FORMULATION, DOSAGE REGIMEN AND MANUFACTURING PROCESS FOR HETERODIMERIC Fc-FUSED PROTEINS

Abstract

To provide a pharmaceutical formulation of a heterodimeric Fc-fused protein.SOLUTION: A pharmaceutical formulation for treating cancer in a subject who has received, as a monotherapy or in combination, treatment with a checkpoint inhibitor antibody or an anti-vascular endothelial growth factor therapy comprises a heterodimeric Fc-fused protein and a pharmaceutically acceptable carrier. The heterodimeric Fc-fused protein comprises first and second Fc regions of an immunoglobulin fragment crystallizable (Fc) pair, and the p40 and p35 subunits of IL-12. The p40 and p35 subunits of IL-12 are linked separately to the first Fc region and the second Fc region or to the second Fc region and the first Fc region, respectively. The p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc regions. The CH3 domains of the first and second Fc regions each comprise one or more mutations promoting heterodimerization.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 013,834, filed April 22, 2020, and U.S. Provisional Patent Application No. 63 / 033,161, filed June 1, 2020, each of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing The contents of the electronically submitted Sequence Listing in an ASCII text file submitted with this application (filename: 3338_259PC03_Seqlisting_ST25.txt; size: 946,336 bytes; and creation date: April 21, 2021) are hereby incorporated by reference in their entirety.

[0003] The present disclosure relates to pharmaceutical formulations and dosage regimens of heterodimeric Fc-fusion proteins and methods of using such proteins for the treatment of cancer. [Background technology]

[0004] Physiologically active proteins generally have a short in vivo half-life. To overcome this drawback, attempts have been made to conjugate them with PEG (polyethylene glycol) or fuse them to an antibody Fc (fragment crystallizable) region. Proteins composed of two or more different subunits exhibit physiological activity by forming a protein complex. When fused to a wild-type Fc domain, an Fc fusion protein can be prepared. Because Fc is essentially a homodimer, this can form a homodimer. Proteins composed of two or more different subunits exhibit physiological activity by forming a protein complex. When fused to heterodimeric Fc regions derived from antibodies of other isotypes, such as IgG1, IgG2, IgG3, and IgG4, heterodimeric Fc fusion proteins can also be formed. In this way, improved Fc fusion protein forms can be formed by fusing one or more subunits of a protein that is composed of two or more different subunits and that exhibits physiological activity by forming a protein complex from the two or more subunits to the termini of a heterodimeric Fc variant region.

[0005] Fc heterodimerization is a technique in which mutations are introduced into two different CH3 domains of Fc by genetic engineering, so that these two Fc fragments form a heterodimer with a tertiary structure very similar to that of naturally occurring antibodies, but with minimal sequence variation (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 7,695,936 Summary of the Invention [Problem to be solved by the invention]

[0007] The invention described in this disclosure provides designs for improving Fc fusion protein configurations, in which two subunits of a heterodimeric protein are linked to two Fc domains with different heterodimerization domains by introducing linkers of various lengths or by introducing mutations into the CH2 and CH3 domains of the Fc. Further inventions described in this disclosure provide pharmaceutical formulations of such proteins, methods for making such proteins and formulations, and methods for treating cancer using such proteins. [Means for solving the problem]

[0008] The present invention generally relates to pharmaceutical formulations comprising certain heterodimeric Fc-fusion proteins comprising an IL12 subunit, processes for preparing such proteins and pharmaceutical formulations, and dosage regimens for using such heterodimeric Fc-fusion proteins and pharmaceutical formulations in the treatment of cancer, such as locally advanced or metastatic solid tumors.

[0009] Thus, in one aspect, provided herein is a pharmaceutical formulation comprising a heterodimeric Fc fusion protein comprising a first polypeptide comprising a first antibody Fc domain polypeptide and a first subunit of a multisubunit cytokine, and a second polypeptide comprising a second antibody Fc domain polypeptide and a second, different subunit of the multisubunit cytokine, citrate, a sugar, a sugar alcohol, and a non-ionic surfactant, at a pH of 6.0 to 7.0, wherein the first and second antibody Fc domain polypeptides each comprise different mutations that promote heterodimerization, and the first subunit and the second, different subunit of the multisubunit cytokine are linked to each other. In some embodiments, the first and / or second antibody Fc domain polypeptides comprise one or more mutations that reduce Fc effector function.

[0010] In some embodiments, the concentration of citrate in the pharmaceutical formulation is about 10 mM to about 30 mM. In certain embodiments, the concentration of citrate in the pharmaceutical formulation is about 20 mM. In some embodiments, the concentration of the sugar in the pharmaceutical formulation is about 3% to about 12% (w / v). In certain embodiments, the concentration of the sugar in the pharmaceutical formulation is about 6% (w / v). In certain embodiments, the sugar is a disaccharide. In certain embodiments, the disaccharide is sucrose. In some embodiments, the concentration of the sugar alcohol in the pharmaceutical formulation is about 0.5% to about 6% (w / v). In certain embodiments, the concentration of the sugar alcohol in the pharmaceutical formulation is about 1% (w / v). In certain embodiments, the sugar alcohol is derived from a monosaccharide. In certain embodiments, the sugar alcohol is mannitol.

[0011] In some embodiments, the concentration of the nonionic surfactant in the pharmaceutical formulation is about 0.005% to about 0.02% (w / v). In certain embodiments, the concentration of polysorbate 80 in the pharmaceutical formulation is about 0.01% (w / v). In certain embodiments, the nonionic surfactant is polysorbate. In certain embodiments, the polysorbate is polysorbate 80.

[0012] In some embodiments, the pH is about 6.1 to about 6.9. In certain embodiments, the pH is about 6.2 to about 6.8. In certain embodiments, the pH is about 6.3 to about 6.7. In some embodiments, the pH is about 6.4 to about 6.6. In certain embodiments, the pH is about 6.5.

[0013] In some embodiments, the pharmaceutical formulation further comprises water, hi certain embodiments, the water is USP Water for Injection.

[0014] In some embodiments, the pharmaceutical formulation comprises the heterodimeric Fc-fusion protein at a bulk concentration of about 1 g / L to about 10 g / L. In certain embodiments, the pharmaceutical formulation comprises the heterodimeric Fc-fusion protein at a bulk concentration of about 2 g / L to about 8 g / L. In certain embodiments, the pharmaceutical formulation comprises the heterodimeric Fc-fusion protein at a bulk concentration of about 4 g / L to about 6 g / L. In certain embodiments, the pharmaceutical formulation comprises the heterodimeric Fc-fusion protein at a bulk concentration of about 5 g / L.

[0015] In some embodiments, the pharmaceutical formulation comprises a protein at an administration concentration of about 0.5 g / L to about 1.5 g / L. In certain embodiments, the pharmaceutical formulation comprises a protein at an administration concentration of about 0.75 g / L to about 1.25 g / L. In certain embodiments, the pharmaceutical formulation comprises a protein at an administration concentration of about 1 g / L.

[0016] In some embodiments, the formulation is designed to be stored at a temperature between 2° C. and 8° C. In some embodiments, the pharmaceutical formulation is a clear, colorless solution and is free of visible particulate matter.

[0017] In some embodiments, the formulation has a T of greater than about 60° C., greater than about 61° C., greater than about 62° C., greater than about 63° C., greater than about 64° C., greater than about 65° C., or greater than about 66° C. as measured by differential scanning fluorimetry. m1 and / or a T of greater than about 70°C, greater than about 71°C, greater than about 72°C, greater than about 73°C, greater than about 74°C, greater than about 75°C, greater than about 76°C, or greater than about 77°C m2 In certain embodiments, the formulation has a thermal stability profile as defined by a T of about 67.0°C. m1 and a T of approximately 77.3°C m2 In certain embodiments, the thermostability profile is as defined by T m1 and / or T m2The thermal stability profile of a pharmaceutical formulation, as defined by, changes by less than about 2°C or less than about 1°C when the pharmaceutical formulation is incubated at 50°C for 1 week compared to the same pharmaceutical formulation incubated at 5°C for 1 week as measured by differential scanning fluorimetry.

[0018] In some embodiments, the formulation has a T of greater than about 60° C., greater than about 61° C., greater than about 62° C., greater than about 63° C., greater than about 64° C., greater than about 65° C., greater than about 66° C., or greater than about 67° C. as measured by differential scanning fluorimetry. agg In certain embodiments, the thermostability profile is as defined by T agg The thermal stability profile of a pharmaceutical formulation, as defined by, changes by less than about 2°C or less than about 1°C when the pharmaceutical formulation is incubated at 50°C for 1 week compared to the same pharmaceutical formulation incubated at 5°C for 1 week as measured by differential scanning fluorimetry.

[0019] In some embodiments, after incubating the pharmaceutical formulation at 5° C. for 1 week, the pH of the pharmaceutical formulation does not change by more than a pH value of about 0.2 or about 0.1. In some embodiments, after incubating the pharmaceutical formulation at 50° C. for 1 week, the pH of the pharmaceutical formulation does not change by more than a pH value of about 0.2 or about 0.1.

[0020] In some embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 15 nm, less than about 14 nm, less than about 13 nm, or less than about 12 nm, as measured by dynamic light scattering at 25° C. In certain embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 11.6 nm. In some embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 20 nm, less than about 19 nm, less than about 18 nm, less than about 17 nm, less than about 16 nm, or less than about 15 nm, as measured by dynamic light scattering at 25° C. after incubating the pharmaceutical formulation at 50° C. for 2 weeks. In certain embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 14.4 nm. In some embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 20 nm, less than about 19 nm, less than about 18 nm, less than about 17 nm, or less than about 16 nm, as measured by dynamic light scattering at 25° C. after the pharmaceutical formulation has been subjected to five freeze-thaw cycles. In a specific embodiment, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of about 15.3 nm.

[0021] In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.30, less than about 0.29, less than about 0.28, or less than about 0.27, as measured by dynamic light scattering at 25° C. In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.26. In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.30, less than about 0.29, less than about 0.28, less than about 0.27, or less than about 0.26, as measured by dynamic light scattering at 25° C. after incubating the pharmaceutical formulation at 50° C. for 2 weeks. In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.25. In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.40, less than about 0.35, or less than about 0.34 when measured by dynamic light scattering at 25° C. after subjecting the pharmaceutical formulation to five freeze-thaw cycles. In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is about 0.33.

[0022] In some embodiments, the purity profile of the pharmaceutical formulation is greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99%, as measured by the area of ​​the main peak as a percentage of the total area detected in a SEC-HPLC analysis. In certain embodiments, the purity profile of the pharmaceutical formulation is about 99.0%, as measured by the area of ​​the main peak as a percentage of the total area detected in a SEC-HPLC analysis.

[0023] In some embodiments, the purity profile of the pharmaceutical formulation is greater than about 75%, greater than about 80%, greater than about 81%, greater than about 82%, greater than about 83%, greater than about 84%, or greater than about 85%, as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis, after incubating the pharmaceutical formulation for 2 weeks at 50° C. In certain embodiments, the purity profile of the pharmaceutical formulation is about 85.2%, as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis.

[0024] In some embodiments, the purity profile of the pharmaceutical formulation, as measured by the area of ​​the main peak as a percentage of the total area detected in a SEC-HPLC analysis, after subjecting the pharmaceutical formulation to five freeze-thaw cycles, is greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, or greater than about 98%. In certain embodiments, the purity profile of the pharmaceutical formulation, as measured by the area of ​​the main peak as a percentage of the total area detected in a SEC-HPLC analysis, is about 98.9%.

[0025] In another aspect, the present disclosure provides a method comprising administering to a subject in need thereof the pharmaceutical formulation as a single dose therapy.

[0026] In another aspect, the present disclosure provides a method comprising administering a pharmaceutical formulation to a subject in need thereof in a multiple dose regimen, with an interval of at least 3 weeks between doses or at least 4 weeks between doses. In some embodiments, the pharmaceutical formulation is administered to the subject once every 3 weeks. In some embodiments, the pharmaceutical formulation is administered to the subject once every 4 weeks. In certain embodiments, the pharmaceutical formulation is administered to the subject once every 6 weeks.

[0027] In some embodiments, the method further comprises discontinuing the multi-dose therapy if the subject experiences disease progression, unacceptable toxicity, or meets withdrawal criteria. In some embodiments, if the subject experiences a complete response (CR) during the multi-dose therapy, the multi-dose therapy is further administered for at least 12 months after confirmation of the complete response. In certain embodiments, the total duration of the multi-dose therapy is 24 months or less. In certain embodiments, the total treatment duration is longer than 24 months.

[0028] In some embodiments, the pharmaceutical formulation is administered by subcutaneous injection.

[0029] In some embodiments, the pharmaceutical formulation is administered to a subject in an amount sufficient to provide the heterodimeric Fc-fusion protein at a dosage of about 0.05 μg / kg to about 1.75 μg / kg of the subject's body weight. In some embodiments, the pharmaceutical formulation is administered to a subject in an amount sufficient to provide the heterodimeric Fc-fusion protein at a dosage of about 0.05 μg / kg, about 0.10 μg / kg, about 0.20 μg / kg, about 0.40 μg / kg, about 0.60 μg / kg, about 0.80 μg / kg, about 1.00 μg / kg, about 1.20 μg / kg, about 1.40 μg / kg, or about 1.75 μg / kg of the subject's body weight. In certain embodiments, the pharmaceutical formulation is administered to a subject in an amount sufficient to provide the heterodimeric Fc-fusion protein at a dosage of greater than 0.00 μg / kg and less than about 0.05 μg / kg of the subject's body weight. In a specific embodiment, the pharmaceutical formulation is administered to a subject in an amount sufficient to provide the heterodimeric Fc-fusion protein at a dosage of greater than about 1.75 μg / kg, based on the subject's body weight.

[0030] In some embodiments, the subject has cancer. In certain embodiments, the subject has a locally advanced or metastatic solid tumor. In certain embodiments, the presence of cancer in the subject is confirmed using Response Evaluation Criteria for Solid Tumors (RECIST), version 1.1. In certain embodiments, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma, primary mediastinal large B-cell lymphoma, bladder cancer, urothelial carcinoma, microsatellite instability-high carcinoma, colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma (RCC), endometrial cancer, cutaneous T-cell lymphoma, and triple-negative breast cancer. In certain embodiments, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma, primary mediastinal large B-cell lymphoma, bladder cancer, urothelial carcinoma, microsatellite instability-high cancer, colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma (RCC), endometrial cancer, cutaneous T-cell lymphoma, and triple-negative breast cancer. In certain embodiments, the subject is anti-PD-1 refractory.

[0031] In some embodiments, the subject has melanoma. In certain embodiments, the subject has been previously treated with an anti-PD-1 antibody for at least 6 weeks. In certain embodiments, the subject has confirmed disease progression while receiving the anti-PD-1 antibody at least 4 weeks after initial diagnosis of disease progression. In certain embodiments, disease progression is confirmed by radiological or clinical findings. In certain embodiments, if the subject has a tumor containing a BRAF-activating mutation, the subject has been previously treated with a BRAF inhibitor.

[0032] In some embodiments, the subject has RCC. In certain embodiments, the RCC has clear cell carcinoma. In certain embodiments, the patient has been previously treated with an anti-PD-1 / PD-L1 antibody and / or anti-vascular endothelial growth factor therapy. In certain embodiments, the subject has received no more than three lines of prior therapy.

[0033] In some embodiments, the subject has urothelial carcinoma. In certain embodiments, the subject has locally advanced or metastatic transitional cell carcinoma of the urothelium. In certain embodiments, the subject has previously been treated with a single line of therapy comprising a platinum-containing regimen and has radiographic progression within six months of the last dose of the platinum-containing regimen. In certain embodiments, the subject has previously received no more than two lines of therapy. In certain embodiments, the subject has not previously received checkpoint inhibitor (e.g., anti-PD-1 or anti-PD-L1 antibody) therapy as monotherapy or in combination with platinum-based chemotherapy.

[0034] In some embodiments, the pharmaceutical formulation is administered to a subject as a monotherapy.

[0035] In some embodiments, the pharmaceutical formulations are administered to a subject as a combination therapy.

[0036] In some embodiments, the method further comprises administering an anti-PD-1 antibody to the subject. In certain embodiments, the anti-PD-1 antibody is pembrolizumab. In certain embodiments, the pembrolizumab is administered intravenously. In certain embodiments, the pembrolizumab is administered at a dose of 200 mg. In certain embodiments, the administration of pembrolizumab occurs prior to each administration of the pharmaceutical formulation. In certain embodiments, the pharmaceutical formulation is administered within 1 hour after completion of administration of pembrolizumab.

[0037] In certain embodiments, the anti-PD-1 antibody is nivolumab. In certain embodiments, nivolumab is administered intravenously. In certain embodiments, nivolumab is administered at a dose of about 480 mg. In certain embodiments, administration of nivolumab occurs before each administration of the pharmaceutical preparation. In certain embodiments, the pharmaceutical preparation is administered within 1 hour after the completion of administration of nivolumab.

[0038] In some embodiments, the combination therapy is for the treatment of a cancer selected from the group consisting of melanoma, NSCLC, SCLC, RCC, classical Hodgkin's lymphoma, HNSCC, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, and esophageal cancer. In some embodiments, the combination therapy is for the treatment of a cancer selected from the group consisting of melanoma, NSCLC, SCLC, RCC, classical Hodgkin's lymphoma, HNSCC, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer, ovarian cancer, and prostate cancer. In some embodiments, the cancer is melanoma. In some embodiments, the melanoma is unresectable. In some embodiments, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is microsatellite instability-high (MSI-H) or mismatch repair-deficient metastatic (dMMR) colorectal cancer.

[0039] In some embodiments, the method further comprises performing a surgical intervention to lyse cancer cells, remove a tumor, or debulk a tumor in the subject. In certain embodiments, the surgical intervention comprises cryotherapy. In certain embodiments, the surgical intervention comprises thermotherapy. In certain embodiments, the surgical intervention comprises administering radiation therapy to the subject. In certain embodiments, the radiation therapy is stereotactic body radiation therapy (SBRT).

[0040] In some embodiments, the method further comprises administering to the subject an NK cell targeted therapy. In certain embodiments, the subject is administered a multispecific binding protein. In some embodiments, the method further comprises administering to the subject a chimeric antigen receptor therapy. In some embodiments, the method further comprises administering to the subject a cytokine therapy. In some embodiments, the method further comprises administering to the subject an innate immune system agonist therapy. In some embodiments, the method further comprises administering to the subject chemotherapy. In some embodiments, the method further comprises administering to the subject a targeted antigen therapy. In some embodiments, the method further comprises administering to the subject an oncolytic virus therapy.

[0041] In another aspect, the disclosure provides a method of detecting toxicity in a subject receiving a pharmaceutical formulation, the method comprising measuring a C-reactive protein (CRP) concentration in the subject's blood, wherein the pharmaceutical formulation comprises a heterodimeric Fc fusion protein and a pharmaceutically acceptable carrier, and the heterodimeric Fc fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, or are linked to the second Fc region and the first Fc region, respectively, and the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization. In some embodiments, if the CRP concentration in the subject's blood is higher than a threshold CRP concentration, the subject is identified as being at risk for developing an adverse drug reaction; and if the CRP concentration in the subject's blood is about the same as or lower than a threshold C-reactive protein concentration, the subject is identified as not being at risk for developing an adverse drug reaction. In certain embodiments, if the CRP concentration in the subject's blood is higher than the threshold CRP concentration, administration of the pharmaceutical formulation is discontinued, a lower dose of the heterodimeric Fc-fusion protein is administered, or therapeutic measures are taken to reduce or alleviate the toxic effects of the formulation in the subject.

[0042] In some embodiments of the pharmaceutical formulations or methods described herein, the first and second antibody Fc domain polypeptides are human IgG1 Fc domain polypeptides. In certain embodiments, the multisubunit cytokine is human IL12. In certain embodiments, the human IgG1 Fc domain polypeptide comprises one or more mutations that reduce Fc effector function. In certain embodiments, the first and second antibody Fc domain polypeptides comprise mutations selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, numbered according to the EU numbering system. In certain embodiments, the first and second antibody Fc domain polypeptides each comprise the mutations L234A, L235A, and P329A. In certain embodiments, the first subunit of the multisubunit cytokine is the p40 subunit of IL12, and the second subunit of the multisubunit cytokine is the p35 subunit of IL12. In certain embodiments of the pharmaceutical formulation or the method, a first subunit of the multi-subunit cytokine comprises the amino acid sequence of SEQ ID NO: 127, and a second subunit of the multi-subunit cytokine comprises the amino acid sequence of SEQ ID NO: 128. In certain embodiments of the pharmaceutical formulation or the method, the second subunit of the multi-subunit cytokine is fused to a second antibody Fc domain by a linker comprising the amino acid sequence of SEQ ID NO: 108. In certain embodiments of the pharmaceutical formulation or the method, the first antibody Fc domain comprises the mutations L234A, L235A, P329A, Y349C, K360E, and K409W, and the second antibody Fc domain comprises the mutations L234A, L235A, P329A, Q347R, S354C, D399V, and F405T. In certain embodiments of the pharmaceutical formulation or the method, the first antibody Fc domain comprises the amino acid sequence of SEQ ID NO: 215 and the second antibody Fc domain comprises the amino acid sequence of SEQ ID NO: 216. In certain embodiments of the pharmaceutical formulation or the method, the first antibody Fc domain peptide comprises the amino acid sequence of SEQ ID NO: 290 and the second antibody Fc domain peptide comprises the amino acid sequence of SEQ ID NO: 291.

[0043] In another aspect, provided herein is a kit comprising one or more containers containing a pharmaceutical formulation, wherein the pharmaceutical formulation comprises a heterodimeric Fc fusion protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or to the second Fc region and the first Fc region, the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization; and a pharmaceutically acceptable carrier, and the one or more containers contain a total of about 0.1 mg to about 2 mg of the heterodimeric Fc fusion protein. In certain embodiments, the one or more containers contain a total of about 0.5 mg to about 2 mg of heterodimeric Fc-fusion protein. In certain embodiments, the one or more containers contain a total of about 1 mg of heterodimeric Fc-fusion protein. In certain embodiments, the kit includes one container containing about 1 mg of heterodimeric Fc-fusion protein. In some embodiments, the pharmaceutical formulation is a lyophilized formulation or a liquid formulation. In certain embodiments, the pharmaceutical formulation is a liquid formulation supplied in a volume of 1 mL.

[0044] In another aspect, the disclosure provides use of a heterodimeric Fc-fusion protein in the manufacture of a medicament for treating cancer, wherein the medicament is prepared as a liquid pharmaceutical formulation comprising about 0.5 g / L to about 1.5 g / L of the heterodimeric Fc-fusion protein in one or more containers, wherein the heterodimeric Fc-fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, or are linked to the second Fc region and the first Fc region, respectively, wherein the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and wherein the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization. In some embodiments, the liquid pharmaceutical formulation comprises about 1.0 g / L of the heterodimeric Fc-fusion protein.

[0045] In another aspect, provided herein is use of a heterodimeric Fc-fusion protein in the manufacture of a medicament for treating cancer, wherein the medicament is prepared as a liquid pharmaceutical formulation comprising about 0.1 mg to about 2 mg of the heterodimeric Fc-fusion protein in one or more containers, the heterodimeric Fc-fusion protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or are linked to the second Fc region and the first Fc region, the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization. In some embodiments, the liquid pharmaceutical formulation comprises 1 mg of the heterodimeric Fc-fusion protein. In some embodiments, the medicament is contained in a single container. In some embodiments, each container contains 1 mg of the heterodimeric Fc-fusion protein. In certain embodiments, the medicament is administered to a subject on day 1 every three weeks. In some embodiments, the medicament is administered to a subject on day 1 every four weeks. In some embodiments, the medicament is administered subcutaneously. In some embodiments, the medicament is administered in a volume of about 0.1 mL to about 1 mL. In certain embodiments, the medicament is administered in a volume of about 1 mL. In some embodiments, the medicament is administered at up to two injection sites. In certain embodiments, the second injection is completed within 10 minutes of the first injection. In some embodiments, the medicament is administered at a dose of about 0.05 mg / kg to about 1.75 mg / kg. In certain embodiments, the medicament is administered at a dose of about 1 mg / kg. In some embodiments, the medicament is diluted in a solution of 0.9% saline (sodium chloride for injection) and 0.01% polysorbate 80 prior to administration.

[0046] In another aspect, provided herein is a method for producing a heterodimeric Fc fusion protein for preparation of a pharmaceutical formulation thereof, the method comprising adding acetic acid for 30 to 90 minutes to a solution containing the heterodimeric Fc fusion protein obtained from a Chinese hamster ovary (CHO) cell culture expressing the heterodimeric Fc fusion protein, wherein the acetate adjusts and maintains the pH of the solution at pH 3.55 to 3.75, and the heterodimeric Fc fusion protein is fused to an immunoglobulin Fc (fragment crystallization). The heterodimeric Fc fusion protein comprises a pair of a first Fc region and a second Fc region (possibly a pair of Fc regions) and p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or to the second Fc region and the first Fc region, and the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and the CH3 domains of the first Fc region and the second Fc region each contain one or more mutations that promote heterodimerization. In a specific embodiment, acetic acid is added to a solution containing the heterodimeric Fc fusion protein for about 60 minutes. In a specific embodiment, the acetic acid adjusts and maintains the pH of the solution at about 3.65. In a specific embodiment, a CHO cell culture expressing the heterodimeric Fc fusion protein is maintained in suspension. In certain embodiments, the CHO cell culture expressing the heterodimeric Fc fusion protein is cultured in a bioreactor for 7 to 21 days. In certain embodiments, the CHO cell culture expressing the heterodimeric Fc fusion protein is cultured in a bioreactor for 14 days. In some embodiments, the CHO cell culture expressing the heterodimeric Fc fusion protein is harvested by depth filtration to produce a CHO harvest medium. In certain embodiments, the depth filtration is a two-stage disposable depth filtration consisting of DOHC and XOHC filters. In certain embodiments, the heterodimeric Fc fusion protein is purified from the CHO harvest medium using protein A capture chromatography, mixed-mode chromatography, and cation exchange chromatography to produce a solution containing the heterodimeric Fc fusion protein.

[0047] In some embodiments, Protein A capture chromatography comprises equilibrating a Protein A resin with 20 mM Tris, 150 mM NaCl at pH 7.5, loading CHO harvest media onto the Protein A resin; washing the loaded Protein A resin with 20 mM Tris, 150 mM NaCl at pH 7.5; washing the loaded Protein A resin with 50 mM acetate at pH 5.4; and eluting the heterodimeric Fc-fusion protein from the Protein A resin with 50 mM acetate, 100 mM arginine at pH 3.7, and collecting by 280 nm UV starting at a 1.25 AU / cm rise and ending at a 1.25 AU / cm fall. In a specific embodiment, acetic acid is added to a solution containing the heterodimeric Fc-fusion protein eluted from the Protein A resin at a concentration of 0.5 M, where the acetic acid acidifies the pH of the solution to pH 3.65 over 60 minutes, followed by neutralizing the solution to pH 5.2 by adding 2 M Tris. In certain embodiments, following acidification and neutralization of the solution, the solution containing the heterodimeric Fc-fusion protein is passed through a 0.2 μm filter. In certain embodiments, the filtered solution containing the heterodimeric Fc-fusion protein eluted from the Protein A resin is passed through an X0SP depth filter.

[0048] In some embodiments, the mixed-mode chromatography comprises equilibrating a mixed-mode chromatography column with 50 mM acetate at pH 5.2; loading the X0SP filtered solution onto the mixed-mode chromatography column; washing the loaded mixed-mode chromatography column with 50 mM acetate at pH 5.2; and eluting the heterodimeric Fc-fusion protein from the mixed-mode chromatography column with 50 mM acetate at pH 5.2, 250 mM NaCl, and collecting by 280 nm UV starting at a 0.625 AU / cm rise and ending at a 1.50 AU / cm fall. In certain embodiments, the solution containing the heterodimeric Fc-fusion protein eluted from the mixed-mode chromatography column is passed through a 0.2 μm filter.

[0049] In some embodiments, the cation exchange chromatography comprises equilibrating a cation exchange chromatography resin with 50 mM Tris at pH 7.4; loading the filtered solution eluted from the mixed-mode chromatography column onto the cation exchange chromatography resin; washing the loaded cation exchange chromatography resin with 50 mM Tris at pH 7.4; and eluting the heterodimeric Fc-fusion protein from the cation exchange chromatography resin with a gradient of 50 mM Tris at pH 7.4 and 50 mM Tris at pH 7.4, 0.5 M NaCl, and collecting the heterodimeric Fc-fusion protein by 280 nm UV starting at a 2.5 AU / cm increase and ending at a 4.5 AU / cm decrease. In certain embodiments, the solution containing the heterodimeric Fc-fusion protein eluted from the cation exchange chromatography resin is passed through a 0.2 μm filter. In certain embodiments, the filtered solution containing the heterodimeric Fc-fusion protein eluted from the cation exchange chromatography resin is nanofiltered using a prefilter, a 20 nm nominal filter, and a 0.2 μm membrane.

[0050] In some embodiments, the nanofiltered solution comprising the heterodimeric Fc-fusion protein is ultrafiltered and diafiltered, wherein the ultrafiltration and diafiltration include equilibrating the ultrafiltration system with 50 mM Tris, 265 mM NaCl at pH 7.4; concentrating the nanofiltered solution comprising the heterodimeric Fc-fusion protein to a concentration of about 5.0 g / L; buffer exchanging using 7 diavolumes of 20 mM citrate at pH 6.5; concentrating the diafiltered solution comprising the heterodimeric Fc-fusion protein to a concentration of about 11.0 g / L; and concentrating the concentrated solution comprising the heterodimeric Fc-fusion protein to a concentration of about 5 g / L to about 10 g / L with 20 mM citrate at pH 6.5. and adding 20 mM citrate, 18% (w / v) sucrose, 3% (w / v) mannitol, 0.03% (w / v) polysorbate 80 at pH 6.5 to achieve a final concentration of the ultrafiltration / diafiltration retentate solution comprising the heterodimeric Fc-fusion protein of 20 mM citrate, 6% (w / v) sucrose, 1% (w / v) mannitol, 0.01% (w / v) polysorbate 80. In certain embodiments, the ultrafiltered / diafiltered solution comprising the heterodimeric Fc-fusion protein is passed through a 0.2 μm membrane to produce the bulk drug substance.

