 350 mg up to 500 mg, from about 400 mg to 500 mg, or from about 450 mg to 500 mg. In some embodiments, the amount of 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 anti-PD-1 antibody in the composition is at least about 360 mg or 480 mg. In some embodiments, the dose of 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 anti-PD-1 antibody in the composition is about 360 mg. In another embodiment, the amount of anti-PD1 antibody in the composition is about 480 mg. In some embodiments, the dosage 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. kg or at least about 5 mg/kg. In some embodiments, the dose of anti-PD-1 antibody in the composition is from about 0.5 mg/kg to 5 mg/kg, from about 0.5 mg/kg to 5 mg/kg, from about 0.5 mg/kg up to 3 mg/kg, or from about 0.5 mg/kg to 2 mg/kg. In some embodiments, the dose of anti-PD-1 antibody in the composition is at least about 1 mg/kg. The appropriate dose of the second antibody is calculated using the desired ratio.

In some embodiments, the anti-PD-1 antibody is administered at a subtherapeutic dose, i. a dose of a therapeutic agent that is significantly lower than the usual or FDA-approved dose when administered as monotherapy for the treatment of cancer. The amount of the second antibody in the composition is calculated based on the desired ratio. Doses of nivolumab that are below the typical dose of 3 mg/kg but not below 0.001 mg/kg are sub-therapeutic doses. Sub-therapeutic doses of anti-PD-1 antibody used in the methods of this document are doses above 0.001 mg/kg and below 3 mg/kg. In some embodiments, the subtherapeutic dose is from about 0.001 mg/kg to 1 mg/kg, from about 0.01 mg/kg to 1 mg/kg, from about 0.1 mg/kg to 1 mg/kg, or from about 0.001 mg/kg to 0.1 mg/kg body weight. In some embodiments, the subtherapeutic 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 of body weight. Receptor occupancy data from 15 subjects who received nivolumab at doses ranging from 0.3 mg/kg to 10 mg/kg indicate that PD-1 occupancy was dose-dependent over this dose range. For all doses, the average occupancy rate was 85% (range 70% to 97%) with an average occupancy plateau of 72% (range 59% to 81%). In some embodiments, a dose of 0.3 mg/kg may provide sufficient exposure resulting in maximum biological activity.

In some embodiments, the composition is administered by intravenous infusion once about a week, once about every 2 weeks, once about every 3 weeks, or once about a month. In certain embodiments, the composition is administered once about every 3 weeks. In one embodiment, 360 mg of anti-PD-1 antibody or antigen-binding fragment thereof is administered once every 3 weeks. In another embodiment, 480 mg of the anti-PD-1 antibody or antigen-binding fragment thereof is administered once about every 4 weeks. In some embodiments, the infusion is 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. .

Actual dosage levels of active ingredients in the pharmaceutical compositions of the present invention may be constant or varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and route of administration without causing toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the specific compositions used according to the present invention, the route of administration, the time of administration, the rate of elimination of the particular compound used, the duration of treatment, other drugs, compounds and/or materials used in combination with the specific compounds used. compositions, age, sex, body weight, pathological condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field. The composition according to the present invention can be entered through one or more routes of administration using one or more of the various methods known in this field. One of skill in the art will appreciate that the route and/or mode of administration will vary depending on the desired results.

Sets

Also within the scope of the present invention are kits containing anti-PD-1 antibody/second antibody compositions and instructions for therapeutic use. Kits usually contain a label indicating the intended use of the contents of the kit and instructions for use. The term label includes any description or recorded material supplied with the kit, or material that is otherwise included with the kit. Thus, this invention provides a kit containing: (a) an appropriate dose of the composition disclosed herein, and (b) instructions for using the composition in any of the methods disclosed herein.

The present invention is further illustrated by the following examples, which should not be construed as a further limitation. The contents of all cited references are incorporated herein by reference.

Examples

Several feasibility studies Feasibility Studies were performed to assess the stability of ipilimumab and nivolumab in a single dosage form containing a fixed dose combination (FDRC). In FIG. 1 shows dosage forms of ipilimumab and nivolumab as formulations of their pharmaceutical substances (Drug Substance, DS) or drugs (Drug Product DP), which were used as controls in the following examples where indicated.

Example 1

A feasibility study was performed to evaluate the stability of ipilimumab and nivolumab in a single fixed dose combination (FDRC) dosage form made by mixing individual dosage forms of ipilimumab and nivolumab (FIG. 1) to a final ipilimumab to nivolumab ratio of 1:1 .

DP ipilimumab (BMS-734016) contains 5 mg/ml ipilimumab in 20 mM Tris-HCl, 100 mM NaCl, 1.0% (w/v) mannitol, 100 μM pentetic acid (DTPA) and 0.01% polysorbate 80 (PS80) at pH 7.0 and is presented as 40 ml in a 50 ml vial and 10 ml in a 10 ml vial (FIG. 1). DP nivolumab (BMS-936558) contains 10 mg/ml nivolumab in 20 mM citrate buffer (sodium citrate dihydrate), 50 mM NaCl, 3.0% (w/v) mannitol, 20 µM DTPA, and 0.02% PS80 at pH 6.0 and presented as 10 ml in a 10 ml vial (FIG. 1).

To achieve a 1:1 ratio of ipilimumab to nivolumab, 80 ml ipilimumab DP (2 vials) was mixed with 40 ml nivolumab DP (4 vials) to give a combined product containing 3.3 mg/ml ipilimumab and 3.3 mg/ml nivolumab. 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 at pH 6.2, as shown in Table. 1.

Table 1

Common dosage form EU FDRC (1:1)

Prototype Ratio Final conc. in vial (mg/ml) Tris Citrate pH Mannitol NaCl DTPA PS80 ipi (mg/ml) Nivo (mg/ml) mm mm %w/v mm µM %w/v 1 1:1 3.3 3.3 13.3 6.7 6.2 1.67 83.3 73.3 0.013

The dosage form containing FDRC (1:1) was filtered and aliquoted into 10 cc glass vials (5 ml per vial), stoppered and sealed. The vials were then stored at 5 or 40°C. Samples were analyzed at days 0, 1 week, 2 weeks, 1 month, 2 months, 3 months and 6 months. Day 0 samples were used as controls.

Image analysis. Ways

At each time point, the sample vials were evaluated for appearance, room temperature pH, HIAC, size exclusion chromatography, and capillary isoelectric focusing (cIEF). The HIAC meter (Royco) is an instrument designed to measure the size of solid particles in a liquid by shading.

Size Exclusion Chromatography (SEC) was performed using an Analytical Size Exclusion HPLC (SE-HPLC) using a TSKgel® G3000SWXL column with a TSKgel® Guard SWXL guard column on an Alliance® 2695 HPLC chromatograph with a 2497 dual wavelength ultraviolet (UV) detector (Waters®) using the software product Empower™ 2 Software. To balance the system used 0.1 M NaH ₂ PO ₄ , 0.1 M Na ₂ SO ₄ and 15% acetonitrile (ACN) at pH 6.8 (mobile phase). Samples were analyzed undiluted if the concentration did not exceed 125 mg/mL. If the concentration exceeded 125 mg/ml, the sample was diluted to 50 mg/ml with the appropriate buffer. Samples were transferred to HPLC vials prior to analysis and stored in the HPLC analytical system at

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5±3°C. A total of 100 μg of sample was injected for analysis and eluted isocratically at a column temperature of 22° C. using mobile phase. The flow rate was 1.0 ml/min with an elution time per sample of 20 minutes and a detector wavelength of 280 nm.

Capillary isoelectric focusing (cIEF) was performed using the Protein Simple™ iCE3 system with an Alcott sampler. Samples were analyzed at a concentration of 25 mg/ml with 2 M urea and 0.35% methylcellulose (MC). A 50 mm capillary with an inner diameter of 100 µm was used for separation. The electrolyte solution was 80 mM H ₃ PO ₄ in 0.1% MS and the catholyte solution was 100 mM NaOH in 0.1% MS. Carrier ampholytes were 1% Pharmalyte® 5-8 and 3% Pharmalyte® 8-10.5. The focusing time was 13 minutes with a focusing voltage of 1.5 kV (300 V/cm) applied for the first minute followed by a voltage of 3 kV (600 V/cm) for the remaining 12 minutes. Detection was performed at 280 nm.

Sample analysis. results

Size exclusion chromatography (SEC) was performed on the controls nivolumab DP and ipilimumab DP, and the fixed dose combination formulation (EC FDRC (1:1)) after 3 months storage at 40°C (FIG. 2A). The HMW peak size of the control DP of nivolumab increased by approximately 1.6% and the HMW peak size of the control DP of ipilimumab increased by approximately 0.25% after storage for 3 months at 40°C (FIG. 2A). The HMW peak size of the dosage form containing EC FDRC (1:1) increased by about 0.7% after 3 months storage at 40°C (FIG. 2A). After storage for 6 months at 40°C, the HMW peak size of the dosage form containing EC FDRC (1:1) increased from 0.555% to a final HMW peak size of 2.82%, with an increase of approximately 2.265% (Table .2). After storage for 6 months at 5°C, the HMW peak size of the dosage form containing EC FDRC (1:1) decreased from 0.555% to a final HMW peak size of 0.525% (Table 2).

table 2

Results of analysis of the combined dosage form containing EC FDRC (1:1) by capillary isoelectric focusing and size exclusion chromatography

medicine, remedy cIEF Acid Peak (Ex) cIEF Acid Peak (6 months/ 5°C) cIEF Acid Peak (Change at 5°C) SEC hmw ref SEC hmw 6 months/ 40°С SEC hmw 6 months/ 5°С SEC HMW Change at 40°C SEC HMW Change, at 5 °C Ipilimumab 34 38.16 4.16 0.555 2.82 0.525 2.265 -0.03 Nivolumab 35.09 38.65 3.56

cIEF was performed on controls nivolumab DP and ipilimumab DP, and a dosage form containing EC FDRC (1:1) after storage for 6 months at 5°C (FIG. 2B). Nivolumab control DP acid peak size increased by about 1.3% and ipilimumab control DP acid peak size increased by about 3% after 6 months storage at 5°C (FIG. 2B). For the dosage form containing EC FDRC (1:1), the size of the acid peak of nivolumab increased by approximately 3.56%, i.e. from 35.09% on day-0 (baseline) to 38.65% at 6 months, while ipilimumab acid peak size increased by about 4.16%, i.e. from 34% at day-0 (baseline) to 38.16% at 6 months (Table 2 and Figure 2B).