[0051] In some embodiments, the bulk drug substance is diluted to an 80% drug product solution in a 0.2 μm filtered buffer containing 20 mM citrate, 6% (w / v) sucrose, 1% (w / v) mannitol, and 0.01% (w / v) polysorbate 80 at pH 6.5. In certain embodiments, the bulk drug substance or 80% drug product is diluted to a dosing concentration of 1 mg / mL of heterodimeric Fc-fusion protein in a 0.2 μm filtered buffer containing 20 mM citrate, 6% (w / v) sucrose, 1% (w / v) mannitol, and 0.01% (w / v) polysorbate 80 at pH 6.5.

[0052] A method for treating cancer in a subject who has received treatment with a checkpoint inhibitor antibody for at least six weeks, comprising administering to the subject a pharmaceutical formulation comprising a heterodimeric Fc-fusion protein and a pharmaceutically acceptable carrier, wherein the heterodimeric Fc-fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or to the second Fc region and the first Fc region, and the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization. In certain embodiments, the checkpoint inhibitor antibody is an anti-programmed cell death protein 1 (PD-1) antibody. In certain embodiments, the cancer is melanoma. In certain embodiments, the melanoma is unresectable or metastatic. In certain embodiments, the subject is confirmed to have disease progression while receiving the anti-PD-1 antibody at least 4 weeks after initial diagnosis of disease progression. In certain embodiments, the subject is confirmed to have disease progression while receiving the anti-PD-1 antibody at least 4 weeks after initial diagnosis of disease progression. In certain embodiments, disease progression is confirmed by radiological or clinical findings.

[0053] In another aspect, provided herein is a method of treating cancer in a subject who has previously been treated with a checkpoint inhibitor antibody or an anti-vascular endothelial growth factor therapy, either as monotherapy or in combination, comprising administering to the subject a pharmaceutical formulation comprising a heterodimeric Fc-fusion protein and a pharmaceutically acceptable carrier, wherein the heterodimeric Fc-fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, or are linked to the second Fc region and the first Fc region, respectively, and the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and wherein the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization. In certain embodiments, the checkpoint inhibitor antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0054] In certain embodiments, the cancer is advanced renal cell carcinoma (RCC). In certain embodiments, the RCC is unresectable or metastatic. In certain embodiments, the RCC has a clear cell component. In certain embodiments, the subject has received three or fewer lines of prior therapy. In certain embodiments, the subject has not been treated with a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor comprises an anti-PD-1 antibody or an anti-PD-L1 antibody as monotherapy or in combination with platinum-based chemotherapy.

[0055] In another aspect, provided herein is a method of treating cancer in a subject who has previously been treated with only one platinum-containing regimen, the method comprising administering to the subject a pharmaceutical formulation comprising a heterodimeric Fc-fusion protein and a pharmaceutically acceptable carrier, wherein the heterodimeric Fc-fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, or are linked to the second Fc region and the first Fc region, respectively, and the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization. In certain embodiments, the platinum-containing regimen is platinum in combination with an agent selected from gemcitabine, methotrexate, vinblastine, and doxorubicin. In certain embodiments, the cancer is locally advanced or metastatic transitional cell carcinoma of the urothelial tract. In certain embodiments, the urothelial carcinoma includes one or more of the group consisting of the renal pelvis, ureter, urinary urothelium, and urethra. In certain embodiments, the urothelial carcinoma is inoperable. In certain embodiments, the urothelial carcinoma is characterized by radiographic progression or recurrence within six months after the last dose of the adjuvant platinum-containing regimen.

[0056] In some embodiments, the urothelial carcinoma is considered a failure of a first-line platinum-containing regimen. In certain embodiments, the subject has received no more than two lines of therapy (including a platinum-containing regimen) for the treatment of urothelial carcinoma prior to administration of the pharmaceutical formulation. In certain embodiments, the subject has not received treatment with a checkpoint inhibitor (CPI) as first-line therapy. In certain embodiments, the checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. In certain embodiments, the checkpoint inhibitor is monotherapy or in combination with platinum-based chemotherapy.

[0057] In some embodiments, the pharmaceutical formulation is administered in combination with pembrolizumab. In certain embodiments, pembrolizumab is administered once every three weeks. In certain embodiments, pembrolizumab is administered before administration of the pharmaceutical formulation. In certain embodiments, the pharmaceutical formulation is administered within one hour after completion of administration of pembrolizumab. In some embodiments, pembrolizumab is administered at a dose of 200 mg. In some embodiments, pembrolizumab is administered intravenously. In some embodiments, the combination is for the treatment of a cancer selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, urothelial carcinoma, high microsatellite instability cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma, and endometrial cancer. In some embodiments, the combination is for the treatment of a cancer selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma, primary mediastinal large B-cell lymphoma, urothelial carcinoma, microsatellite instability-high cancer, gastric cancer, esophageal cancer, cervical cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma, and endometrial cancer.

[0058] In some embodiments, the pharmaceutical formulation is administered in combination with nivolumab. In certain embodiments, nivolumab is administered once every four weeks. In some embodiments, nivolumab is administered before administration of the pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is administered within one hour after completion of administration of nivolumab. In some embodiments, nivolumab is administered at a dose of about 480 mg. In some embodiments, nivolumab is administered intravenously. In some embodiments, the combination is for the treatment of a cancer selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), renal cell carcinoma, classical Hodgkin lymphoma, head and neck squamous cell carcinoma (HNSCC), colorectal cancer, hepatocellular carcinoma, bladder cancer, and esophageal cancer. In some embodiments, the combination is for the treatment of a cancer selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), renal cell carcinoma, classical Hodgkin's lymphoma, head and neck squamous cell carcinoma (HNSCC), colorectal cancer, hepatocellular carcinoma, bladder cancer, esophageal cancer, gastric cancer, ovarian cancer, and prostate cancer. In some embodiments, the cancer is melanoma. In certain embodiments, the melanoma is unresectable or metastatic. In some embodiments, the cancer is colorectal cancer. In certain embodiments, the colorectal cancer is microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) metastatic colorectal cancer.

[0059] In some embodiments, the pharmaceutical formulation is administered to the subject on day 1 every three weeks. In some embodiments, the pharmaceutical formulation is administered to the subject on day 1 every four weeks. In some embodiments, the pharmaceutical formulation is administered subcutaneously. In some embodiments, the pharmaceutical formulation is administered in a volume of about 0.1 mL to about 1 mL. In some embodiments, the pharmaceutical formulation is administered in a volume of about 1 mL. In some embodiments, the pharmaceutical formulation is administered at up to two injection sites. In certain embodiments, the second injection is completed within 10 minutes after the first injection.

[0060] In some embodiments, the pharmaceutical formulation is administered at a dose of about 0.05 mg / kg to about 1.75 mg / kg. In certain embodiments, the pharmaceutical formulation is administered at a dose of about 1 mg / kg. In some embodiments, the pharmaceutical formulation is diluted in a solution of 0.9% saline (sodium chloride for injection) and 0.01% polysorbate 80 prior to administration.

[0061] In some embodiments, the presence of cancer is determined using Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. In some embodiments, subjects with a confirmed complete response are treated with the pharmaceutical preparation for at least 12 months after confirmation, unless they meet censoring criteria.

[0062] In another aspect, provided herein is a method of treating a subject having a blood level of C-reactive protein (CRP) monitored, the method comprising administering to the subject a pharmaceutical formulation comprising a heterodimeric Fc-fusion protein and a pharmaceutically acceptable carrier, wherein the heterodimeric Fc-fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or are linked to the second Fc region and the first Fc region, the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization. In some embodiments, if the CRP concentration in the subject's blood is higher than a threshold CRP concentration, the subject is identified as being at risk for developing an adverse drug reaction; and if the CRP concentration in the subject's blood is about the same as or lower than a threshold C-reactive protein concentration, the subject is identified as not being at risk for developing an adverse drug reaction. In some embodiments, if the CRP concentration in the subject's blood is higher than the threshold CRP concentration, (1) administration of the pharmaceutical formulation is discontinued; (2) a lower dose of the heterodimeric Fc-fusion protein is administered; or (3) therapeutic measures are taken to reduce or alleviate the toxic effects of the formulation in the subject.

[0063] In another aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising subcutaneously administering to the subject a pharmaceutical formulation comprising a heterodimeric Fc fusion protein and a pharmaceutically acceptable carrier, wherein the heterodimeric Fc fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or are linked to the second Fc region and the first Fc region, and the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization; and the pharmaceutical formulation comprises citrate; a sugar; a sugar alcohol; and a non-ionic surfactant, and the pH of the formulation is 5.5 to 7.0.

[0064] In some embodiments of the kits, uses, or methods provided herein, the first Fc region and the second Fc region of the heterodimeric Fc-fusion protein are human IgG1 Fc regions. In some embodiments, the human IgG1 Fc region comprises one or more mutations that reduce Fc effector function. In some embodiments of the kits, uses, or methods provided herein, the first Fc region and the second Fc region comprise one or more mutations selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, numbered according to the EU numbering system. In some embodiments, the first Fc region and the second Fc region each comprise the mutations L234A, L235A, and P329A.

[0065] In certain embodiments of the kits, uses, or methods provided herein, the p40 subunit of IL-12 comprises the amino acid sequence of SEQ ID NO: 127, and the p35 subunit of IL-12 comprises the amino acid sequence of SEQ ID NO: 128. In certain embodiments of the kits, uses, or methods provided herein, the p35 subunit of IL-12 is fused to the second Fc region by a linker comprising the amino acid sequence of SEQ ID NO: 108. In some embodiments, the first Fc region comprises mutations L234A, L235A, P329A, Y349C, K360E, and K409W, and the second Fc region comprises mutations L234A, L235A, P329A, Q347R, S354C, D399V, and F405T. In certain embodiments, the first Fc region comprises the amino acid sequence of SEQ ID NO: 215, and the second Fc region comprises the amino acid sequence of SEQ ID NO: 216. In some embodiments, the first Fc region linked to the p40 subunit of IL-12 comprises the amino acid sequence of SEQ ID NO: 290, and the second Fc region linked to the p35 subunit of IL-12 comprises the amino acid sequence of SEQ ID NO: 291.

[0066] In some embodiments of the kits, uses, or methods provided herein, the pharmaceutical formulation comprises (a) citrate; (b) a sugar; (c) a sugar alcohol; and (d) a non-ionic surfactant, and the pH of the formulation is about 6.0 to about 7.0. In some embodiments of the kits, uses, or methods provided herein, the concentration of citrate in the pharmaceutical formulation is about 10 to about 30 mM. In some embodiments, the concentration of citrate in the pharmaceutical formulation is about 20 mM. In some embodiments, the concentration of the sugar in the pharmaceutical formulation is about 3% to about 12% (w / v). In some embodiments, the concentration of the sugar in the pharmaceutical formulation is about 6% (w / v). In some embodiments, the sugar is a disaccharide. In some embodiments, the disaccharide is sucrose. In some embodiments, the concentration of the sugar alcohol in the pharmaceutical formulation is about 0.5% to about 6% (w / v). In some embodiments, the concentration of the sugar alcohol in the pharmaceutical formulation is about 1% (w / v). In some embodiments, the sugar alcohol is derived from a monosaccharide. In some embodiments, the sugar alcohol is mannitol.

[0067] In some embodiments of the kits, uses, or methods provided herein, the concentration of the nonionic surfactant in the pharmaceutical formulation is about 0.005% to about 0.02% (w / v). In some embodiments of the kits, uses, or methods provided herein, the concentration of polysorbate 80 in the pharmaceutical formulation is about 0.01% (w / v). In some embodiments, the nonionic surfactant is polysorbate. In some embodiments of the kits, uses, or methods provided herein, the polysorbate is polysorbate 80. In some embodiments, the pH is about 6.1 to about 6.9. In some embodiments, the pH is about 6.2 to about 6.8. In some embodiments, the pH is about 6.3 to about 6.7. In some embodiments, the pH is about 6.4 to about 6.6. In some embodiments of the kits, uses, or methods provided herein, the pH is about 6.5.

[0068] In some embodiments of the kits, uses, or methods provided herein, the pharmaceutical formulation further comprises water. In some embodiments of the kits, uses, or methods provided herein, the water is USP Water for Injection.

[0069] In some embodiments of the kits, uses, or methods provided herein, the pharmaceutical formulation comprises a heterodimeric Fc-fusion protein at a bulk concentration of about 1 g / L to about 10 g / L. In some embodiments, the pharmaceutical formulation comprises a heterodimeric Fc-fusion protein at a bulk concentration of about 2 g / L to about 8 g / L. In some embodiments, the pharmaceutical formulation comprises a heterodimeric Fc-fusion protein at a bulk concentration of about 4 g / L to about 6 g / L. In some embodiments, the pharmaceutical formulation comprises a heterodimeric Fc-fusion protein at a bulk concentration of about 5 g / L. In some embodiments, the pharmaceutical formulation comprises a protein administration concentration of about 0.5 g / L to about 1.5 g / L. In some embodiments, the pharmaceutical formulation comprises a protein administration concentration of about 0.75 g / L to about 1.25 g / L. In some embodiments, the pharmaceutical formulation comprises a protein administration concentration of about 1 g / L.

[0070] In some embodiments of the kits, uses, or methods provided herein, the pharmaceutical formulation is designed to be stored at a temperature between 2° C. and 8° C. In some embodiments, the pharmaceutical formulation is a clear, colorless solution and is free of visible particulate matter.

[0071] In some embodiments of the kits, uses, or methods provided herein, the pharmaceutical formulation has a T of greater than about 60° C., greater than about 61° C., greater than about 62° C., greater than about 63° C., greater than about 64° C., greater than about 65° C., or greater than about 66° C., as measured by differential scanning fluorimetry. m1 and / or a T of greater than about 70°C, greater than about 71°C, greater than about 72°C, greater than about 73°C, greater than about 74°C, greater than about 75°C, greater than about 76°C, or greater than about 77°C m2 In some embodiments, the formulation has a thermal stability profile as defined by a T of about 67.0°C. m1 and a T of approximately 77.3°C m2 The thermostability profile is as defined by:

[0072] In some embodiments of the kits, uses, or methods provided herein, T m1 and / or T m2 The thermal stability profile of a pharmaceutical formulation, as defined by, changes by less than about 2° C. or less than about 1° C. when the pharmaceutical formulation is incubated at 50° C. for 1 week compared to the same pharmaceutical formulation incubated at 5° C. for 1 week, as measured by differential scanning fluorimetry. In some embodiments, the formulation has a T of greater than 60° C., greater than about 61° C., greater than about 62° C., greater than about 63° C., greater than about 64° C., greater than about 65° C., greater than about 66° C., or greater than about 67° C., as measured by differential scanning fluorimetry. agg In some embodiments, the thermostability profile is as defined by T aggThe thermal stability profile of a pharmaceutical formulation, as defined by, changes by less than about 2°C or less than about 1°C when the pharmaceutical formulation is incubated at 50°C for 1 week compared to the same pharmaceutical formulation incubated at 5°C for 1 week as measured by differential scanning fluorimetry.

[0073] In some embodiments of the kits, uses, or methods provided herein, after incubating the pharmaceutical formulation for 1 week at 5° C., the pH of the pharmaceutical formulation does not change by more than a pH value of about 0.2 or about 0.1. In some embodiments, after incubating the pharmaceutical formulation for 1 week at 50° C., the pH of the pharmaceutical formulation does not change by more than a pH value of about 0.2 or about 0.1.

[0074] In some embodiments of the kits, uses, or methods provided herein, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 15 nm, less than about 14 nm, less than about 13 nm, or less than about 12 nm, as measured by dynamic light scattering at 25° C. In some embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of about 11.6 nm.

[0075] In some embodiments of the kits, uses, or methods provided herein, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 20 nm, less than about 19 nm, less than about 18 nm, less than about 17 nm, less than about 16 nm, or less than about 15 nm, as measured by dynamic light scattering at 25° C. after incubating the pharmaceutical formulation for 2 weeks at 50° C. In some embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of about 14.4 nm.

[0076] In some embodiments of the kits, uses, or methods provided herein, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 20 nm, less than about 19 nm, less than about 18 nm, less than about 17 nm, or less than about 16 nm, as measured by dynamic light scattering at 25° C. after the pharmaceutical formulation has been subjected to five freeze-thaw cycles. In some embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of about 15.3 nm.

[0077] In some embodiments of the kits, uses, or methods provided herein, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.30, less than about 0.29, less than about 0.28, or less than about 0.27, as measured by dynamic light scattering at 25° C. In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is about 0.26.

[0078] In some embodiments of the kits, uses, or methods provided herein, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.30, less than about 0.29, less than about 0.28, less than about 0.27, or less than about 0.26 when measured by dynamic light scattering at 25° C. after incubating the pharmaceutical formulation for 2 weeks at 50° C. In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is about 0.25.

[0079] In some embodiments of the kits, uses, or methods provided herein, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.40, less than about 0.35, or less than about 0.34, as measured by dynamic light scattering at 25° C. after the pharmaceutical formulation has been subjected to five freeze-thaw cycles. In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is about 0.33.

[0080] In some embodiments of the kits, uses, or methods provided herein, the purity profile of the pharmaceutical formulation, as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis, is greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99%. In some embodiments, the purity profile of the pharmaceutical formulation, as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis, is greater than about 99.0%. In some embodiments, the purity profile of the pharmaceutical formulation, as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis, is greater than about 75%, greater than about 80%, greater than about 81%, greater than about 82%, greater than about 83%, greater than about 84%, or greater than about 85% after incubating the pharmaceutical formulation at 50° C. for 2 weeks. In some embodiments, the purity profile of the pharmaceutical formulation is about 85.2% as measured by the area of ​​the main peak as a percentage of the total area detected in a SEC-HPLC analysis.

[0081] In some embodiments of the kits, uses, or methods provided herein, the purity profile of the pharmaceutical formulation, after subjecting the pharmaceutical formulation to five freeze-thaw cycles, is greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, or greater than about 98%, as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis. In some embodiments, the purity profile of the pharmaceutical formulation, when measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis, is about 98.9%.

[0082] In summary, the present invention provides heterodimeric Fc-fusion protein constructs of multisubunit proteins that may exhibit longer serum half-lives, improved yields during production, increased stability during storage, and / or improved efficacy when used as therapeutic agents compared to native / native multisubunit proteins. [Brief explanation of the drawings]

[0083] [Figure 1-1]

[0023] Figure 1 illustrates various features of an exemplary heterodimeric Fc-fusion protein comprising a first subunit of a multisubunit protein linked by a linker to a first antibody Fc domain polypeptide and a second, different subunit of the multisubunit protein linked by another linker to a second antibody Fc domain polypeptide, where the subunits are linked by two disulfide bonds. Figure 1A shows a general schematic illustrating the various components of an exemplary heterodimeric Fc-fusion protein. Figure 1B shows an exemplary heterodimeric Fc-fusion protein comprising the IL12 subunits p40 and p35, a linker, and an Fc domain having a mutation. Figure 1C shows a schematic illustrating exemplary mutations that may be present in the heterodimeric Fc-fusion protein of Figure 1A or Figure 1B. Figure 1D shows a schematic illustrating exemplary disulfide bonds that may form in the heterodimeric Fc-fusion protein of Figure 1A, Figure 1B, or Figure 1C. [Figure 1-2]

[0023] Figure 1 illustrates various features of an exemplary heterodimeric Fc-fusion protein comprising a first subunit of a multisubunit protein linked by a linker to a first antibody Fc domain polypeptide and a second, different subunit of the multisubunit protein linked by another linker to a second antibody Fc domain polypeptide, where the subunits are linked by two disulfide bonds. Figure 1A shows a general schematic illustrating the various components of an exemplary heterodimeric Fc-fusion protein. Figure 1B shows an exemplary heterodimeric Fc-fusion protein comprising the IL12 subunits p40 and p35, a linker, and an Fc domain having a mutation. Figure 1C shows a schematic illustrating exemplary mutations that may be present in the heterodimeric Fc-fusion protein of Figure 1A or Figure 1B. Figure 1D shows a schematic illustrating exemplary disulfide bonds that may form in the heterodimeric Fc-fusion protein of Figure 1A, Figure 1B, or Figure 1C. [Figure 2-1]Graphs depicting tumor growth curves for individual mice inoculated with CT26 tumor cells and treated weekly with recombinant murine IL-12 (rmIL-12) (FIG. 2A), DF-mIL-12-Fc wt (FIG. 2B), DF-mIL-12-Fc si (FIG. 2C), or mIgG2a isotype control. [Figure 2-2] Graphs depicting tumor growth curves for individual mice inoculated with CT26 tumor cells and treated weekly with recombinant murine IL-12 (rmIL-12) (FIG. 2A), DF-mIL-12-Fc wt (FIG. 2B), DF-mIL-12-Fc si (FIG. 2C), or mIgG2a isotype control. [Figure 3] 1 is a graph showing Kaplan-Meier survival curves for mice inoculated with CT26 tumor cells and treated weekly with rmIL-12, DF-mIL-12-Fc wt, DF-mIL-12-Fc si, or mIgG2a isotype control. [Figure 4-1] Graphs showing tumor growth curves for individual mice inoculated with CT26 tumor cells and treated weekly with 1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc wt (FIG. 4A), 1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc si (FIG. 4B), 0.1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc wt (FIG. 4C), 0.1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc si (FIG. 4D), or mIgG2a isotype control. [Figure 4-2] Graphs showing tumor growth curves for individual mice inoculated with CT26 tumor cells and treated weekly with 1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc wt (FIG. 4A), 1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc si (FIG. 4B), 0.1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc wt (FIG. 4C), 0.1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc si (FIG. 4D), or mIgG2a isotype control. [Figure 4-3]Graphs showing tumor growth curves for individual mice inoculated with CT26 tumor cells and treated weekly with 1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc wt (FIG. 4A), 1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc si (FIG. 4B), 0.1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc wt (FIG. 4C), 0.1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc si (FIG. 4D), or mIgG2a isotype control. [Figure 5] Kaplan-Meier survival curves for mice inoculated with CT26 tumor cells and treated weekly with 1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc wt, 1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc si, 0.1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc wt, 0.1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc si, or mIgG2a isotype control. [Figure 6-1] 6A-6C are graphs showing tumor growth curves for individual mice inoculated with B16F10 melanoma cells and treated weekly with rmIL-12 (FIG. 6A), DF-mIL-12-Fc wt (FIG. 6B), DF-mIL-12-Fc si (FIG. 6C), or mIgG2a isotype control. [Figure 6-2] 6A-6C are graphs showing tumor growth curves for individual mice inoculated with B16F10 melanoma cells and treated weekly with rmIL-12 (FIG. 6A), DF-mIL-12-Fc wt (FIG. 6B), DF-mIL-12-Fc si (FIG. 6C), or mIgG2a isotype control. [Figure 7] 1 is a graph showing Kaplan-Meier survival curves for mice inoculated with B16F10 melanoma cells and treated weekly with rmIL-12, DF-mIL-12-Fc wt, DF-mIL-12-Fc si, or mIgG2a isotype control. [Figure 8-1]Graphs showing tumor growth curves for individual mice inoculated with B16F10 melanoma cells and treated weekly with 0.5 μg rmIL-12 and a molar equivalent of DF-mIL-12-Fc wt (FIG. 8A), 0.5 μg rmIL-12 and a molar equivalent of DF-mIL-12-Fc si (FIG. 8B), 0.1 μg rmIL-12 and a molar equivalent of DF-mIL-12-Fc wt (FIG. 8C), 0.1 μg rmIL-12 and a molar equivalent of DF-mIL-12-Fc si (FIG. 8D), or mIgG2a isotype control. [Figure 8-2] Graphs showing tumor growth curves for individual mice inoculated with B16F10 melanoma cells and treated weekly with 0.5 μg rmIL-12 and a molar equivalent of DF-mIL-12-Fc wt (FIG. 8A), 0.5 μg rmIL-12 and a molar equivalent of DF-mIL-12-Fc si (FIG. 8B), 0.1 μg rmIL-12 and a molar equivalent of DF-mIL-12-Fc wt (FIG. 8C), 0.1 μg rmIL-12 and a molar equivalent of DF-mIL-12-Fc si (FIG. 8D), or mIgG2a isotype control. [Figure 9] Kaplan-Meier survival curves for mice inoculated with B16F10 melanoma cells and treated weekly with 0.5 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc wt, 0.5 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc si, 0.1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc wt, 0.1 μg rmIL-12 plus the molar equivalent of DF-mIL-12-Fc si, or mIgG2a isotype control. [Figure 10-1] FIG. 1 is a graph showing the IL-12 response to treatment with DF-hIL-12-Fc si (DF IL-12-Fc) or recombinant human IL-12 (rhIL-12) using a HEK-Blue IL-12 reporter assay. [Figure 10-2] FIG. 1 is a graph showing IFNγ production by peripheral blood mononuclear cells (PBMCs) in response to treatment with DF-hIL-12-Fc si (DF IL-12-Fc) and rhIL-12. [Figure 11]1 is a graph showing the relative plasma concentrations of DF-hIL-12-Fc si, rhIL-12, and IFNγ in cynomolgus monkey K2 EDTA plasma after a single intravenous administration of 10 μg / kg equimolar amounts of DF-hIL-12-Fc si or wild-type rhIL-12. [Figure 12-1] Figures 12A-12B are graphs showing the PK / PD profiles of rmIL-12 (Figure 12A) and DF-mIL-12-Fc si (Figure 12B) in naive Balb / c mice. Figure 12A shows the PK / PD profile of rmIL-12 in naive Balb / c mice, and Figure 12B shows the PK / PD profile of DF-mIL-12-Fc si in naive Balb / c mice. Serum IL-12 and IFNγ levels were analyzed by ELISA. Figure 12C shows the PK / PD profile of intravenously administered DF-mIL-12-Fc si in naive Balb / c mice. Figure 12D shows the PK / PD profile of intraperitoneally administered DF-mIL-12-Fc si in naive Balb / c mice. Figure 12E is a graph showing the PK / PD profile of subcutaneously administered DF-mIL-12-Fc si in naive Balb / c mice. Mean serum levels represent the mean ± SEM. [Figure 12-2]Figures 12A-12B are graphs showing the PK / PD profiles of rmIL-12 (Figure 12A) and DF-mIL-12-Fc si (Figure 12B) in naive Balb / c mice. Figure 12A shows the PK / PD profile of rmIL-12 in naive Balb / c mice, and Figure 12B shows the PK / PD profile of DF-mIL-12-Fc si in naive Balb / c mice. Serum IL-12 and IFNγ levels were analyzed by ELISA. Figure 12C shows the PK / PD profile of intravenously administered DF-mIL-12-Fc si in naive Balb / c mice. Figure 12D shows the PK / PD profile of intraperitoneally administered DF-mIL-12-Fc si in naive Balb / c mice. Figure 12E is a graph showing the PK / PD profile of subcutaneously administered DF-mIL-12-Fc si in naive Balb / c mice. Mean serum levels represent the mean ± SEM. [Figure 12-3] Figures 12A-12B are graphs showing the PK / PD profiles of rmIL-12 (Figure 12A) and DF-mIL-12-Fc si (Figure 12B) in naive Balb / c mice. Figure 12A shows the PK / PD profile of rmIL-12 in naive Balb / c mice, and Figure 12B shows the PK / PD profile of DF-mIL-12-Fc si in naive Balb / c mice. Serum IL-12 and IFNγ levels were analyzed by ELISA. Figure 12C shows the PK / PD profile of intravenously administered DF-mIL-12-Fc si in naive Balb / c mice. Figure 12D shows the PK / PD profile of intraperitoneally administered DF-mIL-12-Fc si in naive Balb / c mice. Figure 12E is a graph showing the PK / PD profile of subcutaneously administered DF-mIL-12-Fc si in naive Balb / c mice. Mean serum levels represent the mean ± SEM. [Figure 13-1]Figure 13 shows graphs showing tumor growth curves of B16F10 tumor-bearing mice treated with DF-mIL-12-Fc si, anti-PD-1, or a combination thereof. Mice were treated intraperitoneally with 0.5 μg isotype control or 0.5 μg DF-mIL-12-Fc si (Figure 13A), isotype control or anti-PD-1 (Figure 13B), and isotype control or DF-mIL-12-Fc si / anti-PD-1 (Figure 13C). As indicated above, animals were injected with DF-mIL-12-Fc si once weekly and anti-PD-1 twice weekly. Tumor growth was assessed for 60 days. Graphs show tumor growth curves for individual mice. [Figure 13-2] Figure 13 shows graphs showing tumor growth curves of B16F10 tumor-bearing mice treated with DF-mIL-12-Fc si, anti-PD-1, or a combination thereof. Mice were treated intraperitoneally with 0.5 μg isotype control or 0.5 μg DF-mIL-12-Fc si (Figure 13A), isotype control or anti-PD-1 (Figure 13B), and isotype control or DF-mIL-12-Fc si / anti-PD-1 (Figure 13C). As indicated above, animals were injected with DF-mIL-12-Fc si once weekly and anti-PD-1 twice weekly. Tumor growth was assessed for 60 days. Graphs show tumor growth curves for individual mice. [Figure 14] Figure 14A shows the survival rate and body weight of B16F10 tumor-bearing mice treated with DF-mIL-12-Fc si, anti-PD-1, or a combination of these. Mice were treated with isotype, DF-mIL-12-Fc si, anti-PD-1, or a combination of DF-mIL-12-Fc si and anti-PD-1. Animals were injected with 0.5 μg DF-mIL-12-Fc si once a week and 200 μg anti-PD-1 or isotype twice a week. Figure 14A shows the Kaplan-Meier survival curve. Figure 14B shows the mouse body weight as the mean ± standard deviation. [Figure 15-1]15A-C are graphs showing tumor growth curves of B16F10 tumor-bearing mice treated with DF-mIL-12-Fc si, mcFAE-C26.99 TriNKET, or a combination thereof. Mice were treated intraperitoneally with 150 μg isotype control or 0.5 μg DF-mIL-12-Fc si (FIG. 15A), isotype control or 150 μg TriNKET (FIG. 15B), and isotype control or DF-mIL-12-Fc si / TriNKET (FIG. 15C). As indicated above, animals were injected with DF-mIL-12-Fc si once weekly and TriNKET three times weekly. Tumor growth was assessed for 72 days. Graphs show tumor growth curves for individual mice. [Figure 15-2] 15A-C are graphs showing tumor growth curves of B16F10 tumor-bearing mice treated with DF-mIL-12-Fc si, mcFAE-C26.99 TriNKET, or a combination thereof. Mice were treated intraperitoneally with 150 μg isotype control or 0.5 μg DF-mIL-12-Fc si (FIG. 15A), isotype control or 150 μg TriNKET (FIG. 15B), and isotype control or DF-mIL-12-Fc si / TriNKET (FIG. 15C). As indicated above, animals were injected with DF-mIL-12-Fc si once weekly and TriNKET three times weekly. Tumor growth was assessed for 72 days. Graphs show tumor growth curves for individual mice. [Figure 16] Figure 16A shows the survival rate and body weight of B16F10 tumor-bearing mice treated with DF-mIL-12-Fc si, mcFAE-C26.99 TriNKET, or a combination of these. Mice were treated with isotype, DF-mIL-12-Fc si, TriNKET, or a combination of DF-mIL-12-Fc si and TriNKET. Animals were injected with 0.5 μg DF-mIL-12-Fc si once a week and 150 μg TriNKET or isotype three times a week. Figure 16A shows the Kaplan-Meier survival curve. Figure 16B shows the mouse body weight as the mean + standard deviation. [Figure 17]FIG. 16 is a graph showing tumor growth curves of complete responder (CR) mice from the B16F10 tumor model experiment in FIG. 15 treated with DF-mIL-12-Fc si / TriNKET combination therapy (n=3) and re-challenged by implantation of 2×10 B16F10 melanoma cells. [Figure 18-1] Graph showing tumor growth curves for individual mice inoculated with CT26 tumor cells and administered a single dose of DF-mIL-12-Fc si or mIgG2a isotype. [Figure 18-2] 1 is a graph showing the body weight±standard deviation of mice inoculated with CT26 tumor cells and administered weekly doses of DF-mIL-12-Fc si, mIgG2a isotype, or rmIL-12. [Figure 18-3] Graph showing tumor growth curves of individual re-challenged mice that were either naive or had previously been complete responders (CRs) when administered a single dose of DF-mIL-12-Fc si in the CT26 tumor model. [Figure 19] 19A and 19B are graphs showing tumor growth curves for individual mice inoculated with CT26 tumor cells and administered weekly doses of DF-mIL-12-Fc si or mIgG2a isotype either intraperitoneally (IP) (FIG. 19A) or subcutaneously (SC) (FIG. 19B). [Figure 20] 1 is a graph showing tumor growth curves for individual mice inoculated with B16F10 melanoma cells and administered a single dose of DF-mIL-12-Fc si or mIgG2a isotype. [Figure 21] 21A and 21B are graphs showing tumor growth curves for individual mice inoculated with B16F10 melanoma cells and administered weekly doses of DF-mIL-12-Fc si or mIgG2a isotype either intraperitoneally (IP) (FIG. 21A) or subcutaneously (SC) (FIG. 21B). [Figure 22] 22A and 22B are graphs showing tumor growth curves for individual mice inoculated with CT26 tumor cells and administered intraperitoneally either a single dose (FIG. 22A) or weekly doses (FIG. 22B) of DF-mIL-12-Fc si or mIgG2a isotype at the molar equivalent of 1 μg of rmlL-12. [Figure 23] Figure 23A shows graphs depicting tumor growth curves for individual mice inoculated with CT26 tumor cells and administered a single weekly dose of DF-mIL-12-Fc si subcutaneously. Figure 23A shows graphs depicting tumor growth curves for individual mice inoculated with CT26-Tyrp1 tumor cells and treated once (weekly) with either 2 μg mIgG2a isotype control or 1 μg DF-mIL-12-Fc si. Figure 23B shows graphs depicting tumor growth curves for individual mice inoculated with CT26-Tyrp1 tumor cells and treated once (weekly) with either 2 μg mIgG2a isotype control or 2 μg DF-mIL-12-Fc si. [Figure 24-1] 24A-C are graphs showing IFNγ (FIG. 24A), CXCL9 (FIG. 24B), and CXCL10 (FIG. 24C) levels in blood (left) and tumor (right) samples 72 hours after a single dose of DF-mIL-12-Fc si in C57BL / 6 mice bearing B16F10 tumors. [Figure 24-2] 24A-C are graphs showing IFNγ (FIG. 24A), CXCL9 (FIG. 24B), and CXCL10 (FIG. 24C) levels in blood (left) and tumor (right) samples 72 hours after a single dose of DF-mIL-12-Fc si in C57BL / 6 mice bearing B16F10 tumors. [Figure 25-1] 25A-C are line graphs showing the pharmacokinetics of DF-hIL-12-Fc si in cynomolgus monkeys treated with a single subcutaneous dose of 1 μg / kg (FIG. 25A), 2 μg / kg (FIG. 25B), or 4 μg / kg (FIG. 25C) of DF-hIL-12-Fc si. 2240, 2241, 2740, 2741 (FIG. 25A); 3240, 3241, 3740, 3741 (FIG. 25B); 4240, 4241, 4740, 4741 (FIG. 25C) represent individual cynomolgus monkey subjects. [Figure 25-2]25A-C are line graphs showing the pharmacokinetics of DF-hIL-12-Fc si in cynomolgus monkeys treated with a single subcutaneous dose of 1 μg / kg (FIG. 25A), 2 μg / kg (FIG. 25B), or 4 μg / kg (FIG. 25C) of DF-hIL-12-Fc si. 2240, 2241, 2740, 2741 (FIG. 25A); 3240, 3241, 3740, 3741 (FIG. 25B); 4240, 4241, 4740, 4741 (FIG. 25C) represent individual cynomolgus monkey subjects. [Figure 26-1] 26A, 26C, and 26E show IFNγ and IP10 / CXCL10 concentrations in cynomolgus monkeys treated with a single subcutaneous dose of DF-hIL-12-Fc si. Figures 26A, 26C, and 26E show IFNγ concentrations / expression levels in cynomolgus monkeys treated with 1 μg / kg, 2 μg / kg, and 4 μg / kg of DF-hIL-12-Fc si, respectively. Figures 26B, 26D, and 26F show IP10 / CXCL10 concentrations / expression levels in cynomolgus monkeys treated with 1 μg / kg, 2 μg / kg, and 4 μg / kg of DF-hIL-12-Fc si, respectively. 1240, 1740, 2240, 2241, 2740, 2741 (Figures 26A-B); 1240, 1740, 3240, 3241, 3740, 3741 (Figures 26C, 26D, 26F); 1240, 1740, 4240, 4241, 4740, 4741 (Figure 26E) represent individual cynomolgus monkey subjects. [Figure 26-2]26A, 26C, and 26E show IFNγ and IP10 / CXCL10 concentrations in cynomolgus monkeys treated with a single subcutaneous dose of DF-hIL-12-Fc si. Figures 26A, 26C, and 26E show IFNγ concentrations / expression levels in cynomolgus monkeys treated with 1 μg / kg, 2 μg / kg, and 4 μg / kg of DF-hIL-12-Fc si, respectively. Figures 26B, 26D, and 26F show IP10 / CXCL10 concentrations / expression levels in cynomolgus monkeys treated with 1 μg / kg, 2 μg / kg, and 4 μg / kg of DF-hIL-12-Fc si, respectively. 1240, 1740, 2240, 2241, 2740, 2741 (Figures 26A-B); 1240, 1740, 3240, 3241, 3740, 3741 (Figures 26C, 26D, 26F); 1240, 1740, 4240, 4241, 4740, 4741 (Figure 26E) represent individual cynomolgus monkey subjects. [Figure 26-3] 26A, 26C, and 26E show IFNγ and IP10 / CXCL10 concentrations in cynomolgus monkeys treated with a single subcutaneous dose of DF-hIL-12-Fc si. Figures 26A, 26C, and 26E show IFNγ concentrations / expression levels in cynomolgus monkeys treated with 1 μg / kg, 2 μg / kg, and 4 μg / kg of DF-hIL-12-Fc si, respectively. Figures 26B, 26D, and 26F show IP10 / CXCL10 concentrations / expression levels in cynomolgus monkeys treated with 1 μg / kg, 2 μg / kg, and 4 μg / kg of DF-hIL-12-Fc si, respectively. 1240, 1740, 2240, 2241, 2740, 2741 (Figures 26A-B); 1240, 1740, 3240, 3241, 3740, 3741 (Figures 26C, 26D, 26F); 1240, 1740, 4240, 4241, 4740, 4741 (Figure 26E) represent individual cynomolgus monkey subjects. [Figure 27] Graph showing tumor growth curves for individual mice inoculated with breast cancer cells and administered weekly doses of monotherapy (isotype control, DF-mIL-12-Fc si, Doxil® (chemotherapy), or radiation at 10 Gy) or combination therapy (DF-mIL-12-Fc si in combination with Doxil® or radiation). [Figure 28-1] Figure 28A is a graph showing tumor growth curves for individual mice inoculated with CT26-Tyrp1 tumor cells and treated (every other week) with either an isotype control or an anti-PD-1 antibody. Figure 28B is a graph showing tumor growth curves for Balb / c mice inoculated with CT26-Tyrp1 tumor cells and treated (every other week) with either an isotype control or an anti-PD-1 antibody in addition to weekly treatment with 1 μg of DF-mIL-12-Fc si. [Figure 28-2] Figure 28A is a graph showing tumor growth curves for individual mice inoculated with CT26-Tyrp1 tumor cells and treated (every other week) with either an isotype control or an anti-PD-1 antibody. Figure 28B is a graph showing tumor growth curves for Balb / c mice inoculated with CT26-Tyrp1 tumor cells and treated (every other week) with either an isotype control or an anti-PD-1 antibody in addition to weekly treatment with 1 μg of DF-mIL-12-Fc si. [Figure 29] Figure 29A is a graph showing tumor growth curves of treated (Tr) tumors in individual mice inoculated with CT26-Tyrp1 tumor cells and treated intratumorally once (weekly) with either isotype control or DF-mIL-12-Fc si. Figure 29B is a graph showing tumor growth curves of untreated (NT) CT26-Tyrp1 tumors in individual mice described in Figure 29A. [Figure 30] Figure 30A is a graph showing tumor growth curves for individual mice inoculated with CT26-Tyrp1 tumor cells and treated once with either 2 μg mIgG2a isotype control or 2 μg DF-mIL-12-Fc si. Figure 30B is a graph showing mean tumor growth curves for individual mice inoculated with CT26-Tyrp1 tumor cells and treated with 2 μg mIgG2a isotype control, 1 μg DF-mIL-12-Fc si (weekly), 2 μg DF-mIL-12-Fc si (weekly), or 2 μg DF-mIL-12-Fc si (single). [Figure 31-1]Figure 31A is a graph showing IFNγ production after PHA stimulation of PBMCs treated with DF hIL-12-Fc-si with the L234A, L235A, and P329A mutations (LALAPA) or the L234A, L235A, and P329G mutations (LALAPG). Figure 31B shows flow cytometry histograms of binding by fluorophore-conjugated hIgG1 to THP-1 cells in the presence or absence of DF hIL-12-Fc-si with the LALAPA or LALAPG mutations. [Figure 31-2] Figure 31A is a graph showing IFNγ production after PHA stimulation of PBMCs treated with DF hIL-12-Fc-si with the L234A, L235A, and P329A mutations (LALAPA) or the L234A, L235A, and P329G mutations (LALAPG). Figure 31B shows flow cytometry histograms of binding by fluorophore-conjugated hIgG1 to THP-1 cells in the presence or absence of DF hIL-12-Fc-si with the LALAPA or LALAPG mutations. [Figure 32] FIG. 1 is a process flow diagram showing the steps for preparing DF hIL-12-Fc si. [Figure 33] FIG. 1 is a process flow diagram showing the steps for preparing a pharmaceutical formulation containing DF-hIL-12-Fc si. [Figure 34] Figure 34A is a graph showing the ultraviolet-visible spectroscopy (UV-Vis) calculated concentration of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers, and Figure 34B is a graph showing the pH of various pharmaceutical formulations containing DF-IL-12-Fc si. [Figure 35] 35A and 35B are photographs of the appearance of certain pharmaceutical formulations containing DF-hIL-12-Fc si after one week of incubation at 5° C. (FIG. 35A) and 50° C. (FIG. 35B). [Figure 36-1] 36A and 36B are graphs showing the mean Tm1 (FIG. 36A) and Tm2 (FIG. 36B) of DF-hIL-12-Fc si in various pharmaceutical formulations after 1 week of incubation at 5° C. [Figure 36-2] Graphs showing the mean Tm1 (FIG. 36C) and Tm2 (FIG. 36D) of DF-hIL-12-Fc si in various pharmaceutical formulations after 1 week of incubation at 50° C. [Figure 36-3] 36E-36F) and 50°C (Figures 36G-36H). [Figure 36-4] 36E-36F) and 50°C (Figures 36G-36H). [Figure 37-1] 37A and 37B are graphs showing the mean Tag of DF-hIL-12-Fc si in various pharmaceutical formulations after one week of incubation at 5° C. (FIG. 37A) and 50° C. (FIG. 37B). [Figure 37-2] 37C-37D) and 50°C (Figures 37E-37F) show representative DSF aggregation curves of DF-hIL-12-Fc si in various pharmaceutical formulations after 1 week of incubation at 5°C (Figures 37C-37D) and 50°C (Figures 37E-37F). [Figure 37-3] 37C-37D) and 50°C (Figures 37E-37F) show representative DSF aggregation curves of DF-hIL-12-Fc si in various pharmaceutical formulations after 1 week of incubation at 5°C (Figures 37C-37D) and 50°C (Figures 37E-37F). [Figure 38] 38A and 38B are graphs showing the calculated UV-Vis concentration of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers after one week of incubation at 5° C. (FIG. 38A) and 50° C. (FIG. 38B). [Figure 39] 39A and 39B are graphs showing the pH of various pharmaceutical formulations containing DF-hIL-12-Fc si after one week of incubation at 5° C. (FIG. 39A) and 50° C. (FIG. 39B). [Figure 40-1]Figure 40A shows the Z-average hydrodynamic diameter (Figure 40A) and polydispersity index (Figure 40B) of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers after 1 week of incubation at 5° C. Figures 40C-40D show the average monomer diameter and average monomer fraction %Pd of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers after 1 week of incubation at 5° C. [Figure 40-2] Figure 40A shows the Z-average hydrodynamic diameter (Figure 40A) and polydispersity index (Figure 40B) of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers after 1 week of incubation at 5° C. Figures 40C-40D show the average monomer diameter and average monomer fraction %Pd of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers after 1 week of incubation at 5° C. [Figure 40-3] Figures 40E-40F are graphs showing the Z-average hydrodynamic diameter (Figure 40E) and polydispersity index (Figure 40F) of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers after 1 week of incubation at 50° C. Figures 40G-40H are graphs showing the average monomer diameter (Figure 40G) and average monomer fraction %Pd (Figure 40H) of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers after 1 week of incubation at 50° C. [Figure 40-4] Figures 40E-40F are graphs showing the Z-average hydrodynamic diameter (Figure 40E) and polydispersity index (Figure 40F) of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers after 1 week of incubation at 50° C. Figures 40G-40H are graphs showing the average monomer diameter (Figure 40G) and average monomer fraction %Pd (Figure 40H) of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers after 1 week of incubation at 50° C. [Figure 40-5]Figures 40I-40J show representative dynamic light scattering (DLS) traces used to calculate the data depicted in Figures 40A-40D, and Figures 40K-40L show representative DLS traces used to calculate the data depicted in Figures 40E-40H. [Figure 40-6] Figures 40I-40J show representative dynamic light scattering (DLS) traces used to calculate the data depicted in Figures 40A-40D, and Figures 40K-40L show representative DLS traces used to calculate the data depicted in Figures 40E-40H. [Figure 41] FIG. 1 is a graph showing the % purity of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers after 1 week of incubation at 50° C., as measured by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). [Figure 42] FIG. 1 is a graph showing the % purity of DF-hIL-12-Fc si in various pharmaceutical formulations containing different buffers after 1 week of incubation at 50° C., as measured by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). [Figure 43] Figure 43A is a graph showing the calculated UV-Vis concentration of 1 mg / mL DF hIL-12-Fc-si in various pharmaceutical formulations containing different buffers and excipients. With reference to Figure 43A, 1 represents buffer exchange; 2 represents 2-8°C; 3 represents 50°C; and 4 represents freeze / thaw. Figure 43B is a graph showing the calculated UV-Vis concentration of 10 mg / mL DF hIL-12-Fc-si in various pharmaceutical formulations containing different buffers and excipients. With reference to Figure 43B: 1 represents buffer exchange; 2 represents 2-8°C; 3 represents 50°C; and 4 represents freeze / thaw. [Figure 44-1] 44A and 44B show the mean Tm1 (FIGS. 44A and 44B) and Tm2 (FIGS. 44C and 44D) of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations. [Figure 44-2]44A and 44B show the mean Tm1 (FIGS. 44A and 44B) and Tm2 (FIGS. 44C and 44D) of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations. [Figure 44-3] 44A and 44B show the mean Tm1 (FIGS. 44A and 44B) and Tm2 (FIGS. 44C and 44D) of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations. [Figure 45-1] Representative DSF melting curves of DF hIL-12-Fc-si in various pharmaceutical formulations are shown. [Figure 45-2] Representative DSF melting curves of DF hIL-12-Fc-si in various pharmaceutical formulations are shown. [Figure 46-1] 46A and 46B are graphs showing the mean Tagg of DF hIL-12-Fc-si at 1 mg / mL (FIG. 46A) and 10 mg / mL (FIG. 46B) in various pharmaceutical formulations. [Figure 46-2] Representative DSF aggregation curves of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations. [Figure 46-3] Representative DSF aggregation curves of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations. [Figure 47-1] Representative DLS traces showing the size distribution of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations subjected to different stress conditions are shown. [Figure 47-2] Representative DLS traces showing the size distribution of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations subjected to different stress conditions are shown. [Figure 48-1]Figures 48A-48H show the Z-average diameter (Figures 48A-48B), polydispersity index (PDI) (Figures 48C-48D), monomer diameter (48E-48F), and monomer %Pd (Figures 48G-48H) of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations subjected to different stress conditions. In Figures 48A-48H, 1 represents 2-8°C, 2 represents 50°C, and 3 represents freeze-thaw. [Figure 48-2] Figures 48A-48H show the Z-average diameter (Figures 48A-48B), polydispersity index (PDI) (Figures 48C-48D), monomer diameter (48E-48F), and monomer %Pd (Figures 48G-48H) of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations subjected to different stress conditions. In Figures 48A-48H, 1 represents 2-8°C, 2 represents 50°C, and 3 represents freeze-thaw. [Figure 48-3] Figures 48A-48H show the Z-average diameter (Figures 48A-48B), polydispersity index (PDI) (Figures 48C-48D), monomer diameter (48E-48F), and monomer %Pd (Figures 48G-48H) of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations subjected to different stress conditions. In Figures 48A-48H, 1 represents 2-8°C, 2 represents 50°C, and 3 represents freeze-thaw. [Figure 48-4] Figures 48A-48H show the Z-average diameter (Figures 48A-48B), polydispersity index (PDI) (Figures 48C-48D), monomer diameter (48E-48F), and monomer %Pd (Figures 48G-48H) of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations subjected to different stress conditions. In Figures 48A-48H, 1 represents 2-8°C, 2 represents 50°C, and 3 represents freeze-thaw. [Figure 49-1]49A-49D are graphs showing the purity of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations subjected to different stress conditions. Figures 49A-49B show the %Main peak after incubation at 50°C. Figures 49C-49D show the %Main peak after incubation at 2-8°C or after freeze-thaw cycles. Referring to Figures 49C-49D, 1 represents 2-8°C, and 2 represents freeze-thaw. [Figure 49-2] 49A-49D are graphs showing the purity of DF hIL-12-Fc-si at 1 mg / mL and 10 mg / mL in various pharmaceutical formulations subjected to different stress conditions. Figures 49A-49B show the %Main peak after incubation at 50°C. Figures 49C-49D show the %Main peak after incubation at 2-8°C or after freeze-thaw cycles. Referring to Figures 49C-49D, 1 represents 2-8°C, and 2 represents freeze-thaw. [Figure 50-1] 5A-5H are graphs showing particle counts by size in various pharmaceutical formulations containing DF hIL-12-Fc-si subjected to different stress conditions, as measured by high accuracy liquid particle (HIAC) analysis. Figures 50A-50B show counts of particles ≧2 μm, Figures 50C-50D show counts of particles ≧5 μm, Figures 50E-50F show counts of particles ≧10 μm, and Figures 50G-50H show counts of particles ≧25 μm. Referring to Figures 50A-50H, 1 represents 2-8°C, 2 represents 50°C, and 3 represents freeze-thaw. [Figure 50-2] 5A-5H are graphs showing particle counts by size in various pharmaceutical formulations containing DF hIL-12-Fc-si subjected to different stress conditions, as measured by high accuracy liquid particle (HIAC) analysis. Figures 50A-50B show counts of particles ≧2 μm, Figures 50C-50D show counts of particles ≧5 μm, Figures 50E-50F show counts of particles ≧10 μm, and Figures 50G-50H show counts of particles ≧25 μm. Referring to Figures 50A-50H, 1 represents 2-8°C, 2 represents 50°C, and 3 represents freeze-thaw. [Figure 50-3]5A-5H are graphs showing particle counts by size in various pharmaceutical formulations containing DF hIL-12-Fc-si subjected to different stress conditions, as measured by high accuracy liquid particle (HIAC) analysis. Figures 50A-50B show counts of particles ≧2 μm, Figures 50C-50D show counts of particles ≧5 μm, Figures 50E-50F show counts of particles ≧10 μm, and Figures 50G-50H show counts of particles ≧25 μm. Referring to Figures 50A-50H, 1 represents 2-8°C, 2 represents 50°C, and 3 represents freeze-thaw. [Figure 50-4] 5A-5H are graphs showing particle counts by size in various pharmaceutical formulations containing DF hIL-12-Fc-si subjected to different stress conditions, as measured by high accuracy liquid particle (HIAC) analysis. Figures 50A-50B show counts of particles ≧2 μm, Figures 50C-50D show counts of particles ≧5 μm, Figures 50E-50F show counts of particles ≧10 μm, and Figures 50G-50H show counts of particles ≧25 μm. Referring to Figures 50A-50H, 1 represents 2-8°C, 2 represents 50°C, and 3 represents freeze-thaw. [Figure 51] FIG. 51 is a schematic diagram showing the Phase 1 and Phase 2 study design in which DF hIL-12-Fc-si will be used as monotherapy (FIG. 51A) and in combination therapy with pembrolizumab (FIG. 51B). [Figure 52] FIG. 52B is a schematic diagram showing the Phase 1 and Phase 2 study design in which DF hIL-12-Fc-si will be used as monotherapy (FIG. 52A) and in combination therapy with nivolumab (FIG. 52B). DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention provides improved heterodimeric Fc-fusion proteins, pharmaceutical formulations comprising such proteins, and therapeutic methods using such proteins and pharmaceutical formulations, including methods for the treatment of cancer.

[0085] To facilitate the understanding of this invention, a number of terms and phrases are defined below.

[0086] The terms "a" and "an" as used herein mean "one or more" and include the plural forms unless the context requires otherwise.

[0087] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, etc.), and more preferably humans.

[0088] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present invention) sufficient to produce a beneficial or desired result (e.g., a desired prophylactic or therapeutic effect). An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treating" includes any effect that results in the improvement of a condition, disease, disorder, etc., such as alleviation, reduction, modulation, amelioration, or elimination, or the alleviation of symptoms thereof.

[0089] As used herein, the term "pharmaceutical formulation" refers to a combination of an active agent with an inert or active carrier that makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0090] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), and various wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0091] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention that, upon administration to a subject, is capable of providing the compound of the present invention or its active metabolite or residue. As known to those of skill in the art, "salts" of the compounds of the present invention can be derived from inorganic or organic acids and bases. Exemplary acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene 2-sulfonic acid, benzenesulfonic acid, and the like. Other acids, such as oxalic acid, even though not themselves pharmaceutically acceptable, may be utilized in the preparation of salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

[0092] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and bases of formula NW4 + (Wherein W is C 1~4 Examples include compounds in which the aryl group is alkyl.

[0093] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include Na + , NH4 + , and NW4 + (Wherein W is C 1~4 Examples of suitable cations include anions of the compounds of the present invention that are compounded with suitable cations such as alkyl groups.

[0094] For therapeutic use, the salts of compounds of the invention are contemplated as pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0095] Throughout this description, when compositions are described as having, comprising, or including particular ingredients, or when processes and methods are described as having, comprising, or including particular steps, it is contemplated that there are compositions of the invention that consist essentially of, or consist of, the recited ingredients, and that there are processes and methods of the invention that consist essentially of, or consist of, the recited processing steps.

[0096] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Furthermore, if a variable is not accompanied by a definition, the previous definition of that variable takes precedence.

[0097] (a) Protein The present invention provides Fc-fusion protein constructs comprising the amino acid sequences of multisubunit proteins. These fusion protein constructs may exhibit longer serum half-lives, improved yields during production, increased stability during storage, and / or improved efficacy when used as therapeutic agents compared to native / native multisubunit proteins.

[0098] (i) IgG1 Fc fusion protein In one aspect, the invention provides a heterodimeric IgG1 Fc fusion protein comprising a first polypeptide comprising a first antibody IgG1 Fc domain polypeptide and a second polypeptide comprising a second antibody IgG1 Fc domain polypeptide linked to the first antibody Fc domain, wherein the first polypeptide further comprises a first subunit of a multi-subunit protein fused to the first antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO:237 or SEQ ID NO:6; a second, different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide, and the subunits of the multi-subunit protein are linked to each other; and when X1 in SEQ ID NO:237 or SEQ ID NO:6 represents L and / or X2 in SEQ ID NO:237 or SEQ ID NO:6 represents L, at least one of the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide comprises a Q347R mutation to promote heterodimerization.

[0099] In some embodiments, the linker joining the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide within the heterodimeric Fc fusion protein consists of the amino acid sequence of SEQ ID NO:237 or SEQ ID NO:6.

[0100] In certain embodiments, the linker connecting the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide further comprises a spacer peptide, hi certain embodiments, the linker comprises the sequence of SEQ ID NO: 237 or SEQ ID NO: 6 and a spacer peptide.

[0101] In certain embodiments, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker comprising the sequence of SEQ ID NO: 237 or SEQ ID NO: 6 and a spacer peptide. In certain embodiments, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 237 or SEQ ID NO: 6. In certain embodiments, the amino acid sequence of the linker connecting the second different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide is identical to the amino acid sequence of the linker connecting the subunit of the multi-subunit protein to the first antibody Fc domain polypeptide.

[0102] Any spacer peptide described under the heading "Spacer Peptides" can be utilized. For example, in certain embodiments, the spacer peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 107-120. In certain embodiments, the spacer peptide consists of the amino acid sequence set forth in any one of SEQ ID NOs: 107-120. In certain embodiments, the linker linking a subunit of a multi-subunit protein to a first antibody Fc domain polypeptide consists of, or consists essentially of, a spacer peptide disclosed herein and a peptide having the sequence of SEQ ID NO: 237 or SEQ ID NO: 6. In certain embodiments, the linker linking a second, different subunit of a multi-subunit protein to a second antibody Fc domain polypeptide consists of, or consists essentially of, a spacer peptide disclosed herein and a peptide having the sequence of SEQ ID NO: 237 or SEQ ID NO: 6. In certain embodiments, the spacer peptide is N-terminal to either or both linkers.

[0103] In some embodiments, the linker joining the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc-fusion protein comprises the amino acid sequence of SEQ ID NO: 239 or SEQ ID NO: 9. In some embodiments, the linker joining the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc-fusion protein consists of the amino acid sequence of SEQ ID NO: 239 or SEQ ID NO: 9.

[0104] In some embodiments, the linker joining the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 239. In some embodiments, the linker joining the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc fusion protein consists of the amino acid sequence SEQ ID NO: 239.

[0105] In some embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 244. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 244.

[0106] In some embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 10.

[0107] In some embodiments, the linker joining a subunit of a multi-subunit protein to a first antibody Fc domain polypeptide in a heterodimeric Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 238 or SEQ ID NO: 7. In some embodiments, the linker fusing a first subunit of a multi-subunit protein to a first antibody Fc domain polypeptide in a heterodimeric Fc fusion protein consists of the amino acid sequence of SEQ ID NO: 238 or SEQ ID NO: 7.

[0108] In some embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 241. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 241.

[0109] In some embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 242. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 242.

[0110] In some embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 243. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 243.

[0111] In some embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 245. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 245.

[0112] In some embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 246. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 66 or SEQ ID NO: 246.

[0113] In some embodiments, the linker joining the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 240. In some embodiments, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc fusion protein consists of the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 240.

[0114] In some embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 247. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 247.

[0115] In some embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 248. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 248.

[0116] In some embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 249. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 249.