This assay can be used over a wide range of mixed buffer system concentrations, i.e. tris-citrate buffer composition.

Example 2

A feasibility study was performed to evaluate the stability of ipilimumab/nivolumab in a dosage form containing FDRC, obtained by mixing individual dosage forms of ipilimumab and nivolumab to final ratios of 3:1, 1:1 and 1:3 (Table 3). Dosage forms containing FDRC were made by mixing ipilimumab DS at 5 mg/ml and nivolumab DS at 20 mg/ml to a protein ratio of 3:1, 1:1 and 1:3 (Table 3).

Each pooled solution was then stirred with a magnetic stirrer at room temperature for 30 minutes, transferred to vials and stored until stability analysis over time. The vials were stored at 5°C, 25°C and 40°C for up to 12 months.

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Table 3

EU FDRC (3:1; 1:1; 1:3) - combinations of dosage forms containing ipilimumab DP and nivolumab DP

Prototype Sootn Final conc. in vial (mg/ml) pH Mannitol NaCl DTPA PS 80 ipi Nivo % mass/volume mm µM % mass/volume EU: pH 6.6 3:1 4.62 1.54 6.6 1.15 96.15 93.85 0.012 EU: pH 6.0 1:3 2.86 8.57 6.0 1.86 78.57 65.71 0.023 EU: pH 6.2 1:1 4.00 4.00 6.2 1.67 83.33 73.33 0.013

Prototype EU: pH 6.6 at a ratio of ipilimumab to nivolumab 3:1 contained 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 at pH 6.6. Prototype EU: pH 6.0 at a ratio of ipilimumab to nivolumab 1:3 contained 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 at pH 6.0. Prototype EU: pH 6.2 at a ratio of ipilimumab to nivolumab 1:1 contained 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 at pH 6.2.

SEC analysis.

In general, slight increases in HMW and LMW fractions were observed in all 3 prototypes (FIGS. 3A-B). Size exclusion chromatography (SEC) was performed on nivolumab DP control, ipilimumab DP control, and dosage forms containing EC FDRC, EC: pH 6.0 (1:3), EC: pH 6.2 (1:1), and EC: pH 6 .6 (3:1) after storage for 2 months at 40°C (FIGS. 3A and 3B). The control ipilimumab dosage form had an initial HMW peak size of about 0.4% on day 0, which increased by about 0.1% to a final HMW peak size of over 0.5% after 2 months of storage at 40°C (Fig. .3A). The control nivolumab dosage form had an initial HMW peak size of about 0.8% on day 0, which increased by about 0.7% to a final HMW peak size of over 1.5% after 2 months of storage at 40°C (Figure 3A). ). The FDRC EU dosage form: pH 6.0 (1:3) had an initial HMW peak size of about 0.6% on day 0, which increased by about 0.7% to a final HMW peak size of about 1.3% after 2 months of storage at 40°C (Fig. 3A). The FDRC EU dosage form: pH 6.2 (1:1) had an initial HMW peak size of about 0.5% on day 0, which increased by about 0.5% to a final HMW peak size of about 1.0% after 2 months of storage at 40°C (Fig. 3A). The FDRC EC dosage form: pH 6.6 (3:1) had an initial HMW peak size of about 0.5% on day 0, which increased by about 0.3% to a final HMW peak size of about 0.8% after 2 months of storage at 40°C (Fig. 3A).

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

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cIEF analysis cIEF was performed on nivolumab DP control, ipilimumab DP control, and formulations containing EC FDRC, EC: pH 6.0 (1:3), EC: pH 6.2 (1:1) and EC: pH 6, 6 (3:1) after storage for 3 months at 25°C (FIG. 4A), 3 months at 5°C (FIG. 4B), and 1 month at 25°C (FIG. 4C). Nivolumab control DP acid peak size decreased by about 0.05% and ipilimumab control DP acid peak size increased by about 5.59% after 3 months storage at 25°C (FIG. 4A). Acid peak sizes of nivolumab and ipilimumab formulation FDRC EC: pH 6.0 (1:3) increased by about 5% and about 5.7%, respectively, after 3 months storage at 25°C (Fig. 4A) . The acid peak sizes of nivolumab and ipilimumab formulation FDRC EC: pH 6.2 (1:1) increased by about 6.8% and about 6.3%, respectively, after 3 months of storage at 25°C (Fig. 4A). Acid peak sizes of nivolumab and ipilimumab formulation FDRC EC: pH 6.6 (3:1) increased by about 4% and about 7.8%, respectively, after 3 months storage at 25°C (Fig. 4A) . Across all three FDRC formulations, ipilimumab's acid peak size increased by approximately 5.7%-7.8%, or an average of approximately 2.2% per month; and the size of the acid peak of nivolumab increased by about 4-6.8%, or an average of less than 2% (about 1.76%) per month (Fig. 4A).

In FIG. 4B shows the actual change in acid peak size from baseline (day 0) controls for samples stored for 3 months at 5°C using cIEF analysis. The acid peak size of the control DP of nivolumab decreased by about 5.1% and the acid peak size of the control DP of ipilimumab decreased by about 1% after storage for 3 months at 5°C (FIG. 4B). The acid peak sizes of nivolumab and ipilimumab formulation FDRC EC: pH 6.0 (1:3) increased by approximately 0.1% and decreased by approximately 1.5%, respectively, after 3 months of storage at 5°C (Fig. .4B). The acid peak sizes of nivolumab and ipilimumab formulation FDRC EC: pH 6.2 (1:1) increased by about 2.1% and about 0.5%, respectively, after 3 months storage at 5°C (Fig. 4B). FDRC EU formulation: pH 6.6 (3:1) showed no change in ipilimumab acid peak size and a decrease of less than 0.1% in nivolumab acid peak size after 3 months storage at 5°C (Fig. 4B) .

In FIG. 4C shows the actual change in acid peak size from baseline (day 0) controls for samples stored for 1 month at 25°C. Nivolumab control DP acid peak size increased by about 1.05% and ipilimumab control DP acid peak size increased by about 1.16% after 1 month storage at 25°C (FIG. 4C). Acid peak sizes of nivolumab and ipilimumab formulation FDRC EU: pH 6.0 (1:3) increased by about 2.8% and about 1%, respectively, after 1 month storage at 25°C (Fig. 4C) . The acid peak sizes of nivolumab and ipilimumab formulation FDRC EC: pH 6.2 (1:1) increased by about 3.1% and about 1.6%, respectively, after 1 month storage at 25°C (Fig. 4C). The acid peak size of nivolumab formulation FDRC EC: pH 6.6 (3:1) did not change and the acid peak size of ipilimumab increased by about 2.8% after 1 month storage at 25°C (FIG. 4C).

Example 3

A Design of Experiments (DoE) study was performed to identify new drug candidates for ipilimumab/nivolumab. Prototype dosage forms containing FDRC (3:1) ipilimumab/nivolumab were made in selected histidine or citrate buffer systems, as shown in table. 4. All DoE FDRC prototypes were generated at a final ipilimumab/nivolumab concentration of 10 mg/mL in a 3:1 ratio (Table 4). The FDRC Combo 4 prototype contained 20 mM citrate, 50 mM NaCl, 50 μM DTPA, 6% w/v. sucrose and 0.05% wt./about. PS80 at a theoretical pH of 6. The FDRC Combo 5 prototype contained 20 mM histidine, 50 mM NaCl, 50 μM DTPA, 6% w/v. sucrose and 0.05% wt./about. PS80 at a theoretical pH of 6.0. The FDRC Combo 6 prototype contained 20 mM histidine, 50 mM NaCl, 50 μM DTPA, 6% w/v. sucrose and 0.05% wt./about. PS80 at a theoretical pH of 7. The FDRC Combo New prototype contained 20 mM histidine, 50 μM DTPA, 8.5% w/v. sucrose and 0.05% wt./about. PS80 at a theoretical pH of 6. Similar to the existing nivolumab DP formulation, the FDRC Combo 8 prototype contained 20 mM citrate, 50 mM NaCl, 20 μM DTPA, 3% w/v. mannitol and 0.04% w/v. PS80 at a theoretical pH of 6.

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Table 4

DoE FDRC (3:1) - new dosage forms

Prototype (3:1) pH 20 mM buffer Conc., mg/ml NaCl, mM DTPA, μM Sucrose, %w/v Mannitol, % mass / volume PS80, % mass/volume Combo 4 6 Citrate 10 50 50 6 - 0.05 Combo 5 6 G istidine 10 50 50 6 - 0.05 Combo 6 7 G istidine 10 50 50 6 - 0.05 combo new 6 G istidine 10 - 50 8.5 - 0.05 Combo 8 6 Citrate 10 50 20 - 3 0.04

DoE FDRC dosage forms were prepared according to the following Combo New Production Example. Combo New was first prepared by subjecting ipilimumab DS and nivolumab DS (ELN 96488-024 and -025) to ultrafiltration/diafiltration. In particular, a disposable ultrafiltration/diafiltration (UFDF) cassette was used for nivolumab DS and ipilimumab DS. Approximately 250 ml of nivolumab stock DS (~21 mg/ml) was used for UF/DF in diafiltration/concentration mode. Transmembrane pressure (TMP) was set at 15 psi, with a flow rate of 0.3 L/min set for the feed pump. Diafiltration was completed after using 3 L of buffer. The sample in the vessel was then concentrated based on the calculated weight loss and collected in a 250 ml PETG flask. The concentration of nivolumab after ultrafiltration/diafiltration (UFDF) was 30.6 mg/ml. Approximately 500 ml of ipilimumab stock DS (~5.2 mg/ml) was used for UF/DF in diafiltration/concentration mode. The concentration of ipilimumab in the final product was 16.2 mg/ml according to A280.