[0117] In certain embodiments, the Fc domain polypeptide is that of an IgG1 Fc. In some embodiments, the protein of the present invention comprises a first antibody Fc domain polypeptide and a second antibody Fc domain polypeptide, both of which are mutated IgG1 Fc domain polypeptides that promote heterodimerization with each other. For example, when the Fc domain is derived from the Fc of human IgG1, the Fc domain may comprise an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody and may differ at one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439.

[0118] In some embodiments, the antibody constant domain may comprise an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody, and may include any of the following amino acids: Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, They may differ by one or more substitutions selected from the group consisting of T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E. All amino acid positions in the Fc domain or hinge region disclosed herein are numbered according to EU numbering.

[0119] In some embodiments, the first antibody IgG1 Fc domain polypeptide comprises one or more mutations selected from K360E and K409W, and the second antibody IgG1 Fc domain polypeptide comprises one or more mutations selected from Q347R, D399V, and F405T. In some embodiments, the first antibody IgG1 Fc domain polypeptide comprises one or more mutations selected from Q347R, D399V, and F405T, and the second antibody IgG1 Fc domain polypeptide comprises one or more mutations selected from K360E and K409W. In some embodiments, the first antibody IgG1 Fc domain polypeptide comprises mutations K360E and K409W, and the second antibody IgG1 Fc domain polypeptide comprises mutations Q347R, D399V, and F405T. In some embodiments, the first antibody IgG1 Fc domain polypeptide comprises the mutations Q347R, D399V, and F405T, and the second antibody IgG1 Fc domain polypeptide comprises the mutations K360E and K409W.

[0120] In some embodiments, heterodimeric Fc fusion proteins of the invention with IgG1 Fc comprise one or more mutations to reduce binding of the first and / or second polypeptide to an FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB) or a complement component (e.g., C1q). Such mutations are useful for reducing effector function. For example, proteins of the present disclosure comprise L234A and L235A mutations; L234A, L235A, and P329A mutations; L234A, L235A, and P329G mutations; or L234A, L235E, G237A, A330S, and P331S mutations.

[0121] In some embodiments, heterodimeric Fc-fusion proteins of the invention comprise a first antibody IgG4 or IgG1 Fc domain polypeptide and a second antibody IgG4 or IgG1 Fc domain polypeptide, each comprising the mutation P329G or P329A. In a specific embodiment, heterodimeric Fc-fusion proteins of the invention comprise a first antibody IgG4 or IgG1 Fc domain polypeptide and a second antibody IgG4 or IgG1 Fc domain polypeptide, each comprising the mutation P329A.

[0122] In some embodiments, the first IgG1 antibody Fc domain polypeptide and the second, different IgG1 antibody Fc domain polypeptide each have a mutation selected from A330S and P331S, hi some embodiments, the first IgG1 antibody Fc domain polypeptide and the second, different IgG1 antibody Fc domain polypeptide each contain the mutations A330S and P331S.

[0123] In certain embodiments, an additional disulfide bond is introduced between the IgG1 Fc monomers, which improves the stability of the heterodimer. In an exemplary embodiment, a first antibody Fc domain polypeptide fused to a first subunit of a multi-subunit protein contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with a S354C substitution on a second antibody Fc domain polypeptide fused to a second, different subunit of the multi-subunit protein. Alternatively, a first antibody Fc domain polypeptide fused to a first subunit of a multi-subunit protein contains a S354C substitution in the CH3 domain, which forms a disulfide bond with a Y349C substitution on a second antibody Fc domain polypeptide fused to a second, different subunit of the multi-subunit protein.

[0124] Any of the IgG1 antibody Fc domain polypeptides provided in Table 2 below can be utilized in combination with any of the IgG1 hinge sequences provided in Table 1 below (which in the present invention are part of, or the entirety of, a linker joining the protein sequence of a first subunit of a multi-subunit protein to a first IgG1 antibody Fc domain polypeptide, or a linker joining an additional subunit to a second, different IgG1 antibody Fc domain polypeptide). Exemplary IgG1 hinge-Fc domain polypeptides are provided in Table 3 below. In certain embodiments, the first and second polypeptides of the Fc fusion protein comprise the amino acid sequences of SEQ ID NOs: 212 and 212; 213 and 214; 215 and 216; 217 and 218; 214 and 213; 216 and 215; or 218 and 217, respectively. In certain embodiments, the first and second polypeptides of the Fc-fusion protein are, respectively, SEQ ID NOs: 228 and 228; 229 and 230; 231 and 232; 233 and 234; 235 and 236; 230 and 229; 232 and 231; 234 and 233; 236 and 235; 228 and 250; 250 and 228; 250 and 250; 2 255 and 234; 234 and 255; 233 and 256; 256 and 233; 257 and 236; 236 and 257; 258 and 235; or 235 and 258.

[0125] (ii) IgG4 Fc fusion protein In one aspect, the present invention provides an improved multi-subunit protein. In one aspect, the present invention provides a heterodimeric IgG4 Fc fusion protein comprising a first polypeptide comprising a first antibody IgG4 Fc domain polypeptide and a second polypeptide comprising a second, different antibody IgG4 Fc domain polypeptide linked to the first antibody Fc domain polypeptide, wherein the first polypeptide further comprises a first subunit of a multi-subunit protein fused to the first antibody IgG4 Fc domain polypeptide; a second, different subunit of the multi-subunit protein fused to the second antibody IgG4 Fc domain polypeptide, and these subunits of the multi-subunit protein are linked to each other; and the first antibody Fc domain polypeptide and the second antibody IgG4 Fc domain polypeptide each have a different mutation that promotes heterodimerization.

[0126] In some embodiments, in the heterodimeric IgG4 Fc fusion protein, the linker joining the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of the amino acid sequence of SEQ ID NO:1.

[0127] In certain embodiments, the linker connecting the protein sequence of the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide further comprises a spacer peptide, hi certain embodiments, the linker comprises the sequence of SEQ ID NO: 1 and a spacer peptide.

[0128] In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the sequence of SEQ ID NO: 1 and a spacer peptide. In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the amino acid sequence of the linker connecting the second different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide is identical to the amino acid sequence of the linker connecting the subunit of the multi-subunit protein to the first antibody Fc domain polypeptide.

[0129] Any spacer peptide described under the heading "Spacer Peptides" can be utilized. For example, in certain embodiments, the spacer peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 107-120. In certain embodiments, the spacer peptide consists of the amino acid sequence set forth in any one of SEQ ID NOs: 107-120. In certain embodiments, the linker linking the subunits of the multi-subunit protein to the first antibody Fc domain polypeptide consists of, or consists essentially of, a spacer peptide disclosed herein and SEQ ID NO: 1. In certain embodiments, the linker consists of, or consists essentially of a spacer peptide disclosed herein and SEQ ID NO: 1. In certain embodiments, the spacer peptide is N-terminal to the first linker and / or the second linker.

[0130] In some embodiments, the linker joining a subunit of a multi-subunit protein to a first antibody Fc domain polypeptide in a heterodimeric Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the linker fusing a first subunit of a multi-subunit protein to a first antibody Fc domain polypeptide consists of the amino acid sequence of SEQ ID NO: 2.

[0131] In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 3.

[0132] In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 13.

[0133] In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 14.

[0134] In some embodiments, the linker joining the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of the amino acid sequence of SEQ ID NO: 4.

[0135] In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 5.

[0136] In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 63. In some embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 63.

[0137] In some embodiments, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence of SEQ ID NO: 64.

[0138] In certain embodiments, the Fc domain polypeptide is an IgG4 Fc. IgG4 is an unstable dimer that can undergo Fab arm exchange and pair with other IgG4 antibodies in the body. In certain embodiments, an S228P mutation is introduced within the hinge (which in the present invention is part of or the entire linker connecting a first subunit of a multi-subunit protein to a first IgG4 antibody Fc domain polypeptide, or a linker connecting an additional subunit to a second, different IgG4 antibody Fc domain polypeptide), which increases the stability of the hinge region and reduces the likelihood of Fab arm exchange. In certain embodiments, an additional disulfide bond is introduced between Fc domain polypeptide monomers, which improves the stability of the heterodimer. In exemplary embodiments, a first antibody Fc domain polypeptide linked to a first subunit of a multi-subunit protein comprises a Y349C substitution in the CH3 domain that forms a disulfide bond with a S354C substitution on a second antibody Fc domain polypeptide linked to a second, different subunit of the multi-subunit protein that is linked to a second antibody Fc domain polypeptide. Alternatively, a first antibody Fc domain polypeptide linked to a first subunit of a multi-subunit protein comprises a S354C substitution in the CH3 domain that forms a disulfide bond with a Y349C substitution on a second antibody Fc domain polypeptide linked to a second, different subunit of the multi-subunit protein.

[0139] In some embodiments, the proteins of the invention comprise a first antibody Fc domain polypeptide and a second antibody Fc domain polypeptide, both of which are mutated IgG4 Fc domain polypeptides that promote heterodimerization with each other.

[0140] In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises one or more mutations selected from K360E, K370E, and R409W, and the second antibody IgG4 Fc domain polypeptide comprises one or more mutations selected from E357N, Q347R, D399V, and F405T. In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises mutations K370E and R409W, and the second antibody IgG4 Fc domain polypeptide comprises mutations E357N, D399V, and F405T. In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises mutations E357N, D399V, and F405T, and the second antibody IgG4 Fc domain polypeptide comprises mutations K370E and R409W. In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises mutations K360E and R409W, and the second antibody IgG4 Fc domain polypeptide comprises mutations Q347R, D399V, and F405T. In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises mutations Q347R, D399V, and F405T, and the second antibody IgG4 Fc domain polypeptide comprises mutations K360E and R409W.

[0141] In some embodiments, heterodimeric Fc-fusion proteins of the invention with IgG4 Fc comprise one or more mutations in the first and / or second polypeptide to reduce binding to an FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB) or a complement component (e.g., C1q). Such mutations are useful for reducing effector function. For example, proteins of the present disclosure comprise S228P and L235E mutations; S228P, L235E, and P329A mutations; or S228P, L235E, and P329G mutations.

[0142] Any of the IgG4 antibody Fc domain polypeptides provided in Table 2 can be utilized in combination with any of the IgG4 hinge sequences provided in Table 1 (which, in the present invention, are part of, or the entirety of, a linker joining a first subunit of a multi-subunit protein to a first IgG4 antibody Fc domain polypeptide, or a linker joining a second, different subunit of a multi-subunit protein to a second, different IgG4 antibody Fc domain polypeptide). Exemplary IgG4 hinge-Fc domain polypeptides are provided in Table 3. In certain embodiments, the first and second polypeptides of the Fc fusion protein comprise the amino acid sequences of SEQ ID NOs: 205 and 205; 206 and 207; 208 and 209; 210 and 211; 207 and 206; 209 and 208; or 211 and 210, respectively. In certain embodiments, the first and second polypeptides of the Fc fusion protein comprise the amino acid sequences of SEQ ID NOs: 219 and 219; 220 and 221; 222 and 223; 224 and 225; 226 and 227; 221 and 220; 223 and 222; 225 and 224; or 227 and 226, respectively.

[0143] (b) Disulfide bond Some heterodimeric Fc-fusion proteins of the invention comprise a native heterodimeric disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein. For example, in an exemplary embodiment, a heterodimeric Fc-fusion protein of the invention comprises a native heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. Such proteins comprise a native disulfide bond between C74 of p35 and C177 of p40.

[0144] Some heterodimeric Fc-fusion proteins of the invention comprise an artificial or engineered heterodimeric disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein. For example, in an exemplary embodiment, a heterodimeric Fc-fusion protein of the invention comprises an artificial or engineered heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. Such proteins comprise an artificial or engineered disulfide bond between V185C of p35 and Y292C of p40.

[0145] Some heterodimeric Fc-fusion proteins of the invention comprise a native heterodimeric disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein, and an artificial or engineered heterodimeric disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein. For example, in exemplary embodiments, the heterodimeric Fc-fusion proteins comprise a native heterodimeric disulfide bond between the p35 and p40 subunits of IL-12, and an artificial or engineered heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. Such proteins comprise a native disulfide bond between C74 of p35 and C177 of p40, and an artificial or engineered disulfide bond between V185C of p35 and Y292C of p40.

[0146] Some heterodimeric Fc-fusion proteins of the invention are engineered to remove a native disulfide bond and replace it with a non-natural, artificial, or engineered disulfide bond. For example, in an exemplary embodiment, a heterodimeric Fc-fusion protein of the invention comprises IL-12 p35 in which the native C74 has been mutated to serine and IL-12 p40 in which the native C177 has been mutated to serine, thereby removing the native disulfide bond between the p35 and p40 subunits of IL-12. This mutant IL-12 also contains two novel mutations, V185C in p35 and Y292C in p40, thereby introducing a non-natural, artificial, or engineered disulfide bond.

[0147] (c) Sequence of the components of the Fc fusion polypeptide Exemplary heterodimeric Fc-fusion proteins of the invention are constructed comprising any one of the IgG1 or IgG4 Fc variant sequences and any one of the corresponding linker sequences set forth below in Tables 1-2. The fusion protein constructs of the invention may confer a longer serum half-life compared to native / native multi-subunit proteins, improve protein yield during production, enhance stability during storage, and / or improve efficacy when used as therapeutic agents.

[0148] Any of the IgG4 antibody Fc variant domain polypeptides provided in Table 2 below can be utilized in combination with any of the IgG4 hinge sequences provided in Table 1 below. Similarly, any of the IgG1 antibody Fc variant domain polypeptides provided in Table 2 below can be utilized in combination with any of the IgG1 hinge sequences provided in Table 1 below. Exemplary IgG1 hinge-Fc domain polypeptides are provided in Table 3 below.

[0149] [Table 1]

[0150]

Table 2

[0151]

Table 3

[0152]

Table 4

[0153]

Table 5

[0154]

Table 6

[0155]

Table 7

[0156]

Table 8

[0157]

Table 9

[0158]

Table 10

[0159]

Table 11

[0160]

Table 12

[0161]

Table 13

[0162]

Table 14

[0163]

Table 15

[0164] Table 16

[0165]

Table 17

[0166]

Table 18

[0167]

Table 19

[0168] Table 20

[0169] Table 21

[0170] Table 22

[0171] [Table 23]

[0172] [Table 24]

[0173] [Table 25]

[0174] [Table 26]

[0175] [Table 27]

[0176] [Table 28]

[0177] [Table 29]

[0178] (d) IL-12 subunit IL-12 is a multisubunit protein comprising a p40 subunit and a p35 subunit. The amino acid sequence of mature wild-type IL-12 p40 is amino acids 23-328 of GenBank Accession No. NP_002178.2, as set forth below in SEQ ID NO: 127. The amino acid sequence of mature wild-type IL-12 p35 is amino acids 57-253 of GenBank Accession No. NP_000873.2, as set forth below in SEQ ID NO: 128. As used herein, the numbering of amino acid residues in p40 and p35 corresponds to the mature wild-type protein sequence. As used herein, the IL-12 p40 subunit comprises an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 127. As used herein, an IL-12 p35 subunit comprises an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 128.

[0179] In certain embodiments of any one of the foregoing aspects, the p40 and p35 subunits of IL-12 comprise the amino acid sequences of SEQ ID NOs: 121 and 122; 127 and 128; 201 and 202; 203 and 204; 123 and 124; or 125 and 126, respectively. In certain embodiments, a first polypeptide comprises the amino acid sequence of the p40 subunit of IL-12, and a second polypeptide comprises the amino acid sequence of the p35 subunit of IL-12. In certain embodiments, a first polypeptide comprises the amino acid sequence of the p35 subunit of IL-12, and a second polypeptide comprises the amino acid sequence of the p40 subunit of IL-12.

[0180] In certain embodiments, the disclosure includes a heterodimeric Fc-fusion protein comprising a first polypeptide comprising a first antibody Fc domain polypeptide and a second polypeptide comprising a second antibody Fc domain polypeptide, wherein the first polypeptide further comprises a first subunit of IL-12 fused to the first antibody Fc domain polypeptide by a linker; and a second, distinct subunit of IL-12 fused to the second antibody Fc domain polypeptide, wherein the first and second distinct subunits of IL-12 are linked to each other, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain a different mutation that promotes heterodimerization, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are linked to each other, and the first subunit of IL-12 is a p40 subunit with a Y292C substitution and the second, distinct subunit of IL-12 is a p35 subunit with a V185C substitution. In a specific embodiment, the first subunit and the second distinct subunit of IL-12 comprise the amino acid sequences of SEQ ID NOs: 125 and 126, respectively.

[0181] The first subunit and second, different subunit of IL-12 can be fused to any of the antibody Fc domain polypeptides via any linker disclosed herein, thereby forming Fc fusion proteins having sequences including, but not limited to, constructs 120, 120-1, 120-2, 120-3, 120-4, 120-5, 120-6, and 120-7 as set forth in Table 4, and constructs 20, 20-1, 20-2, 20-3, 20-4, 20-5, 20-6, 20-7, 20-8, and 20-9 as set forth in Table 5.

[0182] In certain embodiments, the p40 subunit of IL-12 further comprises a replacement of C177, and the p35 subunit of IL-12 further comprises a replacement of C74. In certain embodiments, C177 in the p40 subunit of IL-12 is replaced by S, and C74 in the p35 subunit of IL-12 is replaced by S. In certain embodiments, the p40 and p35 subunits of IL-12 comprise the amino acid sequences of SEQ ID NOs: 123 and 124, respectively.

[0183] The first subunit and second, different subunit of IL-12 can be fused to any of the antibody Fc domain polypeptides via any linker disclosed herein, thereby forming Fc fusion proteins having sequences including, but not limited to, constructs 119, 119-1, 119-2, 119-3, 119-4, 119-5, 119-6, 119-7, and 119-8 as set forth in Table 4, and constructs 19, 19-1, 19-2, 19-3, 19-4, 19-5, 19-6, 19-7, 19-8, 19-9, and 19-10 as set forth in Table 5.

[0184] [Table 30]

[0185] (e) Spacer peptide Exemplary spacer peptide sequences are provided in Table 7, and exemplary full-length linker sequences are provided in Tables 4 and 5.

[0186] Within a first polypeptide of the invention, in an amino to carboxyl orientation, a first subunit of a multi-subunit protein is fused via a linker to a first antibody Fc domain polypeptide (e.g., an IgG4 antibody Fc variant sequence or an IgG1 antibody Fc variant sequence, as disclosed in Table 2), and within a second polypeptide of the invention, in an amino to carboxyl orientation, a second, different subunit of a multi-subunit protein is fused via a linker to a second antibody Fc domain polypeptide (e.g., an IgG4 antibody Fc variant sequence or an IgG1 antibody Fc variant sequence, as disclosed in Table 2).

[0187] In some embodiments, a first subunit of a multi-subunit protein of the invention is fused to a first antibody Fc domain sequence via a linker, where the linker comprises or consists of a spacer peptide L1 and the amino acid sequence of SEQ ID NO: 1, 2, 4, 6, 7, 9, 11, 237, 238, 239, or 240. In some embodiments, a second, different subunit of a multi-subunit protein is fused to a second antibody Fc domain polypeptide via a linker, where the linker comprises or consists of a spacer peptide L2 and the amino acid sequence of SEQ ID NO: 1, 2, 4, 6, 7, 9, 11, 237, 238, 239, or 240.

[0188] In certain embodiments, L1 and L2 are peptide linkers, for example, L1 and / or L2 comprise 4 to 50 amino acid residues. In certain embodiments, L1 consists of 4 to 50 amino acid residues. In certain embodiments, L1 consists of 4 to 20 amino acid residues. In certain embodiments, L2 consists of 4 to 50 amino acid residues. In certain embodiments, L2 consists of approximately 4 to 20 amino acid residues. In certain embodiments, L1 and L2 each independently consist of approximately 4 to 50 amino acid residues. In certain embodiments, L1 and L2 each independently consist of 4 to 20 amino acid residues.

[0189] In some embodiments, L1 and L2 have optimized length and / or amino acid composition. In some embodiments, L1 and L2 are the same length and have the same amino acid composition. In other embodiments, L1 and L2 are different.

[0190] In certain embodiments, L1 is the same number of amino acids as L2; ​​in certain embodiments, L1 is longer (i.e., has more amino acids) than L2; in certain embodiments, L1 is shorter (i.e., has fewer amino acids) than L2.

[0191] In certain embodiments, L1 and / or L2 are "short," e.g., consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain examples, the spacer peptide consists of about 12 amino acid residues or less. In the case of 0 amino acid residues, the spacer peptide is a peptide bond. In certain embodiments, L1 and / or L2 are "long," e.g., consisting of 15, 20, or 25 amino acid residues. In some embodiments, the spacer peptide consists of about 3 to about 15, e.g., 8, 9, or 10 adjacent amino acid residues. Regarding the amino acid composition of L1 and L2, peptides are selected that confer flexibility to the first and second polypeptides of the protein of the invention, but do not prevent the first and second distinct subunits from binding to each other, and that are resistant to cleavage by proteases. For example, glycine and serine residues generally provide protease resistance. A spacer peptide suitable for linking a first subunit of a multi-subunit protein to the amino acid sequence of SEQ ID NO: 1, 2, 4, 6, 7, 9, 11, 237, 238, 239, or 240 and / or for linking a second, different subunit of a multi-subunit protein to the amino acid sequence of SEQ ID NO: 1, 2, 4, 6, 7, 9, 11, 237, 238, 239, or 240 may be, as part of the linker, a spacer peptide comprising the following structural sequence: (GS) n , (GGS) n , (GGGS) n , (GGSG) n , (GGSGG) n , and (GGGGS)n wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, L1 and / or L2 independently comprise a (GGGGS)4 (SEQ ID NO: 107) or a (GGGGS)3 (SEQ ID NO: 108) sequence as part of the linker. In other embodiments, L1 and / or L2 independently comprise a peptide sequence as part of the linker as set forth in a sequence selected from SEQ ID NOs: 111, 112, 113, 114, 115, 116, 117, 118, 119, and 120, as listed in Table 7. In some embodiments, L1 and / or L2 are independently SEQ ID NO: 108, SEQ ID NO: 109, or SEQ ID NO: 110.

[0192] [Table 31]

[0193] In certain embodiments, L1 comprises the sequence SEQ ID NO: 108 as part of the linker and L2 comprises SEQ ID NO: 109 or SEQ ID NO: 110 as part of the linker. In certain embodiments, L2 comprises the sequence SEQ ID NO: 108 as part of the linker and L1 comprises the sequence SEQ ID NO: 109 or SEQ ID NO: 110 as part of the linker. In certain embodiments, L1 does not comprise a sequence as set forth in SEQ ID NO: 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 as part of the linker.

[0194] In certain embodiments, only L2 comprises, as part of a linker, a sequence as set forth in SEQ ID NO: 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. In certain embodiments, neither L1 nor L2 comprises, as part of a linker, a sequence as set forth in SEQ ID NO: 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120.

[0195] Some heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker comprising SEQ ID NO: 118 links the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide. Some heterodimeric Fc-fusion proteins of the invention comprise a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein a linker comprising SEQ ID NO: 118 links the second, different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide.

[0196] In certain embodiments, some heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker comprising SEQ ID NO: 118 joins the first subunit of the multi-subunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the additional subunit is joined to the second antibody Fc domain polypeptide by a linker that does not comprise SEQ ID NO: 118.

[0197] In certain embodiments, some heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker that does not include SEQ ID NO: 118 joins the first subunit of the multi-subunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second, different subunit of the multi-subunit protein is joined to the second antibody Fc domain polypeptide by a linker that includes SEQ ID NO: 118.

[0198] Some heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker comprising SEQ ID NO: 109 links the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide. Some heterodimeric Fc-fusion proteins of the invention comprise a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein a linker comprising SEQ ID NO: 109 links the second, different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide.

[0199] Some heterodimeric Fc-fusion proteins of the present disclosure comprise a linker comprising SEQ ID NO: 109, which links a first subunit of the multi-subunit protein to a first antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide, and which links a second, different subunit of the multi-subunit protein to a second antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide.

[0200] In certain embodiments, some heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker comprising SEQ ID NO: 109 joins the first subunit of the multi-subunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the additional subunit of the multi-subunit protein is joined to the second antibody Fc domain polypeptide by a linker that does not comprise SEQ ID NO: 109.

[0201] In certain embodiments, some heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker that does not include SEQ ID NO: 109 joins the first subunit of the multi-subunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second, different subunit of the multi-subunit protein is joined to the second antibody Fc domain polypeptide by a linker that includes SEQ ID NO: 109.

[0202] Some heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker comprising SEQ ID NO: 110 links the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide. Some heterodimeric Fc-fusion proteins of the invention comprise a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein a linker comprising SEQ ID NO: 110 links the second, different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide.

[0203] Some heterodimeric Fc-fusion proteins of the present disclosure comprise a linker comprising SEQ ID NO: 110, which links a first subunit of the multi-subunit protein to a first antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide, and which links a second, different subunit of the multi-subunit protein to a second antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide.

[0204] In certain embodiments, some heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker comprising SEQ ID NO:110 joins the first subunit of the multi-subunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second, different subunit of the multi-subunit protein is joined to the second antibody Fc domain polypeptide by a linker that does not comprise SEQ ID NO:110.

[0205] In certain embodiments, some heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker that does not include SEQ ID NO:110 joins the first subunit of the multi-subunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second, different subunit of the multi-subunit protein is joined to the second antibody Fc domain polypeptide by a linker that includes SEQ ID NO:110.

[0206] In certain embodiments, some heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker sequence comprising SEQ ID NO: 110 joins the first subunit of the multi-subunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second, different subunit of the multi-subunit protein is joined to the second antibody Fc domain polypeptide by a linker sequence comprising SEQ ID NO: 109.

[0207] In certain embodiments, some heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker sequence comprising SEQ ID NO: 109 joins the first subunit of the multi-subunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second, different subunit of the multi-subunit protein is joined to the second antibody Fc domain polypeptide by a linker sequence comprising SEQ ID NO: 110.

[0208] (f) Fc domain and substitutions to promote heterodimerization Assembly of the proteins of the invention can be achieved by expressing in the same cell a first polypeptide comprising a first subunit sequence of a multi-subunit protein fused to a first antibody Fc domain polypeptide (e.g., an IgG4 antibody Fc variant sequence or an IgG1 antibody Fc variant sequence, as disclosed in Table 2) and a second polypeptide comprising a second, different subunit sequence of a multi-subunit protein fused to a second antibody Fc domain polypeptide (e.g., an IgG4 antibody Fc variant sequence or an IgG1 antibody Fc variant sequence, as disclosed in Table 2), leading to the assembly of a heterodimeric Fc fusion protein of the invention. The assembled protein comprises a heterodimeric Fc domain polypeptide in which the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are linked to each other. Promotion of preferential assembly of Fc heterodimers can be achieved by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region, as shown in U.S. Patent Application Nos. 13 / 494,870, 16 / 028,850, 11 / 533,709, 12 / 875,015, 13 / 289,934, 14 / 773,418, 12 / 811,207, 13 / 866,756, 14 / 647,480, and 14 / 830,336. For example, mutations may be made in a human IgG1-based CH3 domain to introduce distinct pairs of amino acid substitutions within a first antibody Fc domain polypeptide and a second antibody Fc domain polypeptide that allow these two chains to selectively heterodimerize with one another. All amino acid substitution positions exemplified below are numbered according to the EU index as in Kabat.

[0209] In one scenario, amino acid substitutions in a first antibody Fc domain polypeptide replace the original amino acid with a large amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), and at least one amino acid substitution in a second antibody Fc domain polypeptide replaces the original amino acid with a small amino acid selected from alanine (A), serine (S), threonine (T), or valine (V), such that the large amino acid substitution (protrusion) fits into the surface of the small amino acid substitution (cavity). For example, one antibody Fc domain polypeptide can incorporate a T366W substitution, while the other can incorporate three substitutions including T366S, L368A, and Y407V.

[0210] A first polypeptide comprising a first subunit sequence of a multi-subunit protein of the invention or a second polypeptide comprising a second, different subunit sequence of a multi-subunit protein of the invention can optionally be coupled to an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to an antibody constant region, such as an IgG constant region comprising a hinge, CH2, and CH3 domains with or without a CH1 domain. In some embodiments, the amino acid sequence of the constant region is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to a human antibody constant region, such as a human IgG1, IgG2, IgG3, or IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to an antibody constant region from another mammal, such as rabbit, dog, cat, mouse, or horse. One or more mutations may be incorporated into the constant region compared to a human IgG1 constant region, for example, at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and / or K439.Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[0211] In certain embodiments, mutations that can be incorporated into CH1 of the human IgG1 constant region can be at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that can be incorporated into Cκ of the human IgG1 constant region can be at amino acids E123, F116, S176, V163, S174, and / or T164.

[0212] The amino acid substitutions may be selected from the set of substitutions shown in Table 8 below.

[0213] [Table 32]

[0214] Alternatively, the amino acid substitutions may be selected from the set of substitutions shown in Table 9 below.

[0215] [Table 33]

[0216] Alternatively, the amino acid substitutions may be selected from the set of substitutions shown in Table 10 below.

[0217] [Table 34]

[0218] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 11.

[0219] [Table 35]

[0220] Alternatively, at least one amino acid substitution may be selected from the set of substitutions in Table 12 below, where the position indicated in the first polypeptide column is replaced with any known negatively charged amino acid and the position indicated in the second polypeptide column is replaced with any known positively charged amino acid.

[0221] [Table 36]

[0222] Alternatively, at least one amino acid substitution may be selected from the set in Table 13 below, where the position indicated in the first polypeptide column is replaced by a positively charged amino acid and the position indicated in the second polypeptide column is replaced by any known negatively charged amino acid.

[0223] [Table 37]

[0224] Alternatively, the amino acid substitutions may be selected from the set in Table 14 below.

[0225] [Table 38]

[0226] Alternatively, or in addition, the structural stability of the heterodimeric Fc-fusion proteins of the present invention may be increased by introducing S354C into either the first or second polypeptide chain and Y349C into the opposite polypeptide chain, forming an artificial disulfide bond within the interface of these two polypeptides.

[0227] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, L368, and Y407.

[0228] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, L368, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366.

[0229] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region at one or more positions selected from Y349, E357, S364, L368, K370, T394, D401, F405, and T411.

[0230] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, E357, S364, L368, K370, T394, D401, F405, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411.

[0231] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, D399, S400, and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, N390, K392, K409, and T411.

[0232] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, N390, K392, K409, and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, D399, S400, and Y407.

[0233] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Q347, Y349, K360, and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Q347, E357, D399, and F405.

[0234] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Q347, E357, D399, and F405, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, K360, Q347, and K409.

[0235] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from K370, K392, K409, and K439, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from D356, E357, and D399.

[0236] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from D356, E357, and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from K370, K392, K409, and K439.

[0237] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, E356, T366, and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, L351, L368, K392, and K409.

[0238] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, L351, L368, K392, and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, E356, T366, and D399.

[0239] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a S354C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a Y349C substitution.

[0240] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a Y349C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a S354C substitution.

[0241] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by K360E and K409W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by O347R, D399V, and F405T substitutions.

[0242] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by O347R, D399V, and F405T substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by K360E and K409W substitutions.

[0243] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a T366W substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by T366S, T368A, and Y407V substitutions.

[0244] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T366S, T368A, and Y407V substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a T366W substitution.

[0245] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by T350V, L351Y, F405A, and Y407V substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by T350V, T366L, K392L, and T394W substitutions.

[0246] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by T350V, T366L, K392L, and T394W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by T350V, L351Y, F405A, and Y407V substitutions.

[0247] Those skilled in the art will understand that during protein production and / or storage, lactams can be formed (e.g., naturally or catalyzed by enzymes present during production and / or storage) by cyclic processing of the N-terminal glutamic acid (E) or glutamine (Q). Thus, in some embodiments in which the N-terminal residue of a polypeptide's amino acid sequence is E or Q, the corresponding amino acid sequence in which E or Q is replaced by pyroglutamic acid is also contemplated herein.

[0248] Those skilled in the art will also understand that during protein production and / or storage, the C-terminal lysine (K) of a protein may be removed (e.g., naturally or catalyzed by an enzyme present during production and / or storage). Such removal of K is often observed in proteins that include an Fc domain at their C-terminus. Thus, in some embodiments in which the C-terminal residue of a polypeptide amino acid sequence (e.g., an Fc domain sequence) is K, the corresponding amino acid sequence in which the K has been removed is also contemplated herein.

[0249] (g) Mutations to reduce effector function In one aspect, the present invention provides heterodimeric Fc fusion proteins comprising: (a) a first polypeptide comprising a first antibody Fc domain polypeptide and a first subunit of a multisubunit protein; and (b) a second polypeptide comprising a second antibody Fc domain polypeptide and a second, different subunit of the multisubunit protein, wherein the first and second antibody Fc domain polypeptides each comprise different mutations that promote heterodimerization, the first and / or second antibody Fc domain polypeptides comprise one or more mutations that reduce Fc effector function, and the first subunit and the second, different subunit of the multisubunit protein bind to each other. In certain embodiments, heterodimeric Fc fusion proteins disclosed herein that comprise one or more mutations that reduce Fc effector function exhibit increased tumor growth inhibitory activity compared to their counterparts that lack one or more Fc mutations that reduce effector function. Mutations contemplated herein include amino acid substitutions, insertions, and deletions. All amino acid positions in the Fc domain or hinge region disclosed herein are numbered according to EU numbering.

[0250] In certain embodiments, the first and / or second antibody Fc domain polypeptides comprise one or more mutations that reduce the ability of the Fc domain polypeptides to induce antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). ADCC and ADCP are typically mediated by Fc receptors. For example, in certain embodiments, the first and second antibody Fc domain polypeptides are human IgG (e.g., human IgG1, human IgG2, human IgG3, or human IgG4) antibody sequences. Fc receptors for human IgG, also referred to as Fcγ receptors (FcγR), include, but are not limited to, activating Fcγ receptors FcγRI (CD64), FcγRIIA (CD32A), FcγRIIIA (CD16 or CD16A), and FcγRIIIB (CD16B), and inhibitory Fcγ receptor FcγRIIB (CD32B). Thus, in some embodiments, the heterodimeric Fc-fusion proteins of the invention comprise one or more mutations that reduce binding of the first and / or second polypeptide to an activating FcγR (e.g., FcγRI, FcγRIIA, FcγRIIIA, or FcγRIIIB). In some embodiments, the heterodimeric Fc-fusion proteins of the invention comprise one or more mutations that increase binding of the first and / or second polypeptide to an inhibitory FcγR (e.g., FcγRIIB).

[0251] Fc mutations that reduce binding to activating FcγRs and / or increase binding to inhibitory FcγRs are known in the art. For example, within the hinge and Fc region, CD16 binding is mediated by the hinge region and CH2 domain. For example, in human IgG1, the interaction with CD16 is primarily centered around amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239, and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al., Nature, 406(6793):267-273). Based on these known domains, mutations can be selected to enhance or reduce binding affinity to CD16, such as by using a phage display library or a yeast surface display cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0252] As reviewed in Wang et al., Protein Cell (2018) 9(1):63-73, regions including amino acid positions 232-239, 265-270, 296-299, and 325-332 have been implicated in activating FcγR binding according to the crystal structure of human IgG1 Fc. Wang et al. also disclosed that L235E and F234A / L235A mutations in human IgG4 antibodies, L234A / L235A mutations in human IgG1 antibodies, and N297 mutations (e.g., N297A, N297Q, N297G, or N297D) in IgG antibodies reduce activating FcγR binding. As disclosed in U.S. Patent No. 8,969,526, mutations at position 329 (e.g., P329A, P329G, or P329R) also reduce activating FcγR binding. Additional amino acid positions and mutations (e.g., the E233P mutation) implicated in activating FcγR binding are disclosed in U.S. Pat. No. 7,943,743 and Isaacs et al., J. Immunol. (1998) 161:3862-69.

[0253] Thus, in certain embodiments, the first and second antibody Fc domain polypeptides comprise a mutation (e.g., a substitution compared to human IgG1) at one or more positions selected from positions 233, 234, 235, 297, and 329. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutations E233P; L234A (human IgG1) or F234A (human IgG4); L235A or L235E; N297A, N297Q, N297G, or N297D; and / or P329A, P329G, or P329R. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutations L234A and L235A. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutations L234A, L235A, and P329A. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG4 antibody Fc domain polypeptides comprising the mutation L235E. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutations L235E and P329A.

[0254] In certain embodiments, the first and / or second antibody Fc domain polypeptides comprise one or more mutations that reduce the ability of the Fc domain polypeptides to induce complement-dependent cytotoxicity (CDC). CDC is typically mediated by complement components (e.g., C1). Accordingly, in certain embodiments, the heterodimeric Fc-fusion proteins of the invention comprise one or more mutations that reduce binding of the first and / or second polypeptides to complement components (e.g., C1q).

[0255] Fc mutations that reduce C1q binding are known in the art. For example, as disclosed in U.S. Patent Nos. 5,648,260 and 5,624,821, the amino acid residues at positions 234, 235, 236, 237, 297, 318, 320, and 322 of the Fc have been implicated in C1q binding. As disclosed in Tao et al., J. Exp. Med. (1993) 178:661-667 and Brekke et al., Eur. J. Immunol. (1994) 24:2542-47, the residue Pro at position 331 has been implicated in C1q binding. As disclosed in Idusogie et al., J. Immunol. (2000) 164:4178-84, mutations at Fc positions 270 (e.g., D270A), 322 (K322A), 329 (e.g., P329A), and 331 (e.g., P331A, P331S, or P331G) reduced C1q binding.

[0256] Thus, in certain embodiments, the first and second antibody Fc domain polypeptides comprise a mutation (e.g., a substitution compared to human IgG1) at one or more positions selected from positions 234, 235, 236, 237, 270, 297, 318, 320, 322, 329, and 331. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutations G237A, A330S, P331S, and / or P329A. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutations G237A, A330S, and P331S. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutation P329A.

[0257] Mutations that reduce ADCC and / or ADCP and mutations that reduce CDC can be combined. In certain embodiments, the first and / or second antibody Fc domain polypeptides comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce ADCC and / or ADCP and further comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce CDC. In certain embodiments, the first and second antibody Fc domain polypeptides each comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce ADCC and / or ADCP and further comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce CDC.

[0258] In some embodiments, heterodimeric Fc fusion proteins of the invention with IgG4 Fc comprise one or more mutations in the first and / or second polypeptide to reduce binding to an FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB) or a complement component (e.g., C1q). Such mutations are useful for reducing effector function. For example, proteins of the present disclosure can comprise S228P and L235E mutations; S228P, L235E, and P329A mutations; or S228P, L235E, and P329G mutations.

[0259] In some embodiments, heterodimeric Fc fusion proteins of the invention with IgG1 Fc comprise one or more mutations in the first and / or second polypeptide to reduce binding to an FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB) or a complement component (e.g., C1q). Such mutations are useful for reducing effector function. For example, proteins of the present disclosure can comprise L234A and L235A mutations; L234A, L235A, and P329A mutations; L234A, L235A, and P329G mutations; or L234A, L235E, G237A, A330S, and P331S mutations.

[0260] In some embodiments, the heterodimeric Fc-fusion protein of the invention comprises a first antibody IgG4 or IgG1 Fc domain polypeptide and a second antibody IgG4 or IgG1 Fc domain polypeptide, each having the mutation P329G or P329A.

[0261] In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each have a mutation selected from A330S and P331S.

[0262] In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each have the mutations A330S and P331S.

[0263] In certain embodiments, in the first polypeptide of the heterodimeric Fc-fusion protein of the invention, a first subunit of the multi-subunit protein is fused to a first antibody Fc domain polypeptide by a first linker. In certain embodiments, in the second polypeptide of the heterodimeric Fc-fusion protein of the invention, a second, different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker. The amino acid sequences of linkers suitable for such use are described under the headings "IgG4 Constructs" and "IgG1 Constructs." Additional linker sequences suitable for use in the first and / or second polypeptide include, but are not limited to, wild-type IgG (e.g., human IgG1, human IgG2, human IgG3, or human IgG4) hinge sequences and mutant forms thereof. For example, in certain embodiments, the first and second linkers each comprise the amino acid sequence ESKYGPPCPPCPAPEFXGG (where X is L or E) (SEQ ID NO: 280) or SKYGPPCPPCPAPEFXGG (where X is L or E) (SEQ ID NO: 281). In certain embodiments, the first and second linkers each comprise the amino acid sequence of SEQ ID NO: 282 or SEQ ID NO: 283. In certain embodiments, the first and second linkers each comprise the amino acid sequence of SEQ ID NO: 284 or SEQ ID NO: 285.

[0264] (h) Serum half-life The heterodimeric Fc-fusion proteins of the present invention have pharmacokinetic properties suitable for therapeutic use. For example, in certain embodiments, the heterodimeric Fc-fusion proteins of the present invention have a serum half-life of at least about 50 hours. In certain embodiments, the heterodimeric Fc-fusion proteins of the present invention have a serum half-life of at least about 100 hours.

[0265] In a specific embodiment, 50 hours after intravenous administration to a subject, the serum concentration of a heterodimeric Fc-fusion protein of the present invention is at least 10% of the serum concentration of the protein of the present invention in said subject 1 hour after administration.

[0266] In certain embodiments, heterodimeric Fc-fusion proteins of the present invention have a serum half-life that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% longer than a multi-subunit protein that is not fused to an Fc domain polypeptide. In certain embodiments, heterodimeric Fc-fusion proteins comprising the protein sequence of a multi-subunit protein of the present invention have a serum half-life that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold longer than a multi-subunit protein that is not fused to an Fc domain polypeptide.

[0267] (i) Tumor retention The heterodimeric Fc-fusion proteins of the present invention can optionally incorporate additional features that enhance retention of the protein at tumor sites. For example, in certain embodiments of the present invention, the heterodimeric Fc-fusion proteins further comprise a proteoglycan-binding domain, a collagen-binding domain, and / or a hyaluronic acid-binding domain. In certain embodiments, the heterodimeric Fc-fusion proteins further comprise a proteoglycan-binding domain that binds to one or more proteoglycans present in tumors (e.g., on the surface of tumor cells, in the pericellular matrix of tumors, or in the extracellular matrix of tumors) (e.g., proteoglycans known in the art, e.g., those disclosed in Lozzo et al., Matrix Bio (2015) 42:11-55; and Nikitovic et al., Frontiers in Endocrinology (2018) 9:69). In certain embodiments, the collagen-binding domain binds to one or more collagens present in tumors (e.g., on the surface of tumor cells, in the pericellular matrix of tumors, or in the extracellular matrix of tumors). In certain embodiments, the heterodimeric Fc-fusion protein further comprises a hyaluronic acid (h acid) binding domain that binds to one or more hyaluronic acids present in tumors. Such heterodimeric Fc-fusion proteins exhibit enhanced retention in tumors and can be administered to a subject's tumor at lower doses and / or frequency.

[0268] In certain embodiments, the proteoglycan binding domain contained in the heterodimeric Fc-fusion protein binds to one or more proteoglycans specifically expressed in tumors (e.g., on the surface of tumor cells, in the pericellular matrix of tumors, or in the extracellular matrix of tumors). In certain embodiments, the collagen binding domain contained in the heterodimeric Fc-fusion protein binds to one or more collagens specifically expressed in tumors (e.g., on the surface of tumor cells, in the pericellular matrix of tumors, or in the extracellular matrix of tumors). Such heterodimeric Fc-fusion proteins may be concentrated in tumors after administration (e.g., after intravenous, subcutaneous, or intrapulmonary administration) and have enhanced tumor retention, thereby allowing for lower doses and / or more frequent administration.

[0269] In certain embodiments, the heterodimeric Fc-fusion proteins of the invention further comprise a proteoglycan-binding domain that binds to one or more proteoglycans selected from syndecan, chondroitin sulfate proteoglycan 4 (CSPG4), betaglycan, phosphacan, glypican, perlecan, agrin, collagen (e.g., collagen type IX, XII, XV, or XVIII), hyalectan, aggrecan, versican, neurocan, brevican, and small leucine-rich proteoglycans (SLRPs). Proteoglycans implicated in cancer include, but are not limited to, collagen, syndecan (e.g., syndecan-1 or syndecan-2), serglycin, CSPG4, betaglycan, glypican (e.g., glypican-1 or glypican-3), perlecan, versican, brevican, and SLRPs (e.g., decorin, biglycan, asporin, fibromodulin, and lumican). Thus, in certain embodiments, the proteoglycan binding domain comprised in the heterodimeric Fc-fusion protein binds to one or more proteoglycans selected from syndecan (e.g., syndecan-1 or syndecan-2), serglycin, CSPG4, betaglycan, glypican (e.g., glypican-1 or glypican-3), perlecan, versican, brevican, and SLPR. In certain embodiments, the proteoglycan binding domain comprised in the heterodimeric Fc-fusion protein binds to one or more SLPRs selected from decorin, biglycan, asporin, fibromodulin, and lumican.

[0270] The proteoglycan-binding domain contained in the heterodimeric Fc-fusion protein can be a protein (e.g., an antibody or an antigen-binding fragment thereof), a peptide (e.g., a portion of a proteoglycan-binding protein or a variant thereof), an aptamer, a small molecule, or a combination thereof. Proteoglycan-binding domains are also known in the art. For example, syndecan-binding domains are disclosed in U.S. Patent Nos. 6,566,489, 8,647,828, and 10,124,038; U.S. Patent Application Publication No. 2009 / 0297479; and WO2018199176A1. CSPG4-binding domains are disclosed in U.S. Patent Nos. 9,801,928 and 10,093,745; and U.S. Patent Application Publication Nos. 2016 / 0032007, 2017 / 0342151, and 2018 / 0072811. β-glycan-binding domains are disclosed in U.S. Patent No. 7,455,839. Glypican-binding domains are described in U.S. Patent Nos. 7,919,086, 7,776,329, 8,680,247, 8,388,937, 9,260,492, 9,394,364, 9,790,267, 9,522,940, and 9,409,994; U.S. Patent Application Publication Nos. 2004 / 0236080, 2004 / 0236081, 2004 / 0236082, 2004 / 0236083, 2004 / 0236084, 2004 / 0236086, 2004 / 0236087, 2004 / 0236088, 2004 / 0236089 ... and WO 2017053619A1, WO 2018026533A1, WO 2018165344A1, and WO 2018199318A1. The perlecan binding domain is disclosed in U.S. Patent Application No. 10,166,304.The decorin binding domain is disclosed in U.S. Patent No. 6,517,838 and WO 2000021989A1, WO 2000077041A2, and WO 2000078800A2.

[0271] In certain embodiments, the heterodimeric Fc-fusion proteins of the present invention further comprise a collagen-binding domain. Collagen is a class of proteins, with at least 28 different types identified in vertebrates. As disclosed in Fang et al., Tumor Biol. (2014) 35:2871-82 and Xiong et al., J. Cancer Metasta. Treat. (2016) 2:357-64, each collagen type has its own unique structural characteristics and distribution pattern. Various collagen types, including but not limited to Col3A1, Col5A2, Col6, Col7A1, Col15A1, Col19A1, and Col22A1, have been implicated in cancer. The collagen-binding domain can be a protein (e.g., an antibody or an antigen-binding fragment thereof), a peptide (e.g., a portion of a collagen-binding protein or a variant thereof), an aptamer, a small molecule, or a combination thereof. Collagen binding domains are known in the art and are described, for example, in U.S. Pat. Nos. 5,788,966, 5,587,360, 5,851,794, 5,741,670, 5,849,701, 6,288,214, 6,387,663, 6,908,994, and 7,1 Nos. 69,902, 7,488,792, 7,820,401, 8,956,612, 8,642,728, and 8,906,649, and U.S. Patent Application Publication Nos. 2007 / 0161062, 2009 / 0142345, and 2012 / 0100106.

[0272] In certain embodiments, the heterodimeric Fc-fusion protein of the present invention further comprises a hyaluronic acid-binding domain. The hyaluronic acid-binding domain can be a protein (e.g., an antibody or an antigen-binding fragment thereof), a peptide (e.g., a portion of a hyaluronic acid-binding protein or a variant thereof), an aptamer, a small molecule, or a combination thereof. Hyaluronic acid-binding domains are known in the art and are disclosed, for example, in U.S. Patent Nos. 6,864,235, 8,192,744, 8,044,022, 8,163,498, 8,034,630, 9,217,016, 9,795,686, and 9,751,919, and U.S. Patent Application Publication Nos. 2002 / 0055488 and 2007 / 0259380.

[0273] When present, the proteoglycan-binding domain, collagen-binding domain, and / or hyaluronic acid-binding domain may be located at any position in the heterodimeric Fc-fusion protein. For example, in certain embodiments, when the IL-12 subunit is located N-terminally of an antibody Fc domain polypeptide, the proteoglycan-binding domain, collagen-binding domain, and / or hyaluronic acid-binding domain as disclosed herein may be fused to the C-terminus of a first antibody Fc domain polypeptide and / or to the C-terminus of a second antibody Fc domain polypeptide. In certain embodiments, when the IL-12 subunit is located C-terminally of an antibody Fc domain polypeptide, the proteoglycan-binding domain, collagen-binding domain, and / or hyaluronic acid-binding domain as disclosed herein may be fused to the N-terminus of a first antibody Fc domain polypeptide and / or to the N-terminus of a second antibody Fc domain polypeptide.

[0274] The proteoglycan-binding domain, collagen-binding domain, and / or hyaluronic acid-binding domain, if present, may be fused to the remainder of the heterodimeric Fc-fusion protein via a linker. In certain embodiments, the proteoglycan-binding domain is fused to the remainder of the heterodimeric Fc-fusion protein via a peptide linker. In certain embodiments, the peptide linker comprises a spacer peptide as disclosed herein.

[0275] Exemplary Heterodimeric Fc Fusion Proteins In certain embodiments, heterodimeric Fc-fusion proteins of the invention comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291. In certain embodiments, heterodimeric Fc-fusion proteins of the invention comprising SEQ ID NO: 290 and SEQ ID NO: 291 comprise a Y349C mutation in the CH3 domain of the first antibody Fc domain polypeptide and a S354C mutation in the CH3 domain of the second antibody Fc domain polypeptide. In certain embodiments, heterodimeric Fc-fusion proteins of the invention comprising SEQ ID NO: 290 and SEQ ID NO: 291 comprise different mutations in each Fc domain polypeptide sequence to promote heterodimerization between the Fc domains.

[0276] In certain embodiments, the first polypeptide sequence comprises a first antibody Fc domain polypeptide (human IgG1) sequence comprising K360E and K409W substitutions. In certain embodiments, the second polypeptide sequence comprises a second antibody Fc domain polypeptide (human IgG1) sequence comprising Q347R, D399V, and F405T substitutions. In certain embodiments, the first polypeptide and second polypeptide amino acid sequences comprise one or more mutations to reduce effector function. In certain embodiments, the heterodimeric Fc-fusion protein of the invention comprises L234A, L235A, and P329A mutations.

[0277] In a specific embodiment, in the first polypeptide of the heterodimeric Fc-fusion protein of the invention (SEQ ID NO: 290), the p40 subunit of human IL-12 is fused to a first antibody Fc domain polypeptide by a first linker comprising a first amino acid sequence, and in the second polypeptide of the heterodimeric Fc-fusion protein of the invention (SEQ ID NO: 291), the p35 subunit of human IL-12 is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence.

[0278] SEQ ID NO: 290 is the sequence of the p40 subunit of human IL-12 fused to a human IgG1 Fc domain polypeptide (underlined amino acids). Mutations are shown in bold. [ka]

[0279] SEQ ID NO: 291 is the sequence of the p35 subunit of human IL-12 fused to a human IgG1 Fc domain polypeptide (underlined amino acids). Mutations are shown in bold. [ka]

[0280] The first and second polypeptides represented by amino acid sequences SEQ ID NO: 290 and SEQ ID NO: 291, respectively, form a disulfide bond due to the Y349C mutation sequence (bold and underlined) in the CH3 domain of the first antibody Fc domain polypeptide (human IgG1) of SEQ ID NO: 290 and the S354C mutation sequence (bold and underlined) in the CH3 domain of the second antibody Fc domain polypeptide (human IgG1) of SEQ ID NO: 291, which confers stability to the heterodimeric Fc fusion protein (Fc numbering according to the EU system).

[0281] To promote heterodimerization between the two Fc domain polypeptides of the heterodimeric Fc fusion protein, a first antibody Fc domain polypeptide sequence of SEQ ID NO: 290 (human IgG1) comprises K360E and K409W substitutions in the CH3 domain, and a second, different Fc domain polypeptide sequence of SEQ ID NO: 291 (human IgG1) comprises Q347R, D399V, and F405T substitutions in the CH3 domain (Fc numbering according to the EU system).

[0282] The first antibody Fc domain polypeptide sequence of SEQ ID NO: 290 and SEQ ID NO: 291 and the second, different Fc domain polypeptide sequence (human IgG1) also comprise L234A, L235A, and P329A (LALAPA) mutations to reduce effector function.

[0283] This heterodimeric Fc fusion protein is referred to herein as DF-hIL-12-Fc si.

[0284] (j) Preparation and manufacturing method The proteins of the present invention can be produced using recombinant DNA techniques well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first polypeptide comprising a first subunit sequence of a multi-subunit protein fused to a first antibody Fc domain polypeptide can be cloned into a first expression vector; a second nucleic acid sequence encoding a second polypeptide comprising a second, different subunit sequence of a multi-subunit protein fused to a second antibody Fc domain polypeptide can be cloned into a second expression vector; and the first and second expression vectors can be stably co-transfected into a host cell to produce a multimeric protein.

[0285] To achieve the highest protein yield, different ratios of the first and second expression vectors can be tested to determine the optimal ratio for transfection into host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix.

[0286] Clones can be cultured under conditions suitable for bioreactor scale-up and sustained expression of the proteins of the invention. Proteins can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.

[0287] (i) Drug substance preparation In some embodiments, the heterodimeric Fc-fusion protein of the present disclosure, e.g., DF hIL12-Fc si, is produced in eukaryotic cells, e.g., Chinese hamster ovary (CHO) cells. In certain embodiments, the heterodimeric Fc-fusion protein of the present disclosure, e.g., DF hIL12-Fc si, is produced in CHO cells in suspension culture (e.g., in a shake flask). In certain embodiments, a vial of CHO cells is thawed and passaged two or more times before the protein is produced (e.g., two, three, four, five, or six times). In certain embodiments, a vial of CHO cells is thawed and passaged four times before the protein is produced. In certain embodiments, CHO cells from the fourth passage are used to inoculate the culture medium of the first bioreactor. In certain embodiments, the first bioreactor has a volume of about 40 L, about 45 L, about 50 L, about 55 L, or about 60 L. In certain embodiments, the first bioreactor has a volume of about 50 L. In certain embodiments, CHO cells from the broth of the first bioreactor are used to inoculate the broth of the production bioreactor. In certain embodiments, the production bioreactor has a volume of about 180 L, about 185 L, about 190 L, about 195 L, about 200 L, about 205 L, about 210 L, about 215 L, or about 220 L. In certain embodiments, the final broth volume of the production bioreactor is about 180 L. In certain embodiments, the CHO cells are grown in a growth medium supplemented with L-glutamine (e.g., 6 mM L-glutamine). In certain embodiments, the CHO cells are grown at a temperature of about 37° C. In certain embodiments, the culture conditions are monitored daily (e.g., for glucose, for lactate, for pH).

[0288] In some embodiments, the production bioreactor adjusts the dissolved oxygen in the culture medium by supplementing air and oxygen. In some embodiments, the production bioreactor adjusts the pH by adding carbon dioxide gas and / or sodium carbonate base. In some embodiments, the production bioreactor is spot-checked daily for cell density and viability until a target cell viability is reached. In certain embodiments, the target cell viability is about 10 x 10 6 Live cells / mL, approximately 11×10 6 Live cells / mL, approximately 12×10 6 Live cells / mL, approximately 13×10 6 Live cells / mL, approximately 14×10 6 Live cells / mL, approximately 15×10 6 Live cells / mL, approximately 16×10 6 Live cells / mL, approximately 17×10 6 Live cells / mL, approximately 18×10 6 Live cells / mL, approximately 19×10 6 viable cells / mL, or approximately 20 x 10 6 In certain embodiments, the target cell viability is about 14 x 10 viable cells / mL. 6viable cells / mL. In certain embodiments, after reaching the target cell viability, the temperature is shifted from about 37°C to about 33°C for medium harvest. In certain embodiments, the medium is harvested when the viability is greater than about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, or about 90%. In certain embodiments, the medium is harvested when the viability is greater than about 85%. In certain embodiments, the culture conditions are monitored daily (e.g., for glucose, lactate, and pH) during the culture period. In certain embodiments, the titer of DF hIL12-Fc si is monitored in the culture starting at about day 8 (e.g., day 6, day 7, day 8, day 9, or day 10). In certain embodiments, the culture medium is supplemented with a nutrient feed concentrate, a concentrated glucose solution, and / or an antifoam agent. In some embodiments, the cells are cultured for about 7 to about 21 days, about 8 to about 20 days, about 9 to about 19 days, about 10 to about 18 days, about 11 to about 17 days, about 12 to about 16 days, or about 11 to about 15 days. In certain embodiments, the cells are cultured for about 14 days.

[0289] In some embodiments, the production bioreactor is clarified by depth filtration prior to purification of a protein of the present disclosure, e.g., DF hIL12-Fc si. In certain embodiments, clarification uses a two-stage disposable depth filtration system consisting of DOHC and XOHC filters. In certain embodiments, prior to filtration, the production bioreactor temperature is adjusted to about 18°C ​​and the dissolved oxygen setpoint is increased to about 70% saturation. In certain embodiments, the harvest filter is rinsed with water for injection (WFI) and then equilibrated with buffer. In some embodiments, the cell suspension is passed through the harvest filter using a peristaltic pump, and the product is collected by flushing the filter. In certain embodiments, the pressure is monitored and maintained below about 25 psig (e.g., below about 25 psig, below about 20 psig, or below about 15 psig). The filtrate is then filtered through a 0.45 / 0.2 μm membrane and stored, for example, in a sterile bag.

[0290] In some embodiments, purification of a heterodimeric Fc-fusion protein of the present disclosure, e.g., DF hIL12-Fc si, comprises or consists of three chromatography steps and two virus clearance steps. In certain embodiments, the three chromatography steps comprise or consist of Protein A capture chromatography, mixed-mode chromatography, and cation exchange chromatography. In certain embodiments, a clarified harvest containing a heterodimeric Fc-fusion protein of the present disclosure, e.g., DF hIL12-Fc si, is captured by Protein A capture chromatography (e.g., using a Protein A resin column). In certain embodiments, Protein A capture chromatography removes process-related impurities (e.g., DNA, host cell proteins), media additives, and allows for volume reduction. In certain embodiments, a Protein A resin column is first equilibrated with a buffer containing 20 mM Tris, 150 mM NaCl, pH approximately 7.5. In certain embodiments, after loading, the column is washed with equilibration buffer to remove unbound or loosely bound impurities. In certain embodiments, after the first wash, the column is washed a second time with a buffer containing 50 mM acetate at a pH of about 5.4. In certain embodiments, the second wash lowers the pH and prepares the column for elution. In certain embodiments, DF hIL12-Fc si is eluted with a buffer containing 50 mM acetate and 100 mM arginine at a pH of about 3.7. In certain embodiments, DF hIL12-Fc si is collected at a 280 nm UV wavelength starting at a 1.25 AU / cm rise and then ending at a 1.25 AU / cm fall. In certain embodiments, the eluate is collected in one pool, and each column cycle is treated separately with low-pH viral inactivation.