Then, 20 ml of ipilimumab DS in histidine-sucrose buffer and 7.5 ml of nivolumab DS in histidine-sucrose buffer were added to the D-Tube Dialyzer devices and dialyzed against the Combo New buffer indicated in Table 1. 4, within 24 hours in a cold room using sufficient volume (3 buffer changes). The protein concentration of ipilimumab and nivolumab was then measured by HIAC. Additional ipilimumab DS and/or nivolumab DS and appropriate buffers were then added to bring the final concentration of ipilimumab to 7.5 mg/ml and nivolumab to 2.5 mg/ml (3:1). The remaining prototypes of Combo 4, Combo 5, Combo 6 and Combo 8 were obtained in the same way as Combo New, modified to certain concentrations indicated in table. 5.

The combined DP dosage forms were then filtered and sterile filled into 10 cc vials (SAP #1215125, lot #2L68780), capped (SAP #1239068, lot #0H49862) and crimped. Some vials were left for control analysis on day 0. The rest were placed under stable conditions at 5°C, 25°C and 40°C until analysis at certain time points.

SEC analysis

SEC was performed on nivolumab DP control, ipilimumab DP control, and DoE FDRC formulations (3:1) Combo New, Combo 4, Combo 5, Combo 6 and Combo 8 after 3 months storage at 40°C (FIG. 5A). The control ipilimumab dosage form had an initial HMW peak size of about 0.4% on day 0, which increased by about 0.2% to a final HMW peak size of about 0.6% after 3 months of storage at 40°C (Figure 5A). ). The control nivolumab dosage form had an initial HMW peak size of about 0.7% on day 0, which increased by about 1.6% to a final HMW peak size of about 2.4% after 3 months of storage at 40°C (Figure 5A). ). The FDRC Combo New dosage form had an initial HMW peak size of about 0.4% on day 0, which increased by about 0.1% to a final HMW peak size of over 0.5% after 3 months of storage at 40°C (Fig. 5A). The FDRC Combo 4 dosage form had an initial HMW peak size of about 0.6% on day 0, which increased by about 0.7% to a final HMW peak size of about 1.3% after 3 months of storage at 40°C (Fig. 5A). The FDRC Combo 5 dosage form had an initial HMW peak size of less than 0.5% on day 0, which increased by about 0.3% to a final HMW peak size of less than 0.8% after 3 months of storage at 40°C ( Fig. 5A). The FDRC Combo 6 dosage form had an initial HMW peak size of about 0.5% on day 0, which increased by about 0.3% to a final HMW peak size of about 0.8% after 3 months of storage at 40°C (Fig. 5A). The FDRC Combo 8 dosage form had an initial HMW peak size of about 0.5% on day 0, which increased by about 1.0% to a final HMW peak size of about 1.5% after 3 months of storage at 40°C (Fig. 5A).

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The same dosage forms were analyzed by SEC after storage for 3 months at 25°C (FIG. 5A). The HMW peak size of the control ipilimumab dosage form and the control nivolumab dosage form each increased by 0.1% or less after storage for 3 months at 25°C (FIG. 5A). HMW peak sizes of FDRC Combo New and Combo 8 dosage forms increased by 0.1% or less, and HMW peak sizes of FDRC Combo 4, Combo 5 and Combo 6 dosage forms decreased by about 0.1% or less after storage for 3 months at 25°C (FIG. 5A).

cIEF cIEF analysis was performed on nivolumab DP control, ipilimumab DP control, and DoE FDRC formulation (3:1) Combo New, Combo 4, Combo 5, Combo 6, and Combo 8 after storage for 3 months at 25°C (Fig. 5B). The acid peak size of the control ipilimumab increased by about 5.59% and the acid peak size of the control nivolumab DP decreased by about 0.05% after 3 months storage at 25°C (FIG. 5B). The acid peak sizes of nivolumab and ipilimumab FDRC Combo New dosage form increased by about 0.64% and 5.98%, respectively, after 3 months storage at 25°C (FIG. 5B). The acid peak sizes of nivolumab and ipilimumab FDRC Combo 4 dosage form increased by about 5.32% and 6.97%, respectively, after 3 months storage at 25°C (FIG. 5B). The acid peak sizes of nivolumab and ipilimumab FDRC Combo 5 dosage form increased by about 0.12% and 5.34%, respectively, after 3 months storage at 25°C (FIG. 5B). The acid peak sizes of nivolumab and ipilimumab FDRC Combo 6 dosage form increased by about 7.01% and 12.19%, respectively, after 3 months storage at 25°C (FIG. 5B). The acid peak sizes of nivolumab and ipilimumab FDRC Combo 8 dosage form increased by about 7.17% after 3 months storage at 25°C (FIG. 5B).

Example 4

A feasibility study was performed to evaluate the FDRC stability of ipilimumab/nivolumab at different ratios of ipilimumab to nivolumab using a modified version of the DoE FDRC (3:1) Combo New formulation described in Example 3 as the base dosage form. Joint platform (PC) dosage forms containing ipilimumab/nivolumab FDRCs were made in ipilimumab to nivolbumab ratios of 3:1, 1:3, and 1:1, as shown in Table 1. 5. All dosage forms were made in histidine buffer and at final concentrations of 50 μm DTPA, 0.05% wt./about. PS80 and 8.0% w/v sucrose (Table 5). Prototype 4 PC FDRC (PC: pH 5.5-1:3) had a ratio of 1:3 and a pH of 5.5; prototype 5 PC FDRC (PC: pH 6.0-1:3) had a ratio of 1:3 and a pH of 6.0; prototype 6 PC FDRC (PC: pH 6.5-1:3) had a ratio of 1:3 and a pH of 6.5; prototype 7 PC FDRC (PC: pH 6.0-1:1) had a ratio of 1:1 and a pH of 6.0; and Prototype 8 PC FDRC (PC: pH 6.0-3:1) had a ratio of 3:1 and a pH of 6.0 (Table 5).

Table 5

Joint Platform Dosage Forms Containing ipilimumab/nivolumab FDRC

Prototype Ratio ultimate CONC, in vial: (mg/ml) pH Mannitol NaCl DTPA PS80 sucrose ipi Nivo % mass/volume mm µM % mass/volume % mass/volume 4 1:3 2.5 7.5 5.5 - - 50 0.05 8.0 5 1:3 2.5 7.5 6.0 - - 50 0.05 8.0 6 1:3 2.5 7.5 6.5 - - 50 0.05 8.0 7 1:1 5 5 6.0 - - 50 0.05 8.0 8 3:1 7.5 2.5 6.0 - - 50 0.05 8.0

SEC analysis

SEC was performed on nivolumab DP control, ipilimumab DP control, and formulations on the FDRC pooled platform (PC) 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 for 3 months at 40°C (FIG. 6A). The HMW peak sizes of the control nivolumab and ipilimumab dosage forms increased by about 1.7% and 0.25%, respectively, after 3 months storage at 40°C (FIG. 6A). HMW Peak Dosage Size

- 37 042223

FDRC PC: pH 6.0-1:1 increased by about 0.5% after storage for 3 months at 40°C (FIG. 6A). The HMW peak size of the FDRC PC dosage form: pH 5.5-1:3 increased by about 1.25% after 3 months storage at 40°C (FIG. 6A). The HMW peak size of the FDRC PC dosage form: pH 6.0-1:3 increased by about 0.75% after 3 months storage at 40°C (FIG. 6A). The HMW peak size of the FDRC PC dosage form: pH 6.5-1:3 increased by about 0.1% after 3 months storage at 40°C (FIG. 6A). The HMW peak size of the FDRC PC dosage form: pH 6.03:1 increased by about 0.25% after 3 months storage at 40°C (FIG. 6A).

The same dosage forms were analyzed by SEC after storage for 3 months at 5°C (Fig. 6B). The control nivolumab dosage form had an initial HMW peak size of about 0.70% on day 0, which increased to a final HMW peak size of about 0.71% after 3 months storage at 5°C (FIG. 6B). The control imilimumab dosage form had an initial HMW peak size of about 0.4% on day 0, which did not change after 3 months of storage at 5°C (FIG. 6B). The FDRC PC dosage form: pH 6.0-1:1 had an initial HMW peak size of about 0.44% on day 0, which increased to a final HMW peak size of about 0.45% after storage for 3 months at 5° C (Fig. 6B). The FDRC PC: pH 5.5-1:3 formulation had an initial HMW peak size of about 0.47% on day 0, which increased to a final HMW peak size of about 0.48% after 3 months storage at 5°C ( Fig. 6B). The FDRC PC dosage form: pH 6.0-1:3 had an initial HMW peak size of about 0.51% on day 0, which did not change after 3 months storage at 5°C (FIG. 6B). The FDRC PC formulation: pH 6.5-1:3 had an initial HMW peak size of about 0.56% on day 0, which increased to a final HMW peak size of about 0.58% after 3 months storage at 5°C ( Fig. 6B). The FDRC PC dosage form: pH 6.0-3:1 had an initial HMW peak size of about 0.37% on day 0, which increased to a final HMW peak size of about 0.39% after storage for 3 months at 5° C (Fig. 6B).

cIEF cIEF analysis was performed on nivolumab DP control, ipilimumab DP control, and FDRC combination platform (PC) 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 for 3 months at 25°C (FIG. 7A) and 3 months at 5°C ( Fig. 7B).