[0291] In certain embodiments, the virus removal step comprises low pH inactivation and nanofiltration. In certain embodiments, the Protein A eluate is incubated at low pH to inactivate potentially present viruses. In certain embodiments, the pH of the eluate is adjusted with acetic acid, e.g., 0.5 M acetic acid, and incubated for at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 60 minutes, at least 65 minutes, at least 70 minutes, at least 75 minutes, at least 80 minutes, at least 85 minutes, or at least 90 minutes. In certain embodiments, the pH of the eluate is adjusted with acetic acid, e.g., 0.5 M acetic acid, and incubated for at least 60 minutes. In certain embodiments, the acetic acid adjusts the pH to about 3.55-3.75, e.g., about 3.60-3.70, or about 3.65. In certain embodiments, the acetic acid adjusts the pH to about 3.65. In certain embodiments, after the low pH incubation, the pH is raised, e.g., with Tris base, e.g., with 2 M Tris base. In certain embodiments, the pH is raised to a neutral pH of about 5.1, about 5.2, or about 5.3. In certain embodiments, the Protein A eluate is filtered through a 0.2 μm filtration assembly. In certain embodiments, low pH inactivation occurs prior to nanofiltration. In certain embodiments, nanofiltration occurs prior to low pH inactivation.

[0292] In some embodiments, after the viral clearance step, the pool is filtered through an intermediate depth filter, such as an X0SP intermediate depth filter. In certain embodiments, the DF hIL12-Fc si has a density of about 500 to about 1000 g / m 2 (For example, about 400 to about 1100 g / m 2 , about 450~1050g / m 2 , about 500~about 1000g / m 2 In certain embodiments, the conductivity of the X0SP pool is adjusted to less than 6.0 mS / cm with acetate at a pH of about 5.2, e.g., 50 mM acetate, prior to mixed-mode chromatography.

[0293] In some embodiments, mixed-mode chromatography is performed to remove high molecular weight (HMW) species. In certain embodiments, the column is equilibrated and loaded with, for example, a buffer containing 50 mM acetate at a pH of about 5.2. In certain embodiments, after loading, the column is washed with, for example, a buffer containing 50 mM acetate and 250 mM NaCl at a pH of about 5.2. In certain embodiments, collection begins with a 0.625 AU / cm rise and ends with a 1.50 AU / cm fall, using 280 nm UV detection. In certain embodiments, after collection, each cycle is passed through a filter train with a final 0.2 μm filter.

[0294] In some embodiments, cation exchange chromatography is performed to remove product-related impurities (e.g., HMW species, low molecular weight (LMW) species) and process-related impurities. In some embodiments, multiple cation exchange chromatography cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cycles) are performed for each product lot. In certain embodiments, the cycles are pooled and diluted, e.g., with a buffer containing 50 mM Tris at pH 7.4. In certain embodiments, the pooled sample is adjusted to a pH of about 7.3, about 7.4, about 7.5, about 7.6, or about 7.7 with a basic solution, e.g., Tris, e.g., 2 M Tris base. In certain embodiments, the pooled sample is adjusted to a pH of about 7.5. In certain embodiments, the column is equilibrated with a buffer containing 50 mM Tris at pH 7.4. In certain embodiments, the elution solution comprises a gradient of 50 mM Tris at a pH of about 7.4 (Buffer A) and 50 mM Tris and 0.5 M NaCl at a pH of about 7.4 (Buffer B). In certain embodiments, product collection begins with a 2.5 AU / cm rise and a 4.5 AU / cm fall as detected by UV at 280 nm. In certain embodiments, after collection, each cycle is passed through a filter train with a final 0.2 μm filter.

[0295] In some embodiments, the cycle liquid from the cation exchange chromatography is nanofiltered to remove viruses. In certain embodiments, the eluate is first passed through a prefilter and a nominal filter (e.g., a nominal filter of about 20 nm). In certain embodiments, the system is equilibrated with a buffer, for example, a buffer containing 50 mM Tris and 265 mM NaCl at a pH of about 7.4. In certain embodiments, after loading, the system is rinsed with an equilibration buffer, for example, a buffer containing 50 mM Tris and 265 mM NaCl at a pH of about 7.4. In certain embodiments, the filtrate is filtered through a membrane, for example, a 0.2 μm membrane.

[0296] In some embodiments, the filtrate is subjected to ultrafiltration and diafiltration (UF / DF). In certain embodiments, the ultrafiltration and diafiltration are performed using a membrane with a molecular weight cutoff of about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, or about 40 kDa. In certain embodiments, the ultrafiltration and diafiltration are performed using a membrane with a molecular weight cutoff of about 30 kDa. In certain embodiments, the system is equilibrated with a buffer, for example, a buffer containing 50 mM Tris and 265 mM NaCl at a pH of about 7.4. In certain embodiments, the virus filtrate pool is concentrated to a target of about 5.0 g / L. In certain embodiments, a buffer exchange is performed with at least 7 diavolumes (e.g., 7, 8, or 9 diavolumes) of a buffer containing 20 mM citrate at a pH of about 6.5. In some embodiments, after diafiltration, a second concentration step is performed to a target of about 11.0 g / L. In certain embodiments, the product is diluted with diafiltration buffer to a final target concentration of about 7.5 g / L.

[0297] In some embodiments, a 20 mM citrate, 18% (w / v) sucrose, 3% (w / v) mannitol, 0.03% (w / v) polysorbate 80, pH 6.5 stock solution is spiked into the UF / DF pool to target a final concentration of 20 mM citrate, 6% (w / v) sucrose, 1% (w / v) mannitol, 0.01% (w / v) polysorbate 80 in the drug substance.

[0298] In certain embodiments, the formulated retentate is filtered, e.g., through a 0.2 μm membrane, into a final drug substance storage container. In certain embodiments, the final fill volume is about 1.0 L. In certain embodiments, the final drug substance storage container comprises a 2 L polycarbonate bottle with a polypropylene lid. In certain embodiments, each bottle is aseptically spot-checked, labeled, and frozen at below -65°C (e.g., -65°C, -70°C, -75°C, -80°C, or lower).

[0299] (ii) Preparation of drug products In some embodiments, a frozen drug substance comprising a heterodimeric Fc-fusion protein of the present disclosure, e.g., DF hIL12-Fc si, is thawed in the dark at about 2-8°C for more than 96 hours (e.g., 96 hours, 120 hours, 144 hours, 168 hours, or more). In certain embodiments, a pH 6.0 buffer solution consisting of 20 mM citrate, 6% (w / v) sucrose, 1% (w / v) mannitol, and 0.01% polysorbate 80 (w / v) is prepared. In certain embodiments, the buffer solution is prepared by adding solid sodium citrate dihydrate, citric acid monohydrate, sucrose, and mannitol to water for injection (WFI) and mixing until dissolved. In certain embodiments, the citrate salt in the drug product comprises or consists of solid sodium citrate dihydrate and / or citric acid monohydrate. In some embodiments, a polysorbate 80 stock solution is prepared in WFI and added to the buffer solution. In certain embodiments, the acceptable pH of the buffer is about 6.5±0.4 (e.g., pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, or pH 6.9). In certain embodiments, the buffer is diluted with WFI and tested for an acceptable pH and osmolality of about 6.5±0.4 (e.g., pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, or pH 6.9). In certain embodiments, the buffer is filtered through a membrane, e.g., a 0.2 μm membrane.

[0300] In some embodiments, the weight of the drug substance is used to calculate the target batch volume. In certain embodiments, the drug substance is added to a buffer solution in a carboy (e.g., a 10 L carboy) to about 80% of the calculated batch volume and mixed. In certain embodiments, this 80% drug product solution is tested for an acceptable pH tolerance of about 6.5±0.3 (e.g., pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, or pH 6.8). In certain embodiments, the 80% drug product solution is tested for protein concentration by absorbance at 280 nm using an extinction coefficient of 1.44 L / (g×cm).

[0301] In some embodiments, the buffer components are designed to produce a pH of about 6.5. In certain embodiments, the buffer step may involve titration with 1 N sodium hydroxide or 1 N hydrochloric acid to bring the pH within an acceptable pH range of about 6.5±0.4 (e.g., pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, or pH 6.9). In certain embodiments, the 80% bulk drug product step may involve titration with 1 N sodium hydroxide or 1 N hydrochloric acid to bring the pH within an acceptable pH range of about 6.5±0.3 (e.g., pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, or pH 6.8).

[0302] In some embodiments, the target concentration of a heterodimeric Fc-fusion protein of the present disclosure, e.g., DF hIL12-Fc si, is about 1 mg / mL. In some embodiments, the protein concentration is confirmed by absorbance at 280 nm, with an acceptable criterion of 1.0±0.2 mg / mL (e.g., 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL). In some embodiments, a sample is taken to confirm an acceptable pH and osmolality of about 6.5±0.3 (e.g., pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, or pH 6.8).

[0303] In some embodiments, the compounded bulk drug product solution is passed through a filter, eg, a sterile 0.2 μm filter, for bioburden reduction and placed into a carboy, eg, a 10 L carboy, and held until sterile filtration and filling.

[0304] In some embodiments, filtration is performed by passing the bulk drug product through two filter capsules in series, each consisting of a 0.45 μm polyethersulfone (PES) prefilter membrane and a 0.2 μm PES sterilization membrane. In certain embodiments, the drug product is filtered and placed into sterile disposable filling bags in a Grade B area under the control of a filling suite. In certain embodiments, both sterile filter capsules are tested for integrity by post-filtration bubble point using WFI with an acceptance criterion of >3200 mbar.

[0305] In some embodiments, the bulk drug product solution is filled from disposable bags that are just outside the restricted access barrier system (RABS). In certain embodiments, the product is filled into vials, for example, pre-fabricated 2R borosilicate Type I vials.

[0306] In certain embodiments, the vials are stoppered, for example, with sterile 13 mm rubber ceramic stoppers and crimped with a 13 mm aluminum seal. In certain embodiments, the vial has a fill volume of 1.3 mL ± 5% (i.e., 1.235 mL to 1.365 mL). In certain embodiments, the vials are transferred to storage at 2 to 8°C. In certain embodiments, the vials are stored at 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C.

[0307] (k) Pharmaceutical preparations The present disclosure also features a pharmaceutical composition containing an effective amount of the protein described herein. The composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition to ensure a suitable formulation. The term "excipient" as used herein refers to any non-therapeutic agent added to a formulation to impart desired physical or chemical properties, such as pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability.

[0308] (i) Excipients and pH One or more excipients in the pharmaceutical formulation of the present invention include a buffer. The term "buffer" as used herein refers to one or more components that, when added to an aqueous solution, have the ability to protect the solution from pH fluctuations upon addition of acid or alkali or dilution with a solvent. In addition to phosphate buffers, glycinate, carbonate, citrate, histidine buffers, etc. can be used, in which case sodium, potassium, or ammonium ions can serve as counterions.

[0309] In certain embodiments, the buffer or buffer system comprises at least one buffer having a buffering range that overlaps entirely or partially with the pH range of 5.5 to 7.4. In certain embodiments, the buffer has a pKa of about 6.5±0.5. In certain embodiments, the buffer comprises a citrate buffer. In a specific embodiment, the citrate buffer comprises sodium citrate dihydrate and citric acid monohydrate. In certain embodiments, citrate is present at a concentration of about 5 to about 100 mM, about 10 to about 100 mM, about 15 to about 100 mM, about 20 to about 100 mM, about 5 to about 50 mM, about 10 to about 50 mM, about 15 to about 100 mM, about 20 to about 100 mM, about 5 to about 25 mM, about 10 to about 25 mM, about 15 to about 25 mM, about 20 to about 25 mM, about 5 to about 20 mM, about 10 to about 20 mM, or about 15 to about 20 mM. In certain embodiments, citrate is present at a concentration of about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 50 mM. In certain embodiments, citrate is present at a concentration of 20 mM.

[0310] The pharmaceutical formulation of the present invention may have a pH of 6.0 to 7.0. For example, in certain embodiments, the pharmaceutical formulation has a pH of 6.0 to 7.0 (i.e., 6.5±0.5), 6.1 to 6.9 (i.e., 6.5±0.4), 6.2 to 6.8 (i.e., 6.5±0.3), 6.3 to 6.7 (i.e., 6.5±0.2), 6.4 to 6.6 (i.e., 6.5±0.1), or 6.45 to 6.65 (i.e., 6.5±0.05). In certain embodiments, the pharmaceutical formulation has a pH of 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, or about 7.0. In certain embodiments, the pharmaceutical formulation has a pH of 6.5. Based on scientific rounding rules, a pH between 6.45 and 6.55 should be rounded to 6.0.

[0311] In certain embodiments, the buffer system of the pharmaceutical formulation comprises 10-25 mM citrate at a pH of 6.5±0.2. In certain embodiments, the buffer system of the pharmaceutical formulation comprises 20 mM citrate at a pH of 6.5±0.2. In certain embodiments, the buffer system of the pharmaceutical formulation comprises 10-25 mM citrate at a pH of 6.5±0.05. In certain embodiments, the buffer system of the pharmaceutical formulation comprises 20 mM citrate at a pH of 6.5±0.05.

[0312] One or more excipients in the pharmaceutical formulation of the present invention further comprise a sugar or sugar alcohol. Sugars and sugar alcohols are useful as heat stabilizers in pharmaceutical formulations. In certain embodiments, the pharmaceutical formulation comprises a sugar, such as a monosaccharide (e.g., glucose, xylose, or erythritol), a disaccharide (e.g., sucrose, trehalose, maltose, or galactose), or an oligosaccharide (e.g., stachyose). In specific embodiments, the pharmaceutical formulation comprises sucrose. In certain embodiments, the pharmaceutical formulation comprises a sugar alcohol, for example, a sugar alcohol derived from a monosaccharide (e.g., mannitol, sorbitol, or xylitol), a sugar alcohol derived from a disaccharide (e.g., lactitol or maltitol), or a sugar alcohol derived from an oligosaccharide. In specific embodiments, the pharmaceutical formulation comprises mannitol.

[0313] The amount of sugar or sugar alcohol contained in the formulation can vary depending on the specific context and intended purpose of the formulation. In certain embodiments, the pharmaceutical formulation contains 0% w / v to about 12% w / v, about 1% w / v to about 11% w / v, about 2% w / v to about 10% w / v, about 3% w / v to about 9% w / v, about 3% w / v to about 12% w / v, about 4% w / v to about 8% w / v, or about 5% w / v to about 7% w / v of sugar or sugar alcohol. In certain embodiments, the pharmaceutical formulation comprises 0% to about 2% w / v, about 0.5% to about 1.5% w / v, about 0.6% to about 1.4% w / v, about 0.7% to about 1.3% w / v, about 0.8% to about 1.2% w / v, or about 0.9% to about 1.1% w / v of a sugar or sugar alcohol. In certain embodiments, the pharmaceutical formulation comprises about 0% w / v, about 0.5% w / v, about 1% w / v, about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 8% w / v, about 9% w / v, or about 10% w / v of a sugar or sugar alcohol. In a specific embodiment, the pharmaceutical formulation comprises about 6% w / v sugar or sugar alcohol (e.g., sucrose). In a specific embodiment, the pharmaceutical formulation comprises about 1% w / v sugar or sugar alcohol (e.g., mannitol). In a specific embodiment, the pharmaceutical formulation comprises about 6% w / v sugar or sugar alcohol (e.g., sucrose) and about 1% w / v second sugar or sugar alcohol (e.g., mannitol).

[0314] One or more excipients in the pharmaceutical formulations disclosed herein further comprise a surfactant. The term "surfactant," as used herein, refers to a surface-active molecule having both a hydrophobic portion (e.g., an alkyl chain) and a hydrophilic portion (e.g., carboxyl and carboxylate groups). Surfactants are useful in pharmaceutical formulations to reduce aggregation of therapeutic proteins. Surfactants suitable for use in pharmaceutical formulations are generally non-ionic surfactants, including, but not limited to, polysorbates (e.g., polysorbate 20 or 80); poloxamers (e.g., poloxamer 188); sorbitan esters and derivatives; Triton; sodium laurel sulfate; sodium octyl glycoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl betaine, or cetyl betaine; and lauric acid amidopropyl betaine. Examples of surfactants include cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearamidopropyl betaine (e.g., lauroamidopropyl); myristamidopropyl dimethylamine, palmidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium cocoyl methyl taurate or disodium methyl oleyl taurate; and MONAQUAT™ series (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropyl glycol, and ethylene-propylene glycol copolymers (e.g., Pluronic, PF68, etc.). In certain embodiments, the surfactant is a polysorbate.In certain embodiments, the surfactant is polysorbate 80.

[0315] The amount of nonionic surfactant included in the pharmaceutical formulation of the present invention can vary depending on the specific desired properties of the formulation and the particular circumstances and purposes for which the formulation is intended to be used. In certain embodiments, the pharmaceutical formulation comprises 0.005% to about 0.5%, about 0.005% to about 0.2%, about 0.005% to about 0.1%, about 0.005% to about 0.05%, about 0.005% to about 0.02%, about 0.005% to about 0.01%, about 0.01% to about 0.5%, about 0.01% to about 0.2%, about 0.01% to about 0.1%, about 0.01% to about 0.05%, or about 0.01% to about 0.02% of a nonionic surfactant (e.g., polysorbate 80). In certain embodiments, the pharmaceutical formulation comprises about 0.005%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, or about 0.5% non-ionic surfactant (e.g., polysorbate 80).

[0316] The pharmaceutical formulations of the present invention may further comprise one or more other substances, such as a bulking agent or preservative. A "bulking agent" is a compound that adds mass to the lyophilization mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitating the production of an essentially uniform lyophilized cake that maintains an open-pore structure). Illustrative bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain such bulking agents. Preservatives may reduce bacterial activity and, for example, facilitate the production of multi-use (multi-dose) formulations.

[0317] (ii) Exemplary Formulations In a specific embodiment, a pharmaceutical formulation of the invention comprises a heterodimeric Fc fusion protein, citrate, a sugar (e.g., sucrose), a sugar alcohol (e.g., mannitol), and a polysorbate (e.g., polysorbate 80) at a pH of 6.0 to 7.0.

[0318] In certain embodiments, the pharmaceutical formulation comprises 0.5 to 1.5 mg / mL of the heterodimeric Fc-fusion protein, 10 to 30 mM citrate, 4% w / v to 8% w / v of a sugar (e.g., sucrose), 0.5% w / v to 1.5% w / v of a sugar alcohol (e.g., mannitol), and 0.005% to 0.05% of a polysorbate (e.g., polysorbate 80) at a pH of 6.5 to 7.5. In certain embodiments, the pharmaceutical formulation comprises 0.5 to 1.5 mg / mL of the heterodimeric Fc-fusion protein, 20 mM citrate, 6% w / v of a sugar (e.g., sucrose), 0.5% w / v to 1.5% w / v of a sugar alcohol (e.g., mannitol), and 0.01% of a polysorbate (e.g., polysorbate 80) at a pH of 6.0 to 7.0. In certain embodiments, the pharmaceutical formulation comprises 0.5-1.5 mg / mL of the heterodimeric Fc-fusion protein, 20 mM citrate, 6% w / v of a sugar (e.g., sucrose), 1% w / v of a sugar alcohol (e.g., mannitol), and 0.01% polysorbate (e.g., polysorbate 80) at pH 6.3-6.7. In certain embodiments, the pharmaceutical formulation comprises 0.5-1.5 mg / mL of the heterodimeric Fc-fusion protein, 20 mM citrate, 6% w / v of a sugar (e.g., sucrose), 1% w / v of a sugar alcohol (e.g., mannitol), and 0.01% polysorbate (e.g., polysorbate 80) at pH 6.45-6.55.

[0319] (iii) Stability of Heterodimeric Fc Fusion Proteins and Formulations The pharmaceutical formulations of the present invention exhibit a high degree of stability. A pharmaceutical formulation is stable when the heterodimeric Fc-fusion protein in the formulation retains acceptable physical properties, chemical structure, and / or biological function after storage under specified conditions. In certain embodiments, the pharmaceutical formulation is a clear liquid and does not contain visible particulate matter. In certain embodiments, thermal stability is tested at 5°C, 50°C, and after freeze-thaw cycles (e.g., after five freeze-thaw cycles).

[0320] Stability can be measured by determining the percentage of heterodimeric Fc-fusion proteins in a formulation that retain a native conformation after storage at a specified temperature for a specified length of time. The percentage of protein in the native conformation can be determined, for example, by size exclusion chromatography (e.g., size-exclusion high-performance liquid chromatography, SEC-HPLC), where the protein in the native conformation is either not aggregated (elutes in the high molecular weight fraction) or is degraded (elutes in the low molecular weight fraction). In certain embodiments, after two weeks of incubation at 2-8°C, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% of the heterodimeric Fc-fusion proteins have a native conformation as determined by size exclusion chromatography. In certain embodiments, after freeze-thawing, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% of the heterodimeric Fc-fusion proteins have a native conformation as determined by size exclusion chromatography. In certain embodiments, greater than about 75%, greater than about 76%, greater than about 77%, greater than about 78%, greater than about 79%, greater than about 80%, greater than about 81%, greater than about 82%, greater than about 83%, greater than about 84%, or greater than about 85% of the heterodimeric Fc-fusion proteins have a native conformation as determined by size exclusion chromatography after two weeks of incubation at 50° C. In certain embodiments, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, or less than about 1% of the heterodimeric Fc-fusion proteins form high molecular weight complexes (i.e., have a higher molecular weight than the native protein) as determined by size exclusion chromatography after two weeks of incubation at 2-8° C. In certain embodiments, after incubation at 50°C for 2 weeks, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the heterodimeric Fc-fusion proteins form high molecular weight complexes (i.e., have a higher molecular weight than the native protein) as determined by size exclusion chromatography.In certain embodiments, after freeze-thawing, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, or less than about 1% of the heterodimeric Fc-fusion proteins form high molecular weight complexes (i.e., have a higher molecular weight than the native protein) as determined by size exclusion chromatography. In certain embodiments, after 2 weeks of incubation at 2-8°C, less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, or less than about 1% of the heterodimeric Fc-fusion proteins are degraded (i.e., have a lower molecular weight than the native protein) as determined by size exclusion chromatography. In certain embodiments, after two weeks of incubation at 50° C., less than about 1%, less than about 1.5%, less than about 2%, less than about 2.5%, or less than about 3% of the heterodimeric Fc-fusion proteins are degraded (i.e., have a lower molecular weight than the native protein) as determined by size exclusion chromatography. In certain embodiments, after freeze-thawing, less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, or less than about 1% of the heterodimeric Fc-fusion proteins are degraded (i.e., have a lower molecular weight than the native protein) as determined by size exclusion chromatography.

[0321] SEC-HPLC can provide a measure of the purity of a pharmaceutical formulation by the percentage of protein, e.g., a heterodimeric Fc-fusion protein, that occupies the main peak. The purity profile is determined by the area of ​​the main peak as a percentage of the total area detected in the SEC-HPLC analysis. In some embodiments, the purity profile of the pharmaceutical formulation is greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99%. In certain embodiments, the purity profile of the pharmaceutical formulation is greater than about 99.0%. In some embodiments, the purity profile of the pharmaceutical formulation is greater than about 75%, greater than about 80%, greater than about 81%, greater than about 82%, greater than about 83%, greater than about 84%, or greater than about 85% after incubating the pharmaceutical formulation at 50°C for 2 weeks. In certain embodiments, the purity profile of the pharmaceutical formulation is about 85.2%. In some embodiments, the purity profile of the pharmaceutical formulation is greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 98.5% after subjecting the pharmaceutical formulation to five freeze-thaw cycles. In certain embodiments, the purity profile of the pharmaceutical formulation is about 98.9%.

[0322] Stability can also be measured by determining protein solution parameters by dynamic light scattering. Z-average and polydispersity index (PDI) values ​​indicate the average particle size in solution, and these measurements increase when aggregates are present in the solution. Monomer %Pd values ​​indicate the spread of different monomers detected, with lower values ​​indicating a monodisperse solution, which is preferred. Monomer size detected by DLS is useful for confirming that the predominant population is monomer and for characterizing any higher-order aggregates that may be present. In certain embodiments, the Z-average value of the pharmaceutical formulation does not increase by more than 5%, 10%, or 15% after two weeks of incubation at 2-8°C. In certain embodiments, the Z-average value of the pharmaceutical formulation does not increase by more than two-fold, three-fold, four-fold, or five-fold after freeze-thawing. In certain embodiments, the Z-average value of the pharmaceutical formulation does not increase by more than about 10%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, more than about 150%, or more than about 200% after incubation at 50° C. for 2 weeks. In some embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 15 nm, less than about 14 nm, less than about 13 nm, or less than about 12 nm, as measured by dynamic light scattering at 25° C. In specific embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 11.6 nm, as measured by dynamic light scattering at 25° C. In some embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 20 nm, less than about 19 nm, less than about 18 nm, less than about 17 nm, less than about 16 nm, less than about 15.5 nm, less than about 15 nm, or less than about 14.5 nm, when measured by dynamic light scattering at 25° C. after incubating the pharmaceutical formulation at 50° C. for 2 weeks. In a specific embodiment, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of about 14.4 nm.In some embodiments, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 20 nm, less than about 19 nm, less than about 18 nm, less than about 17 nm, less than about 16.5 nm, less than about 16 nm, or less than about 15.5 nm, as measured by dynamic light scattering at 25° C. after the pharmaceutical formulation has been subjected to five freeze-thaw cycles. In a specific embodiment, the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of about 15.3 nm.

[0323] In certain embodiments, the PDI value of the pharmaceutical formulation does not increase by more than about 2-fold, about 3-fold, about 4-fold, or about 5-fold after incubation at 2-8°C for 2 weeks. In certain embodiments, the PDI value of the pharmaceutical formulation does not increase by more than about 2-fold, about 3-fold, about 4-fold, about 5-fold, or about 6-fold after freeze-thawing. In certain embodiments, the PDI value of the pharmaceutical formulation does not increase by more than about 2-fold, about 3-fold, about 4-fold, about 5-fold, or about 6-fold after incubation at 50°C for 2 weeks. In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.30, less than about 0.29, less than about 0.28, less than about 0.27, less than about 0.26, or less than about 0.25, as measured by dynamic light scattering at 25°C. In certain embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is about 0.26. In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.30, less than about 0.29, less than about 0.28, less than about 0.27, or less than about 0.26, as measured by dynamic light scattering at 25°C after incubating the pharmaceutical formulation at 50°C for two weeks. In certain embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.25. In some embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.40, less than about 0.35, or less than about 0.34, as measured by dynamic light scattering at 25°C after subjecting the pharmaceutical formulation to five freeze-thaw cycles. In certain embodiments, the polydispersity index of the heterodimeric Fc-fusion protein in the pharmaceutical formulation is less than about 0.33.

[0324] Stability can also be measured by determining the thermal stability of a protein solution by differential scanning fluorimetry (DSF). DSF allows for the quantification of changes in the thermal denaturation temperature and stability of a protein under various test conditions, e.g., buffer or pH. In some embodiments, DSF can be used to quantify the change in the thermal unfolding temperature (also known as melting temperature), T m1 and T m2 provide 。 In certain embodiments, the T m1 is greater than about 60°C, greater than about 61°C, greater than about 62°C, greater than about 63°C, greater than about 64°C, greater than about 65°C, or greater than about 66°C. In certain embodiments, the T m1 is greater than about 70° C., greater than about 71° C., greater than about 72° C., greater than about 73° C., greater than about 74° C., greater than about 75° C., greater than about 76° C., or greater than about 77° C. In specific embodiments, the pharmaceutical formulation has a T of about 67.0° C. m1 and a T of approximately 77.3°C m2 In certain embodiments, the thermostability profile is defined by T m1 and / or T m2 changes by less than 2°C, less than 1.5°C, or less than 1°C when the pharmaceutical formulation is incubated at 50°C for 1 week compared to the same pharmaceutical formulation incubated at 5°C for 1 week. In specific embodiments, T m1 changes by less than 1° C. when a pharmaceutical formulation is incubated at 50° C. for 1 week compared to the same pharmaceutical formulation incubated at 5° C. for 1 week. In a specific embodiment, T m2 changes by less than 1°C when a pharmaceutical formulation is incubated at 50°C for 1 week compared to the same pharmaceutical formulation incubated at 5°C for 1 week.

[0325] In some embodiments, the DSF is the temperature at which protein aggregation begins to occur, T agg. In some embodiments, the pharmaceutical formulation agg.is greater than 60°C, greater than about 61°C, greater than about 62°C, greater than about 63°C, greater than about 64°C, greater than about 65°C, greater than about 66°C, or greater than about 67°C. agg changes by less than about 2°C, less than 1.5°C, or less than about 1°C when the pharmaceutical formulation is incubated at 50°C for 1 week compared to the same pharmaceutical formulation incubated at 5°C for 1 week. In certain embodiments, T agg changes by less than about 2°C, less than about 1.5°C, or less than about 1°C when the pharmaceutical formulation is incubated at 50°C for 1 week compared to the same pharmaceutical formulation incubated at 5°C for 1 week. In certain embodiments, T agg changes by less than about 1°C when a pharmaceutical formulation is incubated at 50°C for 1 week compared to the same pharmaceutical formulation incubated at 5°C for 1 week.

[0326] In some embodiments, pH is used to determine the stability of a pharmaceutical formulation. In some embodiments, the pH of the pharmaceutical formulation does not change by more than about 0.25, about 0.2, about 0.15, or about 0.1 after incubating the pharmaceutical formulation at 5° C. for 1 week. In certain embodiments, the pH of the pharmaceutical formulation does not change by more than about 0.2 or about 0.1 after incubating the pharmaceutical formulation at 5° C. for 1 week. In some embodiments, the pH of the pharmaceutical formulation does not change by more than about 0.25, about 0.2, about 0.15, or about 0.1 after incubating the pharmaceutical formulation at 50° C. for 1 week. In certain embodiments, the pH of the pharmaceutical formulation does not change by more than about 0.2 or about 0.1 after incubating the pharmaceutical formulation at 50° C. for 1 week.

[0327] An exemplary method for determining the stability of a heterodimeric Fc-fusion protein in a pharmaceutical formulation is described in Example 24 of the present disclosure.

[0328] (iv) Dosage Form The pharmaceutical formulation can be prepared and stored as a liquid formulation or a lyophilized form. In certain embodiments, the pharmaceutical formulation is a clear, colorless solution free of visible particulate matter. In certain embodiments, the pharmaceutical formulation is a liquid formulation stored at 2-8°C (e.g., 4°C), a frozen formulation stored at -20°C or below, or a frozen formulation stored at -65°C or below. Sugars and / or sugar alcohols are used as lyoprotectants in the formulation.

[0329] Prior to use as a pharmaceutical, the pharmaceutical formulation can be diluted or reconstituted in an aqueous carrier suitable for the route of administration. Other exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution. For example, when the pharmaceutical formulation is prepared for administration, it can be diluted in 0.9% sodium chloride (NaCl) solution. In a specific embodiment, the pharmaceutical formulation is diluted in 0.9% sodium chloride (NaCl) solution containing 0.01% polysorbate 80. In certain embodiments, the diluted pharmaceutical formulation is isotonic and suitable for administration by subcutaneous injection.

[0330] The pharmaceutical formulation contains the heterodimeric Fc fusion protein at a concentration suitable for storage. In certain embodiments, the pharmaceutical formulation contains the heterodimeric Fc fusion protein at a concentration of about 0.1 to about 2 mg / mL, about 0.2 to about 1.8 mg / mL, about 0.3 to about 1.7 mg / mL, about 0.4 to about 1.6 mg / mL, about 0.5 to about 1.5 mg / mL, about 0.6 to about 1.4 mg / mL, about 0.7 to about 1.3 mg / mL, about 0.8 to about 1.2 mg / mL, or about 0.9 to about 1.1 mg / mL. In certain embodiments, the pharmaceutical formulation comprises a heterodimeric Fc-fusion protein at a concentration of about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1 mg / mL, about 2 mg / mL, about 2.5 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, about 5 mg / mL, or about 10 mg / mL.

[0331] In certain embodiments, the pharmaceutical formulation comprises a heterodimeric Fc-fusion protein at a bulk concentration of about 1 g / L to about 10 g / L, about 2 g / L to about 8 g / L, about 4 g / L to about 6 g / L, or about 5 g / L. In certain embodiments, the pharmaceutical formulation comprises a heterodimeric Fc-fusion protein at a bulk concentration of about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 6 g / L, about 7 g / L, about 8 g / L, about 9 g / L, or about 10 g / L. In a specific embodiment, the pharmaceutical formulation comprises a heterodimeric Fc-fusion protein at a bulk concentration of about 5 g / L. In certain embodiments, the pharmaceutical formulation comprises a heterodimeric Fc-fusion protein at a dosage concentration of about 0.5 g / L to about 2 g / L, about 0.75 g / L to about 1.5 g / L, or about 0.9 g / L to about 1.1 g / L. In certain embodiments, the pharmaceutical formulation comprises a heterodimeric Fc-fusion protein at a dosage concentration of about 0.5 g / L, about 0.6 g / L, about 0.7 g / L, about 0.8 g / L, about 0.9 g / L, about 1 g / L, about 1.1 g / L, about 1.2 g / L, about 1.3 g / L, about 1.4 g / L, about 1.5 g / L, or about 2 g / L. In a specific embodiment, the pharmaceutical formulation comprises a heterodimeric Fc-fusion protein at a dosage concentration of about 1 g / L.

[0332] In certain embodiments, the pharmaceutical formulation is packaged in a container (e.g., a vial, bag, pen, or syringe). In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the amount of the heterodimeric Fc fusion protein in the container is suitable for administration as a single dose. In certain embodiments, the amount of the heterodimeric Fc fusion protein in the container is suitable for administration in multiple doses. In certain embodiments, the pharmaceutical formulation comprises the heterodimeric Fc fusion protein in an amount of about 0.1 to about 10 mg. In certain embodiments, the pharmaceutical formulation comprises the heterodimeric Fc fusion protein in an amount of about 0 to about 2 mg, about 0.2 to about 1.8 mg, about 0.3 to about 1.7 mg, about 0.4 to about 1.6 mg, about 0.5 to about 1.5 mg, about 0.6 to about 1.4 mg, about 0.7 to about 1.3 mg, about 0.8 to about 1.2 mg, or about 0.9 to about 1.1 mg. In certain embodiments, the pharmaceutical formulation comprises about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, or about 10 mg of the heterodimeric Fc-fusion protein. In a specific embodiment, the pharmaceutical formulation comprises about 1 mg of the heterodimeric Fc-fusion protein, e.g., DF-hIL-12-Fc si.

[0333] (l) Dosage Regimen and Therapeutic Use In another aspect, the disclosure provides a method of treating cancer, comprising administering to a subject in need thereof a heterodimeric Fc-fusion protein disclosed herein (e.g., DF-hIL-12-Fc si) on day 1 of an initial 3-week treatment cycle. In a specific embodiment, the heterodimeric Fc-fusion protein is administered to the subject only on day 1 of the initial 3-week treatment cycle.

[0334] In certain embodiments, the method comprises administering to a subject in need thereof a heterodimeric Fc-fusion protein in combination with an anti-PD-1 antibody, e.g., pembrolizumab, on day 1 of an initial 3-week treatment cycle. In certain embodiments, the heterodimeric Fc-fusion protein and the anti-PD-1 antibody are administered to the subject only on day 1 of the initial 3-week treatment cycle. In certain embodiments, administration of the PD-1 antibody occurs prior to administration of the heterodimeric Fc-fusion protein.

[0335] In certain embodiments, the method further comprises administering the heterodimeric Fc-fusion protein to the subject in one or more subsequent three-week treatment cycles after the initial treatment cycle, wherein the heterodimeric Fc-fusion protein is administered on day 1 of each subsequent treatment cycle. The subsequent treatment cycles are designed to maintain a constant level of heterodimeric Fc-fusion protein in the subject, wherein the subject receives the heterodimeric Fc-fusion protein once every three weeks or once every four weeks. In certain embodiments, the subject receives the heterodimeric Fc-fusion protein once every three weeks, once every four weeks, once every five weeks, or once every six weeks. In certain embodiments, the subject receives the heterodimeric Fc-fusion protein once every six weeks, i.e., once every other treatment cycle. In certain embodiments, the subject receives at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 subsequent treatment cycles. In certain embodiments, the subject receives subsequent cycles of treatment until the cancer regresses (i.e., a complete response). In certain embodiments, the subject has advanced (i.e., unresectable or metastatic) melanoma. In certain embodiments, the subject has advanced (i.e., unresectable or metastatic) renal cell carcinoma.

[0336] In certain embodiments, the method further comprises administering to the subject, after the initial treatment cycle, the heterodimeric Fc-fusion protein in combination with an anti-PD-1 antibody, e.g., pembrolizumab, for one or more subsequent three-week treatment cycles, wherein the heterodimeric Fc-fusion protein and anti-PD-1 antibody are administered on day 1 of each subsequent treatment cycle. The subsequent treatment cycles are designed to maintain a constant level of the heterodimeric Fc-fusion protein and anti-PD-1 antibody in the subject once every three weeks. In certain embodiments, the subject receives at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 subsequent treatment cycles. In certain embodiments, administration of the anti-PD-1 antibody precedes administration of the heterodimeric Fc-fusion protein. In certain embodiments, the subject receives the subsequent treatment cycles until the cancer regresses (i.e., a complete response). In certain embodiments, the subject has advanced (ie, unresectable or metastatic) urothelial carcinoma.

[0337] In certain embodiments, one or more doses during the initial and subsequent treatment cycles are 0.01 to about 3 μg / kg, about 0.01 to about 0.02 μg / kg, about 0.01 to about 0.05 μg / kg, about 0.05 to about 0.1 μg / kg, about 0.05 to about 0.5 μg / kg, about 0.05 to about 0.75 μg / kg, about 0.05 to about 1 μg / kg, about 0.05 to about 1.5 μg / kg, about 0.05 to about 2 μg / kg, about 0.05 to about 2.5 μg / kg, about 0.05 to about 3 μg / kg, about 0.1 to about 3 μg / kg, about 0 The heterodimeric Fc fusion protein may be administered in an amount of about 0.1 to about 1 μg / kg, about 0.5 to about 1 μg / kg, about 0.1 to about 2 μg / kg, about 0.5 to about 2 μg / kg, about 0.1 to about 0.5 μg / kg, about 0.1 to about 0.25 μg / kg, about 0.2 to about 1 μg / kg, about 0.2 to about 2 μg / kg, about 1 to about 1.2 μg / kg, about 1 to about 1.5 μg / kg, about 1 to about 2 μg / kg, about 1 to about 2.5 μg / kg, about 0.5 to about 2.5 μg / kg, about 1 to about 3 μg / kg, or about 0.5 to about 3 μg / kg. In certain embodiments, one or more doses during the initial and subsequent treatment cycles are about 0.01 μg / kg, about 0.02 μg / kg, about 0.03 μg / kg, about 0.04 μg / kg, about 0.05 μg / kg, about 0.1 μg / kg, about 0.15 μg / kg, about 0.2 μg / kg, about 0.25 μg / kg, about 0.3 μg / kg, about 0.35 μg / kg, about 0.4 μg / kg, about 0.45 μg / kg, about 0.5 μg / kg, about The heterodimeric Fc-fusion protein comprises an amount selected from the group consisting of 0.6 μg / kg, about 0.7 μg / kg, about 0.8 μg / kg, about 0.9 μg / kg, about 1 μg / kg, about 1.2 μg / kg, about 1.25 μg / kg, about 1.3 μg / kg, about 1.4 μg / kg, about 1.5 μg / kg, about 1.75 μg / kg, about 2 μg / kg, about 2.5 μg / kg, about 3 μg / kg, about 4 μg / kg, or about 5 μg / kg.

[0338] In certain embodiments, the doses during the initial and subsequent treatment cycles are each 0.01 to about 3 μg / kg, about 0.01 to about 0.02 μg / kg, about 0.01 to about 0.05 μg / kg, about 0.05 to about 0.1 μg / kg, about 0.05 to about 0.5 μg / kg, about 0.05 to about 0.75 μg / kg, about 0.05 to about 1 μg / kg, about 0.05 to about 1.5 μg / kg, about 0.05 to about 2 μg / kg, about 0.05 to about 2.5 μg / kg, about 0.05 to about 3 μg / kg, about 0.1 to about 3 μg / kg, about 0.05 ... The heterodimeric Fc fusion protein may be administered in an amount of about 0.1 to about 1 μg / kg, about 0.5 to about 1 μg / kg, about 0.1 to about 2 μg / kg, about 0.5 to about 2 μg / kg, about 0.1 to about 0.5 μg / kg, about 0.1 to about 0.25 μg / kg, about 0.2 to about 1 μg / kg, about 0.2 to about 2 μg / kg, about 1 to about 1.2 μg / kg, about 1 to about 1.5 μg / kg, about 1 to about 2 μg / kg, about 1 to about 2.5 μg / kg, about 0.5 to about 2.5 μg / kg, about 1 to about 3 μg / kg, or about 0.5 to about 3 μg / kg. In certain embodiments, each of the doses during the initial and subsequent treatment cycles is about 0.01 μg / kg, about 0.02 μg / kg, about 0.03 μg / kg, about 0.04 μg / kg, about 0.05 μg / kg, about 0.1 μg / kg, about 0.15 μg / kg, about 0.2 μg / kg, about 0.25 μg / kg, about 0.3 μg / kg, about 0.35 μg / kg, about 0.4 μg / kg, about 0.45 μg / kg, about 0.5 μg / kg, about 0 0.6 μg / kg, about 0.7 μg / kg, about 0.8 μg / kg, about 0.9 μg / kg, about 1 μg / kg, about 1.2 μg / kg, about 1.25 μg / kg, about 1.3 μg / kg, about 1.4 μg / kg, about 1.5 μg / kg, about 1.75 μg / kg, about 2 μg / kg, about 2.5 μg / kg, about 3 μg / kg, about 4 μg / kg, or about 5 μg / kg of the same amount of heterodimeric Fc fusion protein.

[0339] In certain embodiments, the doses during the initial and subsequent treatment cycles are each 0.01 to about 3 μg / kg, about 0.01 to about 0.02 μg / kg, about 0.01 to about 0.05 μg / kg, about 0.05 to about 0.1 μg / kg, about 0.05 to about 0.5 μg / kg, about 0.05 to about 0.75 μg / kg, about 0.05 to about 1 μg / kg, about 0.05 to about 1.5 μg / kg, about 0.05 to about 2 μg / kg, about 0.05 to about 2.5 μg / kg, about 0.05 to about 3 μg / kg, about 0.1 to about 3 μg / kg, about 0.05 ... The heterodimeric Fc fusion protein may be administered in an amount of about 0.1 to about 1 μg / kg, about 0.5 to about 1 μg / kg, about 0.1 to about 2 μg / kg, about 0.5 to about 2 μg / kg, about 0.1 to about 0.5 μg / kg, about 0.1 to about 0.25 μg / kg, about 0.2 to about 1 μg / kg, about 0.2 to about 2 μg / kg, about 1 to about 1.2 μg / kg, about 1 to about 1.5 μg / kg, about 1 to about 2 μg / kg, about 1 to about 2.5 μg / kg, about 0.5 to about 2.5 μg / kg, about 1 to about 3 μg / kg, or about 0.5 to about 3 μg / kg. In certain embodiments, each of the doses during the initial and subsequent treatment cycles is about 0.01 μg / kg, about 0.02 μg / kg, about 0.03 μg / kg, about 0.04 μg / kg, about 0.05 μg / kg, about 0.1 μg / kg, about 0.15 μg / kg, about 0.2 μg / kg, about 0.25 μg / kg, about 0.3 μg / kg, about 0.35 μg / kg, about 0.4 μg / kg, about 0.45 μg / kg, about 0.5 μg / kg, about 0 0.6 μg / kg, about 0.7 μg / kg, about 0.8 μg / kg, about 0.9 μg / kg, about 1 μg / kg, about 1.2 μg / kg, about 1.25 μg / kg, about 1.3 μg / kg, about 1.4 μg / kg, about 1.5 μg / kg, about 1.75 μg / kg, about 2 μg / kg, about 2.5 μg / kg, about 3 μg / kg, about 4 μg / kg, or about 5 μg / kg of the same amount of heterodimeric Fc fusion protein.

[0340] In certain embodiments, the heterodimeric Fc-fusion protein is administered subcutaneously. For example, in certain embodiments, the heterodimeric Fc-fusion protein is administered by subcutaneous injection, e.g., using a prefilled pen or syringe. In certain embodiments, the heterodimeric Fc-fusion protein is administered in a volume of about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1 mL, about 1.1 mL, or about 1.2 mL. In certain embodiments, the heterodimeric Fc-fusion protein is administered in a volume of about 1 mL. In certain embodiments, the heterodimeric Fc-fusion protein is administered at up to two injection sites (e.g., at one injection site or at two injection sites). In a specific embodiment, the heterodimeric Fc-fusion protein is administered in a single injection. In a specific embodiment, the heterodimeric Fc-fusion protein is administered in two injections, with the second injection being completed within 10 minutes of the first injection.

[0341] In certain embodiments, the anti-PD-1 antibody, e.g., pembrolizumab, is administered intravenously. In certain embodiments, the anti-PD-1 antibody is administered intravenously prior to administration of the heterodimeric Fc-fusion protein. In certain embodiments, the anti-PD-1 antibody is administered intravenously within 1 hour (e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 1 hour) before administration of the heterodimeric Fc-fusion protein. In certain embodiments, the PD-1 antibody is administered intravenously simultaneously with administration of the heterodimeric Fc-fusion protein.

[0342] Types of cancer that may be treated with the heterodimeric Fc-fusion proteins or pharmaceutical preparations disclosed herein include, but are not limited to, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma, primary mediastinal large B-cell lymphoma, bladder cancer, urothelial carcinoma, high-microsatellite instability carcinoma, colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma (RCC), endometrial cancer, cutaneous T-cell lymphoma, or triple-negative breast cancer. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a locally advanced or metastatic solid tumor. In certain embodiments, the cancer is melanoma. In certain embodiments, the cancer is renal cell carcinoma. In certain embodiments, the cancer is urothelial bladder cancer. In certain embodiments, the subject has clinical or radiological evidence of disease. In certain embodiments, the subject has measurable disease as determined by Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. In certain embodiments, the pharmaceutical preparation disclosed herein is administered as monotherapy. In certain embodiments, the pharmaceutical preparation disclosed herein is administered as combination therapy. In certain embodiments, subjects with confirmed complete response (CR) are treated with the pharmaceutical preparation for at least 12 months after confirmation, unless censoring criteria are met. In certain embodiments, the total duration of the multi-dose therapy is 24 months or less (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months, 24 months). In certain embodiments, the total duration of the multi-dose therapy is more than 24 months.

[0343] In certain embodiments, the subject treated by the methods disclosed herein has advanced melanoma. In certain embodiments, the subject has been treated with an anti-PD-1 antibody for at least 6 weeks and has confirmed disease progression. In certain embodiments, the subject has a BRAF-activating mutation, has been administered a BRAF inhibitor, and has confirmed disease progression after last-line treatment. In certain embodiments, the disease progression is confirmed by radiological or clinical findings. In certain embodiments, the subject does not have a BRAF-activating mutation.

[0344] In certain embodiments, the subject treated by the methods disclosed herein has advanced renal clear cell carcinoma (RCC). In certain embodiments, the subject has a clear cell component. In certain embodiments, the subject has previously received treatment with a checkpoint inhibitor, e.g., an anti-PD-1 / PD-L1 antibody, or VEGF therapy as monotherapy. In certain embodiments, the subject has previously received treatment with a checkpoint inhibitor, e.g., an anti-PD-1 / PD-L1 antibody, in combination with VEGF therapy. In certain embodiments, the subject has previously received treatment with a checkpoint inhibitor, e.g., an anti-PD-1 / PD-L1 antibody, in combination with platinum-based chemotherapy. In certain embodiments, the subject has not previously received treatment with a checkpoint inhibitor, e.g., an anti-PD-1 / PD-L1 antibody. In certain embodiments, the subject has previously received more than three lines of therapy.

[0345] In certain embodiments, the subject treated by the methods disclosed herein has advanced urothelial carcinoma. In certain embodiments, the advanced urothelial carcinoma is metastatic or unresectable. In certain embodiments, the subject has histologically or cytologically confirmed locally advanced or metastatic transitional cell carcinoma of the urothelium (including, but not limited to, the renal pelvis, ureter, urothelium, and urethra). In certain embodiments, the subject has received only one platinum-containing regimen (e.g., platinum plus another agent, such as gemcitabine, methotrexate, vinblastine, doxorubicin, etc.). In some embodiments, the subject has received only one platinum-containing regimen for inoperable locally advanced or metastatic urothelial carcinoma and has radiographic progression or recurrence within six months of the last dose of the adjuvant platinum-containing regimen. In certain embodiments, the subject has not received treatment with a checkpoint inhibitor (CPI) (e.g., anti-PD-1 or anti-PD-L1) as monotherapy or in combination with platinum-based chemotherapy. In certain embodiments, the subject has received no more than two lines of first-line therapy. In certain embodiments, the urothelial carcinoma is considered a failure of a first-line platinum-containing regimen.

[0346] In some embodiments, the drug delivery formulation for use in the method of treating cancer or the method of inhibiting tumor growth is about 0.01 to about 3 μg / kg, about 0.01 to about 0.02 μg / kg, about 0.01 to about 0.05 μg / kg, about 0.05 to about 0.1 μg / kg, about 0.05 to about 0.5 μg / kg, about 0.05 to about 0.75 μg / kg, about 0.05 to about 1 μg / kg, about 0.05 to about 1.5 μg / kg, about 0.05 to about 2 μg / kg, about 0.05 to about 2.5 μg / kg, about 0.05 to about 3 μg / kg, about 0.1 to about 3 μg / kg. This includes administration of the heterodimeric Fc fusion protein at a dose of about 0.1 to about 1 μg / kg, about 0.5 to about 1 μg / kg, about 0.1 to about 2 μg / kg, about 0.5 to about 2 μg / kg, about 0.1 to about 0.5 μg / kg, about 0.1 to about 0.25 μg / kg, about 0.2 to about 1 μg / kg, about 0.2 to about 2 μg / kg, about 1 to about 1.2 μg / kg, about 1 to about 1.5 μg / kg, about 1 to about 2 μg / kg, about 1 to about 2.5 μg / kg, about 0.5 to about 2.5 μg / kg, about 1 to about 3 μg / kg, or about 0.5 to about 3 μg / kg. In certain embodiments, the drug delivery formulation for use in the methods of treating cancer or inhibiting tumor growth is about 0.01 μg / kg, about 0.02 μg / kg, about 0.03 μg / kg, about 0.04 μg / kg, about 0.05 μg / kg, about 0.1 μg / kg, about 0.15 μg / kg, about 0.2 μg / kg, about 0.25 μg / kg, about 0.3 μg / kg, about 0.35 μg / kg, about 0.4 μg / kg, about 0.45 μg / kg, about 0.5 μg / kg, about 0.6 μg / kg, about 0.7 μg / kg, about 0.8 μg / kg, about 0.9 μg / kg, about 10 μg / kg, about 11 μg / kg, about 12 μg / kg, about 13 μg / kg, about 14 μg / kg, about 15 μg / kg, about 16 μg / kg, about 17 μg / kg, about 18 μg / kg, about 19 μg / kg, about 20 μg / kg, about 21 μg / kg, about 22 μg / kg, about 23 μg / kg, about 24 μg / kg, about 25 μg / kg, about 26 μg / kg, about 27 μg / kg, about 28 μg / kg, about 29 μg / kg, about 30 μg / kg, about 31 μg / kg, about 32 μg / kg, about 33 μg / kg, about 34 μg / kg, about 35 μg / kg, about 36 μg / kg, about 37 μg / kg, about 38 μg / kg, about 39 μg / kg, about 40 μg / kg, about 41 μg / kg, about 42 μg / kg, about 4 The method comprises administering a dose of the heterodimeric Fc-fusion protein selected from the group consisting of about 0.6 μg / kg, about 0.7 μg / kg, about 0.8 μg / kg, about 0.9 μg / kg, about 1 μg / kg, about 1.2 μg / kg, about 1.25 μg / kg, about 1.3 μg / kg, about 1.4 μg / kg, about 1.5 μg / kg, about 1.75 μg / kg, about 2 μg / kg, about 2.5 μg / kg, about 3 μg / kg, about 4 μg / kg, or about 5 μg / kg. In certain embodiments, the administered dose is based on the subject's body weight. In certain embodiments, the drug delivery formulation for use in the method of treating cancer or inhibiting tumor growth further comprises administering an anti-PD-1 antibody, e.g., pembrolizumab or nivolumab.

[0347] In some embodiments, a drug delivery formulation for use in a method for treating cancer or a method for inhibiting tumor growth comprises the heterodimeric Fc-fusion protein in an amount of about 0 to about 2 mg, about 0.2 to about 1.8 mg, about 0.3 to about 1.7 mg, about 0.4 to about 1.6 mg, about 0.5 to about 1.5 mg, about 0.6 to about 1.4 mg, about 0.7 to about 1.3 mg, about 0.8 to about 1.2 mg, or about 0.9 to about 1.1 mg. In certain embodiments, a drug delivery formulation for use in a method for treating cancer or a method for inhibiting tumor growth comprises the heterodimeric Fc-fusion protein in an amount selected from the group consisting of about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, or about 10 mg. In certain embodiments, the drug delivery formulation for use in the method of treating cancer or inhibiting tumor growth further comprises administering an anti-PD-1 antibody, e.g., pembrolizumab or nivolumab. In a specific embodiment, the drug delivery formulation for use in the method of treating cancer or inhibiting tumor growth further comprises administering 200 mg of an anti-PD-1 antibody, e.g., pembrolizumab or nivolumab.

[0348] In some embodiments, the drug delivery formulation for use in the method of treating cancer or inhibiting tumor growth in a subject with advanced melanoma comprises a dose of about 0.01 to about 3 μg / kg, about 0.01 to about 0.02 μg / kg, about 0.01 to about 0.05 μg / kg, about 0.05 to about 0.1 μg / kg, about 0.05 to about 0.5 μg / kg, about 0.05 to about 0.75 μg / kg, about 0.05 to about 1 μg / kg, about 0.05 to about 1.5 μg / kg, about 0.05 to about 2 μg / kg, about 0.05 to about 2.5 μg / kg, about 0.05 to about 3μg / kg, about 0.1 to about 3μg / kg, about 0.1 to about 1μg / kg, about 0.5 to about 1μg / kg, about 0.1 to about 2μg / kg, about 0.5 to about 2μg / kg, about 0.1 to about 0.5μg / kg, about 0.1 to about 0.25μg / kg, about 0.2 to about 1μg / kg, about 0.2 to about 2μg / kg, about 1 to about 1.2μg / kg, about 1 to about 1.5μg / kg, about 1 to about 2μg / kg, about 1 to about 2.5μg / kg, about 0.5 to about 2.5μg / kg, about 1 to about 3μg / kg, or about 0.5 to about 3μg / kg. In certain embodiments, the drug delivery formulation for use in the method of treating cancer or inhibiting tumor growth in a subject with advanced melanoma is at a concentration of about 0.01 μg / kg, about 0.02 μg / kg, about 0.03 μg / kg, about 0.04 μg / kg, about 0.05 μg / kg, about 0.1 μg / kg, about 0.15 μg / kg, about 0.2 μg / kg, about 0.25 μg / kg, about 0.3 μg / kg, about 0.35 μg / kg, about 0.4 μg / kg, about The antibody is administered at a dose selected from the group consisting of 0.45 μg / kg, about 0.5 μg / kg, about 0.6 μg / kg, about 0.7 μg / kg, about 0.8 μg / kg, about 0.9 μg / kg, about 1 μg / kg, about 1.2 μg / kg, about 1.25 μg / kg, about 1.3 μg / kg, about 1.4 μg / kg, about 1.5 μg / kg, about 1.75 μg / kg, about 2 μg / kg, about 2.5 μg / kg, about 3 μg / kg, about 4 μg / kg, or about 5 μg / kg.

[0349] In some embodiments, a drug delivery formulation for use in a method for treating cancer or inhibiting tumor growth in a subject with advanced melanoma comprises the heterodimeric Fc-fusion protein in an amount of about 0 to about 2 mg, about 0.2 to about 1.8 mg, about 0.3 to about 1.7 mg, about 0.4 to about 1.6 mg, about 0.5 to about 1.5 mg, about 0.6 to about 1.4 mg, about 0.7 to about 1.3 mg, about 0.8 to about 1.2 mg, or about 0.9 to about 1.1 mg. In certain embodiments, a drug delivery formulation for use in a method for treating cancer or inhibiting tumor growth in a subject with advanced melanoma comprises the heterodimeric Fc-fusion protein in an amount selected from the group consisting of about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, or about 10 mg.

[0350] In some embodiments, the drug delivery formulation for use in the method of treating cancer or inhibiting tumor growth in a subject with advanced RCC comprises about 0.01 to about 3 μg / kg, about 0.01 to about 0.02 μg / kg, about 0.01 to about 0.05 μg / kg, about 0.05 to about 0.1 μg / kg, about 0.05 to about 0.5 μg / kg, about 0.05 to about 0.75 μg / kg, about 0.05 to about 1 μg / kg, about 0.05 to about 1.5 μg / kg, about 0.05 to about 2 μg / kg, about 0.05 to about 2.5 μg / kg, about 0.05 to about 3 μg / kg, about 0.05 to about 3 μg / kg, about 0.05 to about 4 μg / kg, about 0.05 to about 5 μg / kg, about 0.05 to about 6 μg / kg, about 0.05 to about 7 μg / kg, about 0.05 to about 8 μg / kg, about 0.05 to about 9 μg / kg, about 0.05 to about 10 μg / kg, about 0.05 to about 11 μg / kg, about 0.05 to about 12 μg / kg, about 0.05 to about 13 μg / kg, about 0.05 to about 14 μg / kg, about 0.05 to about 15 μg / kg, about 0.05 to about 16 μg / kg, about 0.05 to about 17 μg / kg, about 0.05 to about 18 μg / kg, about 0.05 to about 19 μg / kg, about 0.05 to about 20 μg / kg, about 0.05 to about 21 μg / kg, about 0 μg / kg, about 0.1 to about 3 μg / kg, about 0.1 to about 1 μg / kg, about 0.5 to about 1 μg / kg, about 0.1 to about 2 μg / kg, about 0.5 to about 2 μg / kg, about 0.1 to about 0.5 μg / kg, about 0.1 to about 0.25 μg / kg, about 0.2 to about 1 μg / kg, about 0.2 to about 2 μg / kg, about 1 to about 1.2 μg / kg, about 1 to about 1.5 μg / kg, about 1 to about 2 μg / kg, about 1 to about 2.5 μg / kg, about 0.5 to about 2.5 μg / kg, about 1 to about 3 μg / kg, or about 0.5 to about 3 μg / kg. In certain embodiments, the drug delivery formulation for use in the method of treating cancer or inhibiting tumor growth in a subject with advanced RCC is about 0.01 μg / kg, about 0.02 μg / kg, about 0.03 μg / kg, about 0.04 μg / kg, about 0.05 μg / kg, about 0.1 μg / kg, about 0.15 μg / kg, about 0.2 μg / kg, about 0.25 μg / kg, about 0.3 μg / kg, about 0.35 μg / kg, about 0.4 μg / kg, about 0 The antibody is administered at a dose selected from the group consisting of about 0.45 μg / kg, about 0.5 μg / kg, about 0.6 μg / kg, about 0.7 μg / kg, about 0.8 μg / kg, about 0.9 μg / kg, about 1 μg / kg, about 1.2 μg / kg, about 1.25 μg / kg, about 1.3 μg / kg, about 1.4 μg / kg, about 1.5 μg / kg, about 1.75 μg / kg, about 2 μg / kg, about 2.5 μg / kg, about 3 μg / kg, about 4 μg / kg, or about 5 μg / kg.