After storage for 3 months at 25°C, the acid peak size of control nivolumab decreased by about 0.05% and the acid peak size of control ipilimumab increased by about 5.59% (FIG. 7A). The acid peak sizes of nivolumab and ipilimumab formulation FDRC PC: pH 6.0-1:1 increased by about 2.6% and 7%, respectively, after 3 months storage at 25°C (FIG. 7A). The acid peak sizes of nivolumab and ipilimumab formulation FDRC PC: pH 5.5-1:3 increased by about 2.1% and 5.9%, respectively, after 3 months storage at 25°C (FIG. 7A). The acid peak sizes of nivolumab and ipilimumab formulation FDRC PC: pH 6.0-1:3 increased by about 3.7% and 6.8%, respectively, after 3 months storage at 25°C (FIG. 7A). The acid peak sizes of nivolumab and ipilimumab formulation FDRC PC: pH 6.5-1:3 increased by about 5.9% and 6.3%, respectively, after 3 months storage at 25°C (FIG. 7A). The acid peak sizes of nivolumab and ipilimumab formulation FDRC PC: pH 6.0-3:1 increased by approximately 1.3% and 6.2%, respectively, after 3 months storage at 25°C (FIG. 7A). In all FDRC PC dosage forms stored for 3 months at 25°C, ipilimumab acid peak size increased by about 5.9-7.0%, or an average of about 2.0% per month (Fig. 7A). The acid peak size of nivolumab FDRC FC formulation increased by about 1.3-5.9%, or a maximum of about 2% per month (Figure 7A).

After storage for 3 months at 5°C, the acid peak size of control nivolumab decreased by about 5.2%, and the acid peak size of control ipilimumab decreased by about 1% (FIG. 7B). The acid peak sizes of nivolumab and ipilimumab formulation FDRC PC: pH 6.0-1:1 decreased by about 2% and increased by about 2.2%, respectively, after 3 months storage at 5°C (FIG. 7B). The acid peak sizes of nivolumab and ipilimumab formulation FDRC PC: pH 5.5-1:3 decreased by about 1.1% and about 0.3%, respectively, after 3 months storage at 5°C (FIG. 7B). The acid peak sizes of nivolumab and ipilimumab formulation FDRC PC: pH 6.0-1:3 each decreased by about 0.2% after 3 months storage at 5°C (FIG. 7B). The acid peak sizes of nivolumab and ipilimumab formulation FDRC PC: pH 6.5-1:3 increased by about 0.5% and decreased by about 3.1%, respectively, after 3 months storage at 5°C (Fig. 7B). The acid peak sizes of nivolumab and ipilimumab formulation FDRC PC: pH 6.0-3:1 increased by about 0.1% and decreased by about 0.2%, respectively, after 3 months storage at 5°C (Fig. 7B). In sum, the acid peak sizes of nivolumab and ipilimumab in 1:3 dosage forms in the pH range of 5.5-6.5 showed no significant change after storage for 3 months at 5°C, and there was no significant change in ipilimumab and nivolumab at 3 different ratios (Fig. 7B).

- 38 042223

Example 5

A feasibility study was performed to evaluate the stability of ipilimumab/nivolumab FDRC (1:1) in several dosage forms based on nivolumab DP, as indicated in table. 6. These formulations were developed by modifying the nivolumab DP formulation (FIG. 1). A total of 24 vials of imilimumab DP and nivolumab DP were buffer exchanged from the original DP formulations with buffer formulations containing 20 mM citric acid and 50 mM NaCl at pH 6.0 (Prototype A) using centrifuge filters with a cut-off threshold for molecular weight of 50 kDa. Prototypes B-D was obtained in the same way to obtain the characteristics shown in table. 6. Prototype A contained ipilimumab 7.5 mg/ml, nivolumab 7.5 mg/ml, citrate 20 mM, NaCl 50 mM, 3.0% w/v. mannitol, 100 μM pentetic acid (DTPA) and 0.02% PS80 at pH 6.0. Prototype A was identical to nivolumab DP, except that Prototype A contained 100 μM pentetic acid, while nivolumab DP contained 20 μM pentetic 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 pentetic 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 pentetic 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 pentetic acid (DTPA) and 0.02% PS80 at pH 6.0. Table 6. Dosage forms containing FDRC (1:1) based on nivolumab DP.____________________________________

Prototype A: Ipi/Nivo: 7.5 + 7.5 in Citrate (20 mM), pH 6.0, 50 mM NaCl, 3.0% w/v mannitol, 100 µM pentetic acid, 0.02% PS80 Prototype B: Ipi/Nivo: 7.5 + 7.5 in Citrate (20 mM), pH 6.5, 50 mM NaCl, 3.0% w/v mannitol, 100 µM pentetic acid, 0.02% PS80 Prototype C: Ipi/Nivo: 7.5 + 7.5 in Citrate (20 mM), pH 6.5, 100 mM NaCl, 1.0% w/v mannitol, 100 µM pentetic acid, 0.02% PS80 Prototype D: Ipi/Nivo: 7.5 + 7.5 in Citrate (20 mM), pH 6.0, 50 mM NaCl, 6% w/v sucrose, 100 µM pentetic acid, 0.02% PS80

^Note: Prototype A is similar to the nivolumab DP formulation except that the concentration of pentetic acid (DTPA) is the same as the ipilimumab DP formulation (see Figure 1).

Prototypes A, B, C and D FDRC were filtered through a 0.2 micron filter and filled into 10 cc SCHOTT® vials (1 or 2 ml per vial), capped and sealed. They were then placed under stable conditions for up to 12 months prior to stability analysis by appearance, pH measurement, SEC, HIAC and cIEF.

SEC analysis

SEC was performed on nivolumab DP control, ipilimumab DP control, and FDRC prototypes A, B, C and D (1:1) based on nivolumab DP after 1 month storage at 40°C (FIG. 8). The HMW peak sizes of the nivolumab and ipilimumab control dosage forms increased by approximately 0.38% and 0.02%, respectively, after 1 month storage at 40°C (FIG. 8). The HMW peak size of the FDRC Prototype A dosage form increased by about 0.36% after 1 month storage at 40°C (FIG. 8). The HMW peak size of the prototype B FDRC dosage form increased by about 0.41% after storage for 1 month at 40°C (FIG. 8). The HMW peak size of the prototype C FDRC dosage form increased by about 0.37% after storage for 1 month at 40°C (FIG. 8). The HMW peak size of the prototype D FDRC dosage form increased by about 0.24% after storage for 1 month at 40°C (FIG. 8). The control nivolumab dosage form and the FDRC prototype dosage forms A and B each contained 3% w/v. mannitol, while the dosage form of the control ipilimumab and the dosage form of prototype C

FDRC contained 1% mannitol and the FDRC Prototype D formulation did not contain mannitol (see Table 6).

cIEF cIEF analysis was performed on nivolumab DP control, ipilimumab DP control, and FDRC prototype formulations A, B, C and D (1:1) based on nivolumab DP after 3 months storage at 25°C (FIG. 9). The acid peak size of nivolumab increased by about 7.5% and the acid peak size of ipilimumab increased by about 8.8% after storage for 3 months at 25°C (FIG. 9). The acid peak sizes of nivolumab and ipilimumab FDRC Prototype A dosage form each increased by about 9.4% after 3 months of storage at 25° C. (FIG. 9). The acid peak sizes of nivolumab and ipilimumab in the FDRC prototype formulation B increased with

- 39 042223 measured by 8.2% and 13.8%, respectively, after storage for 3 months at 25°C (Fig. 9). The acid peak sizes of nivolumab and ipilimumab FDRC Prototype C dosage form increased by approximately 8.7% and 10.2%, respectively, after 3 months of storage at 25°C (FIG. 9). The acid peak sizes of nivolumab and ipilimumab FDRC prototype D dosage form increased by approximately 10.1% and 9%, respectively, after 3 months of storage at 25°C (FIG. 9). The effect of NaCl on acid peak change can be observed by comparing the control ipilimumab formulation and FDRC prototype C formulation, each containing 100 mM NaCl, with the nivolumab control formulation and FDRC prototype formulations A, B, and D, each containing 50 mM NaCl (Fig. 9; Table 6).

Example 6

A drug product containing a fixed dose ratio combination (FDRC) of nivolumab and ipilimumab was made in a ratio of 1:3. The ipilimumab/nivolumab FDRC combination was made from the commercial pharmaceutical substances of ipilimumab and nivolumab. See figure 1. The ipilimumab pharmaceutical substance is an aqueous solution 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 at pH 7.0. The pharmaceutical substance of nivolumab is an aqueous solution 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% (wt. /v) polysorbate 80 at pH 6.0. Both pharmaceutical substances of ipilimumab and nivolumab were stored at a temperature of 2-8°C.

A drug product containing the FDRC (3:1) combination of nivolumab and ipilimumab was made by combining the pharmaceutical substances of ipilimumab and nivolumab in a protein ratio of ipilimumab to nivolumab of 3:1. Stability change data up to 6 months showed that the FDRC-containing drug product was stable when stored at 2 to 8°C, the intended storage condition. The FDRC-containing drug product is a sterile, non-pyrogenic, disposable, preservative-free, isotonic aqueous solution intended for intravenous (IV) administration. The FDRC-containing drug product may be administered neat at a total protein concentration of 6.2 mg/mL or further diluted with 0.9% Sodium Chloride Injection, USP or 5% Dextrose Injection, USP to desired concentrations. The FDRC-containing drug product is packaged in a colorless type I neutral glass vial or molded vial and sealed with a butyl rubber stopper covered with FluroTec® film. The composition of FDRC is presented in table. 7.

Table 7

Comparison of drug formulations containing FDRC, ipilimumab and nivolumab

Component Function Quantity (Concentration) FDRC ipilimumab Nivolumab Unit. Ipilimumab (BMS734016) Active Ingredient 4.62 5 - mg/ml Nivolumab (BMS936558) Active Ingredient 1.54 - 10 mg/ml Tris hydrochloride buffer agent 18.5 20 - mm sodium citrate dihydrate buffer agent 1.5 - 20 mm Sodium chloride Tonicity modifier 96.2 100 50 mm Mannitol Tonicity modifier 1.2 1.0 h, o % (w/v) Penthetic acid ¹ Metal ion chelator 93.9 100.0 20.0 µM Polysorbate 80 surface active 0.012 0.010 0.020 % (weight/ substance volume) pH at 20 °C - 25 °C ² pH adjustment 6.0 7.0 (at 4°C) 6.0 pH units Water for injection Solvent q.s.

^x Also known as diethylenetriaminepentaacetic acid (DTPA) ² Dilute solutions of hydrochloric acid and sodium hydroxide can be used to

-40042223 pH adjustment during the manufacture of ipilimumab and nivolumab DS. Solution pH not adjusted during DP fabrication

Stability of DP FDRC samples made as shown in Table. 7 were evaluated under expected (5°C), accelerated (25°C) and stress (40°C) storage conditions.

The main degradation pathways for ipilimumab and nivolumab were identified as aggregation (HMW fractions detected by SE-HPLC), fragmentation (LMW fractions detected by SEHPLC), and deamidation (options differing in acid charge detected by CEX or iCIEF) as shown in Examples 1-5 . These changes were assessed by SE-HPLC and iCIEF in the FDRC stability change study. In addition, particulate matter and binding activities were assessed by HIAC and ELISA binding assay, respectively, in these studies at given time points.

The results obtained from the studies showed that in DP FDRC levels of pooled HMW fractions, pooled LMW fractions, nivolumab acid-charged variants, ipilimumab acid-charged variants, and particulate matter remained largely unchanged after 6 months of storage at from 2 to 8°C.

Studies conducted after 6 months of storage under accelerated conditions at 25°C showed that the rate of formation of HMW fractions is comparable between DP FDRC, DP nivolumab and DP ipilimumab. The rate of formation of LMW fractions in DP FDRC was 0.15% per month, which is comparable to the rate of formation of LMW fractions in ipilimumab DP of 0.18% per month, since FDRC predominantly consists of ipilimumab. The rate of formation of acid variants in nivolumab in FDRC DP was 1.98% per month, which is comparable to the rate of formation of acid variants in nivolumab DP of 1.76% per month. The rate of formation of ipilimumab acid variants in FDRC and ipilimumab DP is considered comparable at 2.4% and 1.9% per month, respectively. The level of mechanical inclusions remained basically unchanged.

Studies conducted after 3 months of storage at 40°C showed that similar but more significant changes were observed in DP FDRC under these conditions.

Data from the use-time study demonstrated the stability, compatibility, and equivalence of dosing solutions made from FDRC DP and individual nivolumab and ipilimumab DPs combined in IV bags.

Thus, comparable rates of formation of critical quality indicators (CQAs) such as HMW fractions, LMW fractions, and acidic variants under stressed and accelerated conditions, as well as minor changes in these CQAs under recommended storage conditions, point to the potential development of FDRC DPs with commercialized DS. nivolumab and ipilimumab. Each of the above studies is described in more detail below.

Aggregation and Fragmentation Detected by SE-HPLC

The degree of aggregation (HMW fractions) and fragmentation (LMW fractions) of FDRC was assessed by SE-HPLC. The HMW fractions, monomer and LMW fractions of ipilimumab were co-eluted with the HMW fractions, monomer and LMW fractions of nivolumab, respectively. The results shown in table. 8 are presented as percent of the combined monomer area, pooled HMW fractions, and pooled LMW fractions of ipilimumab and nivolumab. The levels of pooled HMW fractions tested were 0.5% at baseline and remained largely unchanged after 6 months of storage at 5 and 25°C (0.5 to 0.6% change) and increased to 1 .0% after 3 months of storage at 40°C. The pooled LMW fractions tested were 0.1% at baseline and remained largely unchanged after 6 months of storage at 5°C (0.1% to 0.2% change), increased to 1.0 % after 6 months of storage at 25°C, and increased to 2.4% after 3 months of storage at 40°C.

Table 8

Combined monomer, HMW fractions and LMW fractions in FDRC detected by SE-HPLC

Time (Month) Combined monomer (%) Combined HMW Fractions (%) Combined LMW Fractions (%) 5°C 25°C 40°C 5°C 25°C 40°C 5°C 25°C 40°C Ref. 99.3 99.3 99.3 0.5 0.5 0.5 0.1 0.1 ο,ι 1 NT 99.3 98.9 NT 0.6 0.6 NT 0.2 0.4 2 99.3 99.2 97.5 0.5 0.5 0.8 0.2 oh, s 1.7 3 99.3 98.8 96.7 0.5 0.6 ι, ο 0.2 0.6 2.4 6 99.3 98.4 NT 0.6 0.7 NT 0.2 ι, ο NT

-41 042223

NT = not tested

These results were compared with commercial formulations of control ipilimumab (5 mg/mL) and nivolumab (10 mg/mL) DPs stored at 5°C, 25°C, and 40°C, as well as FDRC DP analyzed in a similar manner with using this modified SEC-HPLC method, as shown in Table. 9 and 10, respectively. Based on the available data, it appears that ipilimumab, nivolumab, and FDRC do not tend to form HMW fractions at the recommended storage temperature of 2-8°C; and the rate of formation of HMW fractions per month is comparable between ipilimumab, nivolumab and FDRC at 25°C and 40°C, as shown in table. 11. More importantly, the rates of formation of HMW and LMW fractions in FDRC are equivalent to the rates of formation of HMW and LMW fractions in ipilimumab, since FDRC is predominantly composed of ipilimumab, and the total protein concentration in FDRC, i.e. 6.2 mg/ml is close to ipilimumab's DP concentration of 5 mg/ml. Comparable rates of formation of CQAs such as HMW and LMW fractions under stressed and accelerated conditions, as well as a slight change in these CQAs under recommended storage conditions, indicate the potential for the development of FDRC DPs.

Table 9

Monomer, HMW fractions and LMW fractions in ipilimumab detected by SE-HPLC

Time (Month) Monomer (%) HMW fraction (%) LMW Fraction (%) 5°C 25°C 40°C 5°C 25°C 40°C 5°C 25°C 40°C Ref. 99.38 99.38 99.38 0.4 0.4 0.4 0.22 0.22 0.22 1 99.37 99.33 98.98 0.4 0.38 0.42 0.23 0.29 0.6 2 99.39 99.26 98.54 0.39 0.395 0.51 0.22 0.325 0.955 3 99.37 99.16 96.65 0.4 0.43 0.635 0.23 0.415 2.54 6 99.29 98.62 NT 0.475 0.535 NT 0.245 1.32 NT

Table 10

Monomer, HMW fractions and LMW fractions in nivolumab detected by SE-HPLC

Time (Month) Monomer (%) HMW fraction (%) LMW Fraction (%) 5°C 25°C 40°C 5°C 25°C 40°C 5°C 25°C 40°C Ref. 98.99 98.99 98.99 0.705 0.705 0.705 0.315 0.315 0.315 1 98.98 98.94 98.35 0.705 0.73 1.115 0.32 0.33 0.55 2 98.98 98.91 97.56 0.705 0.76 1.595 0.305 0.34 0.81 3 98.98 98.79 96.45 0.715 0.815 2.37 0.31 0.395 1.18 6 98.91 98.14 NT 0.795 0.965 NT oh, s 0.425 NT

Table 11

Monomer formation rate/month, HMW fractions and LMW fractions in ipilimumab, nivolumab and FDRC detected by SE-HPLC

Time (Month) Monomer (%) HMW fraction (%) LMW Fraction (%) 5°C 25°C 40°C 5°C 25°C 40°C 5°C 25°C 40°C FDRC 0.00 0.16 0.92 0.02 0.03 0.17 0.02 0.15 0.82 Ipilimumab 0.01 0.13 0.86 0.01 0.02 0.08 0.00 0.18 0.73 Nivolumab 0.01 0.14 0.84 0.02 0.04 0.55 0.00 0.02 0.29

Variants with different charges detected by the iCIEF method. The FDRC charge variant profile was determined by iCIEF analysis. The chromatographic profile contained separated peaks of ipilimumab and nivolumab. The relative areas of the acid peaks, the areas of the main peaks and the areas of the main peaks of ipilimumab are presented in Table. 12, and the relative areas of acid peaks, areas of the main peaks and areas of the main peaks of nivolumab are presented in table. 13. Acid, principal and base peak areas for ipilimumab and nivolumab remained largely unchanged after 6 months of storage at 5°C. Changes in the charge profile were observed at 25 and 40°C for ipilimumab and nivolumab. Degradation was significant at 40°C in a short time interval and therefore was not used for comparison and was considered too aggressive to assess the stability of DP.

-42 042223

Table 12

ipilimumab charge profile in FDRC detected by iCIEF method

Time (Month) Acid Peaks (%) Main Peak (%) Main Peaks (%) 5°C 25°C 40°C 5°C 25°C 40°C 5°C 25°C 40°C Ref. 34.6 34.6 34.6 59.5 59.5 59.5 5.9 5.9 5.9 1 NT 37.2 48.7 NT 56.7 45.2 NT 6.2 6.1 2 34.5 39.6 61.2 57.9 54.5 33.7 7.6 5.9 5.1 3 34.6 42.4 68.3 59.2 51.9 27.4 6.2 5.7 4.3 6 35.6 49.0 NT 59.0 45.9 NT 5.4 5.1 NT

Table 13

Nivolumab charge profile in FDRC detected by iCIEF method_______

Time (Month) Acid Peaks (%) Main Peak (%) Main Peaks (%) 5°C 25°C 40°C 5°C 25°C 40°C 5°C 25°C 40°C Ref. 33.7 33.7 33.7 59.8 59.8 59.8 6.5 6.5 6.5 1 NT 34.3 45.0 NT 59.8 46.3 NT 5.9 8.7 2 34.8 39.1 61.3 59.3 54.5 31.2 5.9 6.5 7.5 3 33.6 37.7 65.9 60.6 55.2 27.9 5.9 7.1 6.2 6 33.7 45.5 NT 59.3 46.4 NT 7.1 8.1 NT

In addition, as previously discussed, the FDRC composition evaluated above consists of tpuc.HCI and sodium citrate dihydrate and therefore tends to change pH with temperature change due to the Tris-HCl amine buffer. Therefore, charge profile changes between FDRC and nivolumab occurred at 25°C according to sample preparation temperature and storage temperature.

Comparison of the acid charge profile of ipilimumab in FDRC, presented in table. 12 against control ipilimumab DP in Tris-HCl buffer (commercial composition) at pH 7 (4°C) at 25°C (Table 14) showed that acid peak formation was comparable at 2.4% and 1.93% in month, respectively, as shown in Fig. 10. This relatively small difference under stress conditions is considered insignificant for the stability of the drug product containing FDRC under the recommended storage conditions (2-8°C), as seen in Table. 12.

Historically, changes in the charge profile of ipilimumab were assessed by the CEX method and thus data were collected to reveal the comparability of the charge profile under different conditions for ipilimumab, however, the likelihood of deamidation, the main degradation pathway of ipilimumab, is potentially low, since the kinetics of deamidation is usually slower at lower pH .

Table 14

ipilimumab charge profile obtained by iCIEF method at 25°C

Time (Month) Acid Peaks (%) Main Peak (%) Main Peaks (%) Lab batch for stability testing Lab batch for stability testing Lab batch for stability testing Ref. 39.71 56.59 3.69 1 42.34 54.84 2.82 3 45.3 51.2 3.5 6 51.6 45 3.4

Comparison of the FDRC acid charge profile of nivolumab shown in Table 1. 15 versus three batches of long-term stability nivolumab (LTSB) at 25°C indicates that acid peak formation is comparable at 1.97% and 1.75% per month, respectively, as shown in FIG. eleven.

- 43 042223

Table 15

Nivolumab charge profile (in GMP lots) obtained by iCIEF method at 25°C

Time (Month) Acid Peaks (%) Main Peak (%) Main Peaks (%) The consignment 2J71008 Batch 2J71191 The consignment 2J71192 The consignment 2J71008 The consignment 2J71191 The consignment 2J71192 The consignment 2J71008 The consignment 2J71191 The consignment 2J71192 Ref. 36.1 36.3 35.2 58.1 56.6 58.8 5.8 7.1 6.0 1 33.0 29.6 36.2 61.8 62.9 57.2 5.2 7.5 6.6 3 40.8 39.3 37.2 52.9 52.4 55.6 6.3 8.3 7.2 6 45.7 44.1 44.3 46.4 47.2 47.8 7.9 8.7 7.9

Mechanical inclusions detected by the HIAC method.

Samples stored at 5 and 25° C. for up to 6 months were tested using a shading particulate count (HIAC) procedure to determine particle size and number according to FDRC DP size. As shown in Table. 16, particulate counts for sizes >2 microns, >5 microns, >10 microns, and >25 microns varied but were within the acceptance criteria specified in USP <787>.

Table 16

Mechanical inclusions according to HIAC

Mechanical inclusions (particles/ml) > 2 µm > 5 µm > 10 µm > 25 µm Initial 161 21 5 0 After 6 months at 5°C 134 43 13 2 After 6 months at 25°C 174 34 6 1

Binding activity as determined by ELISA assays. ELISA assays were used to investigate the specific binding of ipilimumab to the human CTLA-4 receptor and the specific binding of nivolumab to the human PD-1 receptor. The binding activities of ipilimumab and nivolumab in FDRC samples were calculated relative to ipilimumab and nivolumab reference standards, respectively. The binding activities of the FDRC samples after 2 months of storage at 25°C were within the proposed acceptance criterion (70-130%) (Table 17).

Table 17

Binding activities determined by ELISA assays

Storage condition ipilimumab binding activity relative to ipilimumab reference standard (%) Nivolumab Binding Activity Relative to Nivolumab Reference Standard (%) Initial 87 119 After 2 months storage at 25°C 87 78

Tryptic peptide mapping analysis.

Tryptic peptide mapping analysis was performed to measure deamidation and oxidation. Samples were reduced, alkylated and digested with trypsin. Tryptic peptides were separated on a C-18 column and detected with a UV detector at 215 and 280 nm followed by a mass spectrometer (LTQ-Orbitrap-Elite). Comparative quantitative analysis was performed by comparing the peak areas of intact peptides as well as modified peptides on selected ion chromatograms. The results of the analysis are shown in table. 18 and 19.

- 44 042223

Table 18

Tryptic Peptide Mapping - Deamidation

Storage condition Nivo H ₄ ³ (%) Ipi N ₅ ⁴ (%) Ipi N37/Nivo IZ 6 Deaml ⁵ (%) Ipi H37/Nivo IZ 6 Deam2 ⁶ (%) Ipi N37/Nivo IZ 6 Deam3 ⁷ (%) Ipi N37/Nivo IZ 6 Deam4 ⁸ (%) Initial 0.5 2.8 3.4 3.2 1.4 3.6 After 2 months storage at 25°C 0.7 4.6 4.7 4.6 1.5 3.7 After 6 months storage at 25°C 1.0 3.9 5.2 5.9 0.7 2.5

³ Nivo H ₄ = Tryptic heavy chain peptide # 4 for nivolumab ⁴ Ipi H ₅ = Tryptic heavy chain peptide # 5 for ipilimumab ⁵ Ipi H ₃₇ /Nivo H ₃₆ Deaml = Tryptic heavy chain peptide # 37 for ipilimumab (Asn #) and # 36 for nivolumab (Asn #) ⁶ Ipi H ₃₇ / Nivo H ₃₆ Deam2 = Heavy chain tryptic peptide # 37 for ipilimumab (Asn #) and #36 for nivolumab (Asn #) ⁷ Ipi H ₃₇ / Nivo H ₃₆ Deam3 = Tryptic peptide heavy chain #37 for ipilimumab (Asn#) and #36 for nivolumab (Asn#) ⁸ Ipi H ₃₇ /Nivo H ₃₆ Deam4 = Heavy chain tryptic peptide #37 for ipilimumab (Asn#) and #36 for nivolumab (Asn#)

Table 19

Tryptic Peptide Mapping - Oxidation

Storage condition Ipi N ₂ 1/Nivo N ₂ 2 ⁹ (%) Nivo H4 ¹⁰ (%) Ipi N ₃ ¹¹ (%) Initial 4.4 0.2 0.5 After 2 months of storage at 25°C 4.9 0.6 0.5 After 6 months of storage at 25°C h, h oh, s 0.2

⁹ Ipi H ₂ 1/Nivo H ₂ 2 = Tryptic heavy chain peptide #21 for ipilimumab (His/Met #) and #22 for nivolumab (His/Met #) ¹⁰ Nivo H ₄ = Tryptic heavy chain peptide #4 for nivolumab ^p Ipi H ₃ = Heavy chain tryptic peptide #3 for ipilimumab

Sample pH analysis

Measured pH DP FDCR, as shown in table. 20.

Table 20

pH values of DP solutions

Storage conditions 25°C 5°C original 6.58 6.58 2 months 6.59 6.56 6 months 6.44 6.38

Stability during the Use-Time Stability of an FDRC medicinal product.

The study was performed to demonstrate the stability and compatibility of DP FDRC with 0.9% sodium chloride solution, USP (NS), intravenous (IV) infusion bags, intravenous infusion sets and inline filters. After 2 months storage at 5°C, DP FDRC samples were diluted in NS in an IV bag, which was stored at 25°C for 4 hours followed by 20 hours at 5°C. The solution was then infused in an IV bag using an IV set and an inline filter. Samples were collected and analyzed by HIAC, Micro Flow Imaging (MFI), SE-HPLC, CE-SDS, iCIEF, and reverse phase ultra high performance liquid chromatography (RP-UPLC).

The results of the study are presented in table. 21-23. Data showed little or no change from baseline for mechanical inclusions (according to HIAC), aggregation (according to SE-HPLC), fragmentation (according to SE-HPLC), purity (according to CE-SDS), Zarya-45 profile 042223 dosing (according to iCIEF) and ipilimumab/nivolumab protein ratio (according to RP-UPLC) after completion of the compatibility study.

The results indicate that DP FDRC can be diluted with 0.9% sodium chloride, USP to a concentration in the range of 1.5/0.5-4.2/1.4 mg/mL ipilimumab/nivolumab intravenous infusion. The diluted solution in an IV bag can be stored at 5°C for up to 24 hours and for 4 hours out of 24 hours at room temperature (25°C).

Table 21

Stability during the period of use and comparability of the drug product FDRC

Conditions HIAC (particles/ml) MFI (particles/ml) SE-HPL CE-SDS (refurbished) >2 micron >5 micron >10 micron >25 micron 2-10 micron >10 micron >25 micron HMW (%) Monomer (%) LMW (%) Purity (%) 1.5/0.5 mg/ml ipilimumab/nivolumab Original ¹² 138 26 5 0 353 I,5 0 0.4 99.4 0.3 99.6 After 24 hours ¹³ 213 52 14 0 414 18 1.6 0.3 99.3 0.4 99.6 4.2/1.4 mg/ml ipilimumab/nivolumab Initial 345 77 24 1 888 6.5 0 0.4 99.3 0.3 99.6 After 24 hours 193 36 12 1 455 0 0 0.4 99.3 0.3 99.6

¹² Samples taken from IV bag at time zero after dilution with DP FDRC in IV bag ¹³ Samples taken after 24-hour storage and infusion with IV kit and built-in filter

Table 22

Period-of-use stability and comparability of the FDRC medicinal product

Conditions iCIEF-ipilimumab iCIEF - Nivolumab RP-UPLC Acid peaks (%) Heads, peak (%) Fundamentals, Peaks (%) Acid peaks (%) Heads, peak (%) Basics. peaks (%) Ipilimumab/ Nivolumab Protein Ratio 1.5/0.5 mg/ml ipilimumab/nivolumab Come out. 38.9 56.2 4.9 34.3 57.5 8.3 2.8 24 hours 38.0 56.8 5.1 34.2 57.7 8.1 2.8 4.2/1.4 mg/ml ipilimumab/nivolumab Come out. 37.2 58.1 4.7 34.2 58.0 7.8 2.9 24 hours 37.9 57.2 4.9 34.8 57.9 7.3 2.8

Table 23

In-use stability and comparability of the FDRC medicinal product

Conditions A280 (Traditional) A280 (Solo-VPE) pH 1.5/0.5 mg/ml ipilimumab/nivolumab Initial 2.07 1.998 6.36 After 24 hours 1.924 2.012 6.33 4.2/1.4 mg/ml ipilimumab/nivolumab Initial 5.618 5.615 6.38 After 24 hours 5.529 5.647 6.37

-46042223

Use-Time Stability of co-administered drug ipilimumab and nivolumab

A study was performed to demonstrate the stability and compatibility of co-administered DP with 0.9% sodium chloride solution, USP (NS), IV bags, IV sets, and inline filters. Vials containing the monotherapy DP ipilimumab and nivolumab were diluted in NS in an IV bag that was stored at 25°C for 4 hours followed by 20 hours at 5°C. The solution contained in the IV bag was then infused with the IV set and built-in filter. Samples were collected and analyzed using HIAC, micro-flow imaging (MFI), SE-HPLC, CE-SDS, iCIEF reverse phase ultra high performance liquid chromatography (RP-UPLC).

The results of the study are presented in table. 24-26. Data show little or no change from baseline for particulate matter (by HIAC), aggregation (by SE-HPLC), fragmentation (by SE-HPLC), purity (by CE-SDS), charge variant profile (with using iCIEF) and ipilimum/nivolumab protein ratio (using RPUPLC) after completion of the compatibility study. The results indicate that co-administered DP can be diluted with 0.9% sodium chloride, USP to a concentration in the range of 1.5/0.5-4.2/1.4 mg/ml ipilimumab/nivolumab intravenous infusion. The diluted solution in an IV bag can be stored at 5°C for up to 24 hours and for up to 4 hours out of 24 hours at room temperature (25°C).

Table 24

Use-Time Stability and Comparability of co-administered imilimumab and nivolumab

Conditions HIAC (particles/ml) MFI (particles/ml) SE-HPLC CESDS (in recovery condition) >2 micron >5 micron >10 micron >25 micron 2-10 micron >10 micron >25 micron HMW (%) Monomer (%) LMW (%) Purity (%) 1.5/0.5 mg/ml ipilimumab/nivolumab (Non-DEHP Intravia sachets) Come out. ¹⁴ 327 75 10 0 1078 8 1 0.3 99.4 0.2 99.6 After 24 hours ¹⁵ 49 9 1 0 152 3 1 0.3 99.4 oh, s 99.6 1.5/0.5 mg/ml ipilimumab/nivolumab (DEHP Viaflex sachets) Come out. 373 54 8 0 1233 2 0 0.4 99.3 o,h 99.6 After 24 hours 35 5 1 0 71 5 1 0.4 99.4 oh, s 99.6 4.2/1.4 mg/mL ipilimumab/nivolumab (Non-DEHP Intravia sachets) Come out. 862 170 17 0 2979 8 1 0.4 99.3 oh, s 99.6 After 24 hours 112 21 3 0 237 8 0 0.4 99.4 oh, s 99.6 4.2/1.4 mg/ml ipilimumab/nivolumab (DEHP Viaflex sachets) Come out. 431 91 15 0 1400 eleven 1 0.4 99.4 0.3 99.6 After 24 hours 53 eleven 4 0 150 3 1 0.4 99.4 0.3 99.6

¹⁴ Samples collected from IV bags at time zero after diluting co-administered DP in an IV bag ¹⁵ Samples collected after 24 hours of storage and infusion with an IV set and built-in filter

- 47 042223

Table 25

Use-Time Stability and Comparability of Co-Drug Ipilimumab and Nivolumab (continued 2)

Conditions iCIEF - Ipilimumab iCIEF - Nivolumab RP-UPLC Acid Peaks (%) Main Peak (%) Main peaks (%) Acid peaks (%) Main peak (%) Main peaks (%) Ipilimumab / Nivolumab Protein ratio 1.5/0.5 mg/ml ipilimumab/nivolumab (Non-DEHP Intravia sachets) Ref. 36.3 59.9 3.8 35.9 59.1 5.0 2.85 After 24 hours 36.2 59.9 3.9 33.8 60.7 5.5 2.80 1.5/0.5 mg/ml ipilimumab/nivolumab (DEHP Viaflex sachets) Ref. 36.8 59.8 3.4 35.2 58.6 6.2 2.87 After 24 hours 36.2 60.1 3.7 35.3 58.3 6.4 2.79 4.2/1.4 mg/mL ipilimumab/nivolumab (Non-DEHP Intravia sachets) Ref. 36.6 59.8 3.6 34.1 59.8 6.1 2.9 After 24 hours 36.9 59.5 3.6 33.6 60.4 6.0 2.87 4.2/1.4 mg/ml ipilimumab/nivolumab (DEHP Viaflex sachets) Ref. 36.3 59.9 3.8 36.0 57.3 6.7 2.93 After 24 hours 36.7 59.5 3.8 35.2 58.6 6.2 2.92

Table 26

Use-Time Stability and Comparability of Co-Drug Ipilimumab and Nivolumab (continued 3)

Terms | A280 A280 (Solo-VPE) | pH

| (Traditional) | 1.5/0.5 mg/ml ipilimumab/nivolumab (Non-DEHP Intravia sachets) Ref. 1.994 1.941 6.21 After 24 hours 1.898 1.936 6.19 1.5/0.5 mg/ml ipilimumab/nivolumab (DEHP Viaflex bags) Ref. 2.019 1.982 6.2 After 24 hours 1.879 1.986 6.2 4.2/1.4 mg/ml ipilimumab/nivolumab (Non-DEHP Intravia bags) Ref. 5.701 5.481 6.26 After 24 hours 5.338 5.538 6.24 4.2/1.4 mg/ml ipilimumab/nivolumab (DEHP Viaflex bags) Ref. 5.586 5.509 6.26 After 24 hours 5.363 5.499 6.26

Example 7

The Process Performance Qualification (PPQ) phase determined pH and polysorbate 80 cutoffs for the 1:3 fixed dose ratio combination (FDRC) nivolumab-ipilimumab. Quantitative compositions of the medicinal product containing FDRC are presented in table. 27.

- 48 042223

Table 27

Quantitative formulations of a medicinal product containing FDRC

Component Function Composition Quantity (mg) per vial (Nivolumab/ ipilimumab) 30/90 40/120 Nivolumab (BMS936558) Active Ingredient 1.54 mg/ml 31.11 41.58 Ipilimumab (BMS734016) Active Ingredient 4.61 mg/ml 93.32 124.74 sodium citrate dihydrate buffer agent 1.54 mM 9.15 12.23 Tris hydrochloride buffer agent 18.46 mm 58.76 78.55 Sodium chloride Tonicity modifier 96.15 mM 113.50 151.71 Mannitol Tonicity modifier 1.15% w/v 232.30 310.50 Penthetic acid ¹⁶ Metal ion chelator 93.85 µM 0.75 1.00 Polysorbate 80 surface active 0.012% w/v 2.42 3.24 substance Hydrochloric acid Sodium hydroxide ¹⁷ pH regulation q.s. up to pH 6.3 Water for injection Solvent q.s. up to 20.2 ml q.s. up to 27.0 ml

¹⁶ Also known as diethylenetriaminepentaacetic acid ¹⁷ Dilute solutions of hydrochloric acid and sodium hydroxide can be used to adjust the pH during the manufacture of nivolumab and ipilimumab DS. The pH value of the solution is not adjusted during the manufacture of DP FDRC qs = sufficient pH limit

The commercial drug substances (DS) of ipilimumab and nivolumab have acceptance criteria for pH values in the range of 6.6-7.6 (4°C) and 5.5-6.5, respectively. Since the DP FDRC was made without any further manipulation of the parent drug substance (DS), a study was conducted to understand the potential pH range in the DP FDRC due to the variability of the parent DS.

Table 28

pH variability in DP FDRC due to original DS variability

20 mM Tris-HCl 20 mM sodium citrate Final pH value pH (4°C) pH (21°C) pH 6.6 pH 5.5 5.77 5.64 pH 7.0 pH 6.0 6.47 6.32 pH 7.6 pH 6.5 7.34 6.99

Solutions were prepared by adding 42 ml of 20 mM Tris-HCl and 3.5 ml of 20 mM sodium citrate buffer to simulate the manufacture of DP FDRC. The results of this evaluation (Table 28) indicate that the pH range of DP FDRC may be 5.7-7.0 with a target pH of 6.2-6.3 at room temperature. This is highly controlled in the parent drug substances (DS) of nivolumab and ipilimumab, so it is unlikely that FDRC DP will be subjected to extremely high pH values of 5.7 or 7.0. In addition, based on the available CQA data for ipilimumab and nivolumab, the risk for FDRC DP quality scores would be higher in the higher pH range. Based on this understanding, two additional studies were conducted to evaluate the effect of pH on drug product quality (DP) scores, and to understand the effect of variability of various excipients (including pH) in the parent drug substance (DS) on drug product quality scores ( D.P.).

Evaluation of data from testing at various pH values targeted FDRC performance measures that are affected by pH variability, such as charge profile controlled by capillary isoelectric focusing (icIEF) and high molecular weight aggregates controlled by size exclusion chromatography (SEC) . There is no noticeable change in the SEC DP FDRC profile over pH 5.4-6.6 (room temperature) under recommended storage conditions of 2-8°C or up to 3 months at 25°C. Quantifiable changes were observed only under accelerated conditions (40°C), where it was clear that the estimated pH range did not affect the SEC profile (Table 29).

Table 29

Monomer, HMW and LMW fractions (%) due to pH variability

Prototype DP (pH) HMW fraction (%) Monomer (%) LMW Fraction (%) Ref. 1M/40°C ZM/40°С Ref. 1M/40°C ZM/40°C Ref. 1M/40°C ZM/40°C 5.4 1.12 1.25 1.79 98.7 98.2 96.8 0.1 0.53 1.35 5.7 1.19 1.24 1.69 98.7 98.3 97.1 0.1 0.46 1.16 6.0 1.23 1.24 1.65 98.6 98.3 97.2 0.1 0.43 1.07 6.3 1.24 1.2 1.63 98.6 98.3 97.3 0.1 0.42 1.04 6.6 1.33 1.33 1.7 98.6 98.2 97.2 0.1 0.43 1.05

The charge profile of ipilimumab and nivolumab did not show any significant difference beyond the assay error at the recommended storage temperature of 2-8°C after 6 months, as shown in Table 1. 30. The charge profile for ipilimumab and nivolumab was evaluated primarily at a storage temperature of 25°C, since the differences are more pronounced, in contrast to 40°C, when the profile changes dramatically and the molecules are largely destroyed. In FIG. 12 shows the acid profile of ipilimumab and nivolumab in FDRC DP compared to their respective controls at pH 6.0.

Table 30

Acid and principal peak profile (cIEF) due to pH variability after 6 months storage at 5°C

Acid peak profile (%) (% change from baseline after 6M storage at 5°C) Main peak change (%) (% Change from baseline after 6M storage at 5°C) Prototype DP (pH) ipilimumab Nivolumab ipilimumab Nivolumab 5.4 -1.0 -o,z -1.0 -0.4 5.7 0.6 -3.7 1.4 -1.8 6.0 0.6 -1.5 0.8 -1.2 6.3 0.9 -2.3 1.5 -1.6 6.6 1.1 -1.9 1.6 -0.7

In addition, a study of stability depending on pH, conducted using the variables shown in table. 31, in which the estimated pH range varied between 5.8-7.0, resulted in similar observations for the SEC and cIEF profiles.

Table 31

DP sustainability study plan

Prototype Final conc. in vial (mg/ml) pH Tris Citrate NaCI Mannitol PS 80 DTPA Ipilimumab (mg/ml) Nivolumab (mg/ml) mm mm mm % mass/volume % mass/volume µM 1 4.62 1.54 5.8 18.46 1.54 76.92 1.38% 0.005 93.85 2 4.62 1.54 5.8 18.46 1.54 115.38 1.38% 0.020 93.85 3 4.62 1.54 5.8 18.46 1.54 76.92 0.92% 0.020 93.85 4 4.62 1.54 6.4 18.46 1.54 96.15 1.15% 0.012 93.85 5 4.62 1.54 7 18.46 1.54 115.38 1.38% 0.005 93.85 6 4.62 1.54 7 18.46 1.54 76.92 0.92% 0.005 93.85 7 4.62 1.54 6.4 18.46 1.54 96.15 1.15% 0.012 93.85 8 4.62 1.54 5.8 18.46 1.54 115.38 0.92% 0.005 93.85 9 4.62 1.54 7 18.46 1.54 115.38 0.92% 0.020 93.85 10 4.62 1.54 7 18.46 1.54 76.92 1.38% 0.020 93.85

The HMW profile of DP FDRC remained unchanged after 6 months of storage at 2-8°C and 25°C as shown in FIG. 13 (monomer profile shown in FIG. 14), indicating no effect of pH even in the presence of other variables such as sodium chloride concentration, mannitol and PS80. Acid and principal peak profiling of ipilimumab and nivolumab in DP FDRC (FIGS. 15-18) clearly indicates deamidation versus pH under accelerated conditions at 25°C, as indicated by an increase in acid peak profile at higher temperatures. at pH 7.0. However, this effect under accelerated conditions does not lead to a quantifiable difference at the recommended storage temperature of 2-8°C.

The cIEF peak profile and pH effect are shown in FIG. 19 and iCIEF profile: pH range 5.4-6.6 shown in FIG. 20.

Maximum concentration of polysorbate 80: not less than 60 mcg/ml.

The concentration of polysorbate 80 in DP FDRC was determined primarily by the quantitative ratio of pharmaceutical substances (DS) of ipilimumab and nivolumab mixed to make DP FDRC, while the target concentration of PS 80 in DP FDRC is 120 μg/ml, and at the same time the nominal concentration of pharmaceutical substances ( DS) of ipilimumab and nivolumab is 100 µg/mL and 400 µg/mL, respectively. There is no release acceptance criterion for PS 80 for both active substance (DS) nivolumab and ipilimumab; however, the manufacturer of nivolumab DS and ipilimumab DS has an in-house limit of 275-525 µg/mL and 60-140 µg/mL, respectively.

Preliminary analysis of DP scores affected by PS80 variability showed no noticeable change in HMW SEC (%) (FIG. 13), and HIAC particulates in the 10-25 µm range met the USP acceptance criteria. In addition, the manufacturing process of the DP FDRC is designed so that there is no need to flush the filter before filling the DP vial due to the presence of an intermediate reservoir (35-40 L) located after the reserve sterile filters, which is filled before the filling operation begins.

In addition, during DP optimization studies, a range of PS 80 concentrations of 120 μg/mL-1000 μg/mL was evaluated in DP FDRC in the worst case (worst case) shaking at 300 rpm for up to 72 hours on a horizontal shaker. These studies, when analyzed for appearance, showed no visible mechanical inclusions after 72 hours, and the SEC profile for all prototypes did not have noticeable differences from the initial time point. Based on this, it was decided not to change the target concentration of DP FDRC by adding concentrated PS 80 at the DP manufacturing site.

However, to assess the potential risk of particulate or HMW fraction formation due to significantly lower PS80 levels after dilution with infusion solutions such as saline, a study was conducted in which a DP FDRC solution containing PS 80 at a target concentration of 120 µg/mL was subjected to 20-fold dilution (6 μg/ml) with 0.9% sodium chloride solution, and the resulting solutions were evaluated for up to 24 h in appearance, for the presence of particulate matter by the HIAC method and the content of HMW fractions (%) by the SEC method. This study showed that lowering the PS 80 concentration to 6 µg/mL in FDRC DP derived infusion solutions did not cause any change in appearance, HMW profile, or HIAC solution characteristics, supporting the target concentration of 120 µg/mL. . In addition, it is expected that DP FDRC will be diluted ~3-fold with infusion solutions during clinical and commercial administration resulting in a PS 80 concentration of 40 μg/mL. An assumed PS 80 concentration of at least 60 µg/mL would still result in a final infusion solution concentration of 20 µg/mL, which is higher than the 6 µg/mL concentration determined in the dilution study described above.

The present application claims priority to Provisional Application US 62/303855, filed March 4, 2016; 62/269000, filed December 17, 2015; 62/265268, filed December 9, 2015; and 62/149325, filed April 17, 2015, the entire contents of which are incorporated herein by reference.

Claims (11)

CLAIM 1. Pharmaceutical composition for the treatment of an infectious disease or cancer in a subject in need thereof, wherein the pharmaceutical composition contains nivolumab and ipilimumab in a ratio of 1:3 in tris-citrate buffer containing 4.62 mg/ml ipilimumab, 1.54 mg/ml nivolumab, 1.15% mannitol, 96.15 mM NaCl, 93.85 μM DTPA and 0.012% PS80 and having a pH of 5.6 to 6.6. 2. Pharmaceutical composition according to claim 1, wherein the tris-citrate buffer contains sodium citrate dihydrate and tris-hydrochloride (tris-HCl). 3. Pharmaceutical composition according to claim 1 or 2, wherein the Tris-citrate buffer contains 18.5 mM Tris-HCl. 4. Pharmaceutical composition according to any one of claims 1 to 3, wherein the tris-citrate buffer contains 1.5 mM sodium citrate dihydrate. 5. Pharmaceutical composition according to any one of claims 1 to 4, wherein the pH of the composition is 6.0, 6.2, 6.5 or 6.6. - 51 042223 6. Pharmaceutical composition according to any one of claims 1 to 5, wherein the pH is 6.2. 7. Pharmaceutical composition according to any one of claims 1 to 5, wherein the pH is 6.1. 8. Pharmaceutical composition for the treatment of an infectious disease or cancer, wherein the pharmaceutical composition contains nivolumab and ipilimumab in a ratio of 1:3, containing ipilimumab 4.62 mg/ml, nivolumab 1.54 mg/ml, 18.46 mM tris hydrochloride (tris -HCl), 1.54 mm sodium citrate dihydrate, 96.15 mm NaCl, 1.15% w/v. mannitol, 93.85 μm pentetic acid, 0.012% w/v. PS80, and has a pH of about 6.0. 9. Composition according to any one of claims 1 to 8, characterized in that the composition a) is stable at 5°C for at least 1 week; b) is stable at 40°C for at least 1 week; or c) is stable at 25°C for at least 1 week. 10. Pharmaceutical composition according to any one of claims 1 to 9, characterized in that a) exhibits an acid peak change that is less than 10% after storage for 6 months or 3 months at 5°C; b) exhibits an acid peak change that is less than 15% after 3 months storage at 25°C; c) exhibits an acid peak change that is less than 15% after storage for 3 months at 40°C; d) exhibits a high molecular weight peak change that is less than 5% after 3 months storage at 4°C; e) exhibits a high molecular weight peak change that is less than 5% after storage for 2 months or 3 months at 25°C; f) exhibits a high molecular weight peak change that is less than 5% after storage for 2 months or 3 months at 40°C; g) exhibits a major peak change in capillary isoelectric focusing (cIEF) analysis that is less than 5% after 1 month storage at 4°C; h) exhibits a major peak change in capillary isoelectric focusing (cIEF) analysis that is less than 5% after 1 month storage at 25°C; i) exhibits a major peak change in capillary isoelectric focusing (cIEF) analysis that is less than 5% after 1 month storage at 40°C; j) exhibits a low molecular weight peak change that is less than 5% after 2 months storage at 40°C; k) exhibits a low molecular weight peak change that is less than 5% after 2 months storage at 25°C; or l) shows a low molecular weight peak change that is less than 5% after 2 months storage at 4°C. 11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the cancer is selected from melanoma, 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, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's disease 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 ureter, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, condition-induced T-cell lymphoma environmental cancers, including asbestos-induced cancers and any combination of these types of cancer.