[0351] In some embodiments, a drug delivery formulation for use in a method for treating cancer or inhibiting tumor growth in a subject with advanced RCC comprises the heterodimeric Fc-fusion protein in an amount of about 0 to about 2 mg, about 0.2 to about 1.8 mg, about 0.3 to about 1.7 mg, about 0.4 to about 1.6 mg, about 0.5 to about 1.5 mg, about 0.6 to about 1.4 mg, about 0.7 to about 1.3 mg, about 0.8 to about 1.2 mg, or about 0.9 to about 1.1 mg. In certain embodiments, a drug delivery formulation for use in a method for treating cancer or inhibiting tumor growth in a subject with advanced RCC comprises the heterodimeric Fc-fusion protein in an amount selected from the group consisting of about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, or about 10 mg.

[0352] In some embodiments, the drug delivery formulation for use in the method of treating cancer or inhibiting tumor growth in a subject with advanced urothelial carcinoma comprises a dose of about 0.01 to about 3 μg / kg, about 0.01 to about 0.02 μg / kg, about 0.01 to about 0.05 μg / kg, about 0.05 to about 0.1 μg / kg, about 0.05 to about 0.5 μg / kg, about 0.05 to about 0.75 μg / kg, about 0.05 to about 1 μg / kg, about 0.05 to about 1.5 μg / kg, about 0.05 to about 2 μg / kg, about 0.05 to about 2.5 μg / kg, about 0.05 to about 3μg / kg, about 0.1 to about 3μg / kg, about 0.1 to about 1μg / kg, about 0.5 to about 1μg / kg, about 0.1 to about 2μg / kg, about 0.5 to about 2μg / kg, about 0.1 to about 0.5μg / kg, about 0.1 to about 0.25μg / kg, about 0.2 to about 1μg / kg, about 0.2 to about 2μg / kg, about 1 to about 1.2μg / kg, about 1 to about 1.5μg / kg, about 1 to about 2μg / kg, about 1 to about 2.5μg / kg, about 0.5 to about 2.5μg / kg, about 1 to about 3μg / kg, or about 0.5 to about 3μg / kg. In certain embodiments, the drug delivery formulation for use in the method of treating cancer or inhibiting tumor growth in a subject with advanced urothelial carcinoma is about 0.01 μg / kg, about 0.02 μg / kg, about 0.03 μg / kg, about 0.04 μg / kg, about 0.05 μg / kg, about 0.1 μg / kg, about 0.15 μg / kg, about 0.2 μg / kg, about 0.25 μg / kg, about 0.3 μg / kg, about 0.35 μg / kg, about 0.4 μg / kg, about The antibody is administered at a dose selected from the group consisting of 0.45 μg / kg, about 0.5 μg / kg, about 0.6 μg / kg, about 0.7 μg / kg, about 0.8 μg / kg, about 0.9 μg / kg, about 1 μg / kg, about 1.2 μg / kg, about 1.25 μg / kg, about 1.3 μg / kg, about 1.4 μg / kg, about 1.5 μg / kg, about 1.75 μg / kg, about 2 μg / kg, about 2.5 μg / kg, about 3 μg / kg, about 4 μg / kg, or about 5 μg / kg.

[0353] In some embodiments, a drug delivery formulation for use in a method for treating cancer or inhibiting tumor growth in a subject with advanced urothelial carcinoma comprises the heterodimeric Fc-fusion protein in an amount of about 0 to about 2 mg, about 0.2 to about 1.8 mg, about 0.3 to about 1.7 mg, about 0.4 to about 1.6 mg, about 0.5 to about 1.5 mg, about 0.6 to about 1.4 mg, about 0.7 to about 1.3 mg, about 0.8 to about 1.2 mg, or about 0.9 to about 1.1 mg. In certain embodiments, a drug delivery formulation for use in a method for treating cancer or inhibiting tumor growth in a subject with advanced urothelial carcinoma comprises the heterodimeric Fc-fusion protein in an amount selected from the group consisting of about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, or about 10 mg.

[0354] In certain embodiments, a subject treated by a method disclosed herein has not previously received a therapy for the treatment of their cancer. In certain embodiments, a subject treated by a method disclosed herein has not previously received chemotherapy or immunotherapy for the treatment of their cancer. In certain embodiments, a subject treated by a method disclosed herein has previously received a therapy (e.g., chemotherapy or immunotherapy) but continues to experience cancer progression despite the previous therapy. In certain embodiments, a subject treated by a method disclosed herein experienced cancer regression after previous therapy (e.g., chemotherapy or immunotherapy) but later experienced cancer recurrence. In certain embodiments, a subject treated by a method disclosed herein is intolerant to previous therapy (e.g., chemotherapy or immunotherapy).

[0355] In certain embodiments, subjects treated by the methods disclosed herein meet all of the inclusion criteria for a clinical trial cohort (e.g., a dose escalation cohort, a dose expansion cohort, a melanoma cohort, a renal cell carcinoma cohort, a urothelial carcinoma cohort, or a combination therapy cohort with pembrolizumab or nivolumab) described in Examples 26 and 29. In certain embodiments, subjects treated by the methods disclosed herein do not meet any of the exclusion criteria described in Examples 26 and 29.

[0356] The heterodimeric Fc-fusion proteins disclosed herein can be used as monotherapy or in combination with one or more therapies. In certain embodiments, the heterodimeric Fc-fusion proteins are used as monotherapy according to the dosage regimens disclosed herein. In other embodiments, the heterodimeric Fc-fusion proteins are used in combination with one or more therapies, where the heterodimeric Fc-fusion proteins are administered according to the dosage regimens disclosed herein, and the one or more therapies are administered according to dosage regimens known to be suitable for treating a particular subject with a particular cancer. In certain embodiments, the treatment methods disclosed herein are used as adjunctive therapy to surgical resection of the primary lesion. In certain embodiments, surgical intervention of the primary lesion includes lysing the subject's cancer cells, removing the tumor, or debulking the tumor.

[0357] Exemplary therapeutic agents that may be used in combination with the heterodimeric Fc fusion proteins include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindet Examples of agents that may exhibit differences in binding to their cognate receptors or increased or decreased serum half-lives include phenytoin, flutamide, drogenil, butosin, carmofur, razoxane, sizofiran, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-α, interferon-2α, interferon-β, interferon-γ (IFN-γ), colony-stimulating factor-1, colony-stimulating factor-2, denileukin diftitox, interleukin-2, luteinizing hormone-releasing factor, and variants of the foregoing agents that may exhibit differences in binding to their cognate receptors or that have increased or decreased serum half-lives.

[0358] Another class of drugs that can be used as part of a combination therapy in the treatment of cancer are immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of: (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the U.S. Food and Drug Administration for the treatment of melanoma.

[0359] Still other agents that may be used as part of a combination therapy in the treatment of cancer are monoclonal antibody agents that target non-checkpoint targets (e.g., Herceptin) and non-cytotoxic agents (e.g., tyrosine kinase inhibitors).

[0360] Further categories of anti-cancer agents include, for example: (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors, CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors plus 2-chloro-deoxyadenosine, HDAC inhibitors, hedgehog signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, MEK inhibitors (ii) an inhibitor selected from a phosphoinositide 3-kinase inhibitor, a MELK inhibitor, an MTH1 inhibitor, a PARP inhibitor, a phosphoinositide 3-kinase inhibitor, an inhibitor of both PARP1 and DHODH, a proteasome inhibitor, a topoisomerase-II inhibitor, a tyrosine kinase inhibitor, a VEGFR inhibitor, and a WEE1 inhibitor; (iii) an agonist of OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF, and G-CSF.

[0361] In certain embodiments, the cancer treated with a single or more doses of a heterodimeric IL-12-Fc fusion protein (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291) is a metastatic cancer. In certain embodiments, the metastatic cancer is a local, regional, or distant metastatic cancer. In certain embodiments, a single or multiple doses of a heterodimeric IL-12-Fc fusion protein (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291) treats a distant cancer that is not a primary cancer in the organ or tissue of origin and / or is not the direct target of the treatment regimen via the abscopal effect. In certain embodiments, the abscopal effect of the heterodimeric IL-12-Fc fusion protein is enhanced during and / or after a treatment regimen that includes radiation and / or chemotherapy. In certain embodiments, single or multiple doses of a heterodimeric IL-12-Fc fusion protein (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291) treats cancer in a patient by inducing a systemic anti-tumor response as determined, for example, by increased expression of IFNγ, CXCL9, and / or CXCL10 in the patient's serum and / or tumor.

[0362] Combination therapy Another aspect of the present invention provides combination therapy: The pharmaceutical formulations comprising the heterodimeric Fc-fusion proteins described herein can be used in combination with additional therapeutic agents to treat cancer.

[0363] In certain embodiments, heterodimeric Fc-fusion proteins of the invention (e.g., heterodimeric Fc-fusion proteins comprising an IL-12 subunit) are administered as a combination therapy to treat a subject diagnosed with cancer. In certain embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, leukemia, lung cancer, lymphoma, mesothelioma, melanoma, myeloma, ovarian cancer, endometrial cancer, prostate cancer, pancreatic cancer, renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), brain cancer, sarcoma, neuroblastoma, classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, urothelial carcinoma, microsatellite instability-high carcinoma, Merkel cell carcinoma, endometrial carcinoma, cutaneous T-cell lymphoma, triple-negative breast cancer, or head and neck squamous cell carcinoma (HNSCC). In certain embodiments, the cancer is colon cancer. In certain embodiments, the heterodimeric Fc-fusion protein is administered as a combination therapy to a subject diagnosed with colon cancer. In certain embodiments, the cancer is melanoma. In certain embodiments, the heterodimeric Fc-fusion protein is administered as a combination therapy to a subject diagnosed with melanoma. In certain embodiments, the cancer is breast cancer. In certain embodiments, the heterodimeric Fc-fusion protein is administered as a combination therapy to a subject diagnosed with breast cancer.

[0364] In some embodiments, the heterodimeric Fc-fusion proteins of the invention (e.g., heterodimeric Fc-fusion proteins comprising an IL-12 subunit) are used in the treatment of advanced malignancies in combination with another therapeutic agent selected from: cytotoxic chemotherapy; radiation therapy; antibodies targeting molecules involved in anti-tumor immune responses, such as CTLA-4, PD-1, PD-L1, or TGF-β; antibodies that act on tumor-associated antigens by ADCC; multispecific antibodies that bind to NKG2D, CD16, and tumor-associated antigens, optionally administered in combination with antibodies targeting PD-1 or PD-L1; tailored cancer vaccines; oncolytic cancer vaccines; and tailored vaccines administered in combination with antibodies targeting PD-1 or PD-L1.

[0365] In some embodiments, the heterodimeric Fc fusion proteins of the invention (e.g., heterodimeric Fc fusion proteins comprising an IL-12 subunit) are used in the treatment of malignancies (e.g., advanced malignancies) in combination with another therapy, including, but not limited to, NK-targeted therapy (e.g., CAR-NK therapy), antibody therapy, checkpoint inhibitor therapy, additional cytokine therapy, innate immune system agonist therapy, chemotherapy, targeted drug therapy, radiation therapy, adoptive NK therapy, stem cell transplant (SCT) therapy, agonist antibodies, chimeric antigen receptor (CAR) T-cell therapy, T-cell receptor (TCR) modified therapy, multispecific binding proteins (TriNKET), agents that induce cellular senescence, and vaccines and / or oncolytic virus therapy. In some embodiments, the heterodimeric Fc-fusion proteins of the invention are used to treat malignancies (e.g., advanced malignancies) in combination with two or more additional therapies selected from NK-targeted therapy (e.g., CAR-NK therapy), antibody therapy, checkpoint inhibitor therapy, additional cytokine therapy, innate immune system agonist therapy, chemotherapy, targeted drug therapy, radiation therapy, adoptive NK therapy, stem cell transplant (SCT) therapy, agonist antibodies, chimeric antigen receptor (CAR) T-cell therapy, T-cell receptor (TCR)-engineered therapy, multispecific binding proteins (TriNKET), agents that induce cellular senescence, and vaccines and / or oncolytic virus therapy.

[0366] In some embodiments, the heterodimeric Fc fusion proteins of the invention (e.g., heterodimeric Fc fusion proteins comprising IL-12) are used in combination with cancer vaccines or antibodies targeting PD-1 or PD-L1 for the treatment of locally advanced malignancies that can be completely resected.

[0367] The proteins of the present invention can also be used as adjunctive therapy to surgical resection of the primary lesion.

[0368] The amounts and relative timing of administration of the heterodimeric Fc-fusion protein of the present invention (e.g., a heterodimeric Fc-fusion protein comprising IL-12) and additional therapeutic agent may be selected to achieve a desired combined therapeutic effect. For example, when a combination therapy is administered to a patient in need thereof, the therapeutic agents, pharmaceutical formulations comprising therapeutic agents, or pharmaceutical compositions comprising therapeutic agents in the combination may be administered in any order, e.g., sequentially, in parallel, together, simultaneously, etc. Furthermore, for example, the heterodimeric Fc-fusion protein may be administered while the additional therapeutic agent is exerting its prophylactic or therapeutic effect, or vice versa.

[0369] As disclosed herein, the methods of the invention involve the co-administration of a heterodimeric Fc-fusion protein (e.g., a heterodimeric Fc-fusion protein comprising an IL-12 subunit) in combination with an additional therapeutic agent. As disclosed herein, the methods of the invention involve the co-administration of a heterodimeric Fc-fusion protein comprising an IL-12 subunit in combination with an additional therapeutic agent.

[0370] "Co-administered" encompasses methods in which the heterodimeric Fc-fusion protein (e.g., a heterodimeric Fc-fusion protein comprising an IL-12 subunit) and the additional therapeutic agent are given simultaneously, methods in which the heterodimeric Fc-fusion protein and the additional therapeutic agent are given sequentially, and methods in which either the heterodimeric Fc-fusion protein or the additional therapeutic agent, or both, are given intermittently or continuously, or any combination of simultaneous, sequential, intermittent, and / or continuous. Those skilled in the art will recognize that intermittent administration is not necessarily the same as sequential, as intermittent administration also includes the first administration of an agent followed by another administration of that very same agent at a later time. Furthermore, those skilled in the art will understand that intermittent administration also encompasses sequential administration in some embodiments, as intermittent administration does include the first administration of an agent interrupted by the administration of a different agent before the first agent is again administered. Moreover, those skilled in the art will also know that continuous administration can be achieved by a number of routes, including intravenous drip (intravenous infusion) or feeding tube.

[0371] Additionally, and in more general terms, the term "co-administered" encompasses any and all ways in which the separate administration of a heterodimeric Fc-fusion protein and the separate administration of an additional therapeutic agent to a subject overlap within any time frame.

[0372] The frequency of administration of the heterodimeric Fc-fusion protein or additional therapeutic agent to a subject is known in the art as Qnd or qnd, where n is the frequency in days for consecutive administrations of the agent. For example, Q3d would be administration of the agent once every three days. In certain embodiments, the method comprises administering to a subject either the heterodimeric Fc-fusion protein and / or the additional therapeutic agent, or both, or any combination thereof, Q1d, Q2d, Q3d, Q4d, Q5d, Q6d, Q7d, Q8d, Q9d, Q10d, Q14d, Q21d, Q28d, Q30d, Q90d, Q120d, Q240d, or Q365d.

[0373] In certain embodiments, either the heterodimeric Fc-fusion protein and / or the additional therapeutic agent, or both, are administered intermittently. In certain embodiments, the method comprises administering either the heterodimeric Fc-fusion protein or the additional therapeutic agent, or both, to a subject with a delay of at least 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, or 4 weeks between administrations. In certain embodiments, the delayed administration follows a pattern in which one or both of the heterodimeric Fc-fusion protein and / or additional therapeutic agent, or any combination thereof, is administered continuously for a given period of about 10 minutes to about 365 days, followed by no administration for a given period of about 10 minutes to about 30 days.

[0374] In certain embodiments, either the heterodimeric Fc-fusion protein and / or the additional therapeutic agent, or any combination thereof, are administered intermittently while the other is given continuously, hi certain embodiments, a first effective amount of the heterodimeric Fc-fusion protein combination is administered sequentially with a second effective amount of the additional therapeutic agent.

[0375] In certain embodiments, the heterodimeric Fc-fusion protein and the additional therapeutic agent are administered simultaneously. In certain embodiments, a first effective amount of the heterodimeric Fc-fusion protein combination is administered sequentially with a second effective amount of the additional therapeutic agent. In such embodiments, the combination is also referred to as "co-administered," as this term encompasses any and all ways in which a subject is exposed to both components in combination. However, such embodiments are not limited to the combination being provided in only one formulation or composition. It may be more advantageous to deliver certain concentrations of the heterodimeric Fc-fusion protein and the additional therapeutic agent at regular intervals, and therefore the first effective amount and the second effective amount may vary depending on the formulation being administered.

[0376] In certain embodiments, the heterodimeric Fc-fusion protein and the additional therapeutic agent are administered simultaneously or sequentially. In certain embodiments, a first effective amount of the heterodimeric Fc-fusion protein is administered sequentially after a second effective amount of the additional therapeutic agent. In certain embodiments, a second effective amount of the additional therapeutic agent is administered sequentially after the first effective amount of the heterodimeric Fc-fusion protein.

[0377] In certain embodiments, the combination of the heterodimeric Fc-fusion protein (e.g., a heterodimeric Fc-fusion protein comprising an IL-12 subunit) and the additional therapeutic agent is administered in a single formulation. In certain embodiments, the combination is administered in two compositions, wherein a first effective amount of the heterodimeric Fc-fusion protein is administered in a separate formulation from a second effective amount of the additional therapeutic agent. In certain embodiments, the combination is administered in two compositions, wherein a first effective amount of the heterodimeric Fc-fusion protein is administered in a separate formulation from a second effective amount of the additional therapeutic agent. In certain embodiments, the first effective amount of the heterodimeric Fc-fusion protein is administered sequentially after the second effective amount of the additional therapeutic agent. In certain embodiments, the second effective amount of the additional therapeutic agent is administered sequentially after the first effective amount of the heterodimeric Fc-fusion protein. In certain embodiments, the heterodimeric Fc-fusion protein and the additional therapeutic agent are administered; and then both the heterodimeric Fc-fusion protein and the additional therapeutic agent are administered intermittently over a period of at least 24 hours. In certain embodiments, the heterodimeric Fc-fusion protein and the additional therapeutic agent are administered on a non-overlapping, every other day schedule.

[0378] In certain embodiments, the first effective amount of the heterodimeric Fc-fusion protein is administered 4 hours or more after the second effective amount of the additional therapeutic agent. In certain embodiments, the first effective amount of the heterodimeric Fc-fusion protein is administered 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 60 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 24 hours or more, 2 days or more, 4 days or more, 6 days or more, 8 days or more, 10 days or more, 12 days or more, 14 days or more, 21 days or more, or 30 days or more after the second effective amount of the additional therapeutic agent. In certain embodiments, the second effective amount of an additional therapeutic agent is administered 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 60 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, 12 hours or more, 24 hours or more, 2 days or more, 4 days or more, 6 days or more, 8 days or more, 10 days or more, 12 days or more, 14 days or more, 21 days or more, or 30 days or more after the first effective amount of the heterodimeric Fc-fusion protein.

[0379] In certain embodiments, either the heterodimeric Fc-fusion protein and / or additional therapeutic agent(s), or both, are administered by a route selected from the group consisting of intravenous, subcutaneous, cutaneous, oral, intramuscular, and intraperitoneal. In certain embodiments, either the heterodimeric Fc-fusion protein and / or additional therapeutic agent(s), or both, are administered intravenously. In certain embodiments, either the heterodimeric Fc-fusion protein and / or additional therapeutic agent(s), or both, or any combination thereof, are administered orally.

[0380] Those skilled in the art will understand that the unit dosage forms of the present disclosure may be administered in the same or different physical forms, i.e., orally in capsules or tablets and / or by liquid intravenous infusion. Furthermore, the unit dosage form per administration may vary depending on the precise route of administration. Several different dosage forms may exist for either the heterodimeric Fc-fusion protein and / or additional therapeutic agent, or for the combination thereof. Because different medical conditions may require different routes of administration, the same components of the combinations described herein may be quite similar in composition and physical form, but may need to be given in different ways, perhaps at different times, to alleviate the condition. For example, conditions such as persistent nausea, particularly those associated with vomiting, may make oral dosage forms difficult to use, and in such cases, administration may instead or similarly be required in a separate unit dosage form via inhalation, buccal, sublingual, or suppository routes, perhaps even identical to other dosage forms used previously or in the future. The specific dosage form may be a requirement of the particular combination of the heterodimeric Fc-fusion protein and the additional therapeutic agent, as well as issues related to various factors such as chemical stability or pharmacokinetics.

[0381] (i)NK targeting therapy In certain embodiments, heterodimeric Fc-fusion protein therapy is combined with NK-targeting therapy. For example, in certain embodiments, the heterodimeric Fc-fusion protein is co-administered with a therapeutic agent that targets NKp46. In certain embodiments, the therapeutic agent that targets NKp46 also binds to CD16, one or more tumor-associated antigens, or a combination thereof. Exemplary therapeutic agents that target NKp46 are described in further detail in U.S. Patent Application Publication No. 20170198038A1 (herein incorporated by reference for all purposes).

[0382] In certain embodiments, heterodimeric Fc-fusion protein therapy is combined with bispecific and trispecific killer engager (BiKE and TriKE) therapy, e.g., BiKE and TriKE therapy, which target NK cells. BiKE and TriKE are constructed with a single heavy chain (VH chain) and light chain (VL chain) of the variable regions of each antibody of interest. The VH and VL domains are connected by a short, flexible polypeptide linker to prevent dissociation. BiKE and TriKE are described in further detail in U.S. Patent Application Publication Nos. 20180282386A1 and 20180258396A1, which are incorporated by reference for all purposes. BiKE and TriKE may have a binding domain specific for NK cells.

[0383] In certain embodiments, BiKE and TriKE therapy is used in combination with heterodimeric Fc-fusion protein therapy to treat subjects known to have or suspected of having high-risk myelodysplastic syndrome, acute myeloid leukemia, systemic mastocytosis, or mast cell leukemia. In certain embodiments, BiKE and TriKE therapy is administered as a single course of three one-week treatment blocks. In certain embodiments, a treatment block comprises four consecutive 24-hour continuous infusions (approximately 96 hours) followed by a 72-hour rest period. In certain embodiments, BiKE and TriKE therapy is administered at a dose of 5 μg / kg / day, 10 μg / kg / day, 25 μg / kg / day, 50 μg / kg / day, 100 μg / kg / day, or 200 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at a dose of at least 5 μg / kg / day, at least 10 μg / kg / day, at least 25 μg / kg / day, at least 50 μg / kg / day, at least 100 μg / kg / day, or at least 200 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at a dose of at least 1 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at a dose of at least 5 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at a dose of at least 200 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at a dose of at least 500 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at a dose of at least 1000 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at a dose of 200 μg / kg / day or less. In certain embodiments, BiKE and TriKE therapy are administered at a dose of 500 μg / kg / day or less. In certain embodiments, BiKE and TriKE therapy are administered at a dose of 1000 μg / kg / day or less. In certain embodiments, BiKE and TriKE therapy are administered at a dose of 1-200 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at a dose of 5-200 μg / kg / day.In certain embodiments, BiKE and TriKE therapy are administered at a dose of 1-500 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at a dose of 1-1000 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at a dose of 5-500 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at a dose of 5-1000 μg / kg / day. In certain embodiments, BiKE and TriKE therapy are administered at the maximum tolerated dose. In cert...

Claims

1. (a) a heterodimeric Fc fusion protein, (i) a first polypeptide comprising a first antibody Fc domain polypeptide and a first subunit of a multisubunit cytokine; and (ii) a second polypeptide comprising a second antibody Fc domain polypeptide and a second, different subunit of said multisubunit cytokine. A heterodimeric Fc fusion protein comprising: (b) Citrate; (c) sugar; (d) sugar alcohol; and (e) Nonionic surfactant A pharmaceutical formulation having a pH of 6.0 to 7.0 comprising: the first and second antibody Fc domain polypeptides each contain a different mutation that promotes heterodimerization; and A pharmaceutical formulation, wherein said first subunit and a second, different subunit of said multisubunit cytokine are linked to one another.

2. 2. The pharmaceutical formulation of claim 1, wherein the first and / or second antibody Fc domain polypeptide comprises one or more mutations that reduce an Fc effector function.

3. 3. The pharmaceutical formulation of claim 1 or 2, wherein the concentration of citrate in the pharmaceutical formulation is from about 10 mM to about 30 mM.

4. 4. The pharmaceutical formulation of claim 3, wherein the concentration of citrate in the pharmaceutical formulation is about 20 mM.

5. The pharmaceutical formulation of any one of claims 1 to 4, wherein the concentration of the sugar in the pharmaceutical formulation is from about 3% to about 12% (w / v).

6. 6. The pharmaceutical formulation of claim 5, wherein the concentration of the sugar in the pharmaceutical formulation is about 6% (w / v).

7. 7. The pharmaceutical formulation of claim 5 or 6, wherein the sugar is a disaccharide.

8. 8. The pharmaceutical formulation of claim 7, wherein the disaccharide is sucrose.

9. The pharmaceutical formulation of any one of claims 1 to 8, wherein the concentration of the sugar alcohol in the pharmaceutical formulation is from about 0.5% to about 6% (w / v).

10. 10. The pharmaceutical formulation of claim 9, wherein the concentration of the sugar alcohol in the pharmaceutical formulation is about 1% (w / v).

11. The pharmaceutical formulation according to any one of claims 1 to 10, wherein the sugar alcohol is derived from a monosaccharide.

12. 12. The pharmaceutical formulation of claim 11, wherein the sugar alcohol is mannitol.

13. 13. The pharmaceutical formulation of any one of claims 1 to 12, wherein the concentration of the non-ionic surfactant in the pharmaceutical formulation is from about 0.005% to about 0.02% (w / v).

14. 14. The pharmaceutical formulation of claim 13, wherein the concentration of polysorbate 80 in the pharmaceutical formulation is about 0.01% (w / v).

15. 15. The pharmaceutical formulation of claim 13 or 14, wherein the non-ionic surfactant is a polysorbate.

16. 16. The pharmaceutical formulation of claim 15, wherein the polysorbate is polysorbate 80.

17. The pharmaceutical formulation of any one of claims 1 to 16, wherein the pH is from about 6.1 to about 6.

9.

18. 18. The pharmaceutical formulation of claim 17, wherein the pH is from about 6.2 to about 6.

8.

19. 19. The pharmaceutical formulation of claim 18, wherein the pH is from about 6.3 to about 6.

7.

20. 20. The pharmaceutical formulation of claim 19, wherein the pH is from about 6.4 to about 6.

6.

21. 21. The pharmaceutical formulation of claim 20, wherein the pH is about 6.

5.

22. The pharmaceutical formulation of any one of claims 1 to 21, further comprising water.

23. 23. The pharmaceutical formulation of claim 22, wherein the water is USP water for injection.

24. 24. The pharmaceutical formulation of any one of claims 1 to 23, comprising a bulk concentration of the heterodimeric Fc-fusion protein of about 1 g / L to about 10 g / L.

25. 25. The pharmaceutical formulation of claim 24, comprising a bulk concentration of the heterodimeric Fc-fusion protein of about 2 g / L to about 8 g / L.

26. 26. The pharmaceutical formulation of claim 25, comprising a bulk concentration of the heterodimeric Fc-fusion protein of about 4 g / L to about 6 g / L.

27. 27. The pharmaceutical formulation of claim 26, comprising a bulk concentration of the heterodimeric Fc-fusion protein of about 5 g / L.

28. 28. The pharmaceutical formulation of any one of claims 1 to 27, comprising the protein in an administration concentration of about 0.5 g / L to about 1.5 g / L.

29. 30. The pharmaceutical formulation of claim 28, comprising the protein at a dosage concentration of about 0.75 g / L to about 1.25 g / L.

30. 30. The pharmaceutical formulation of claim 29, comprising the protein at an administration concentration of about 1 g / L.

31. 31. The pharmaceutical formulation of any one of 1 to 30, which is designed to be stored at a temperature of about 2°C to about 8°C.

32. 32. The pharmaceutical formulation of any one of claims 1 to 31, which is a clear, colorless solution and is free of visible particulate matter.

33. When measured by differential scanning fluorimetry, (a) a T of greater than about 60° C., greater than about 61° C., greater than about 62° C., greater than about 63° C., greater than about 64° C., greater than about 65° C., or greater than about 66° C. ｍ１ and / or (b) a T of greater than about 70° C., greater than about 71° C., greater than about 72° C., greater than about 73° C., greater than about 74° C., greater than about 75° C., greater than about 76° C., or greater than about 77° C. ｍ２ 33. The pharmaceutical formulation of any one of claims 1 to 32, having a thermal stability profile as defined by:

34. T of about 67.0°C ｍ１ and a T of about 77.3° C. ｍ２ 34. The pharmaceutical formulation of claim 33, having a thermal stability profile as defined by:

35. T ｍ１ and / or T ｍ２ 35. The pharmaceutical formulation of claim 34, wherein the thermal stability profile of the pharmaceutical formulation, as defined by: changes by less than about 2° C. or less than about 1° C. when the pharmaceutical formulation is incubated at 50° C. for 1 week compared to the same pharmaceutical formulation incubated at 5° C. for 1 week as measured by Differential Scanning Fluorometry.

36. A T of greater than about 60° C., greater than about 61° C., greater than about 62° C., greater than about 63° C., greater than about 64° C., greater than about 65° C., greater than about 66° C., or greater than about 67° C. as measured by differential scanning fluorimetry. ａｇｇ 36. The pharmaceutical formulation of any one of 1-35, having a thermal stability profile as defined by:

37. T ａｇｇ 37. The pharmaceutical formulation of claim 36, wherein the thermal stability profile of the pharmaceutical formulation, as defined by: changes by less than about 2° C. or less than about 1° C. when the pharmaceutical formulation is incubated at 50° C. for 1 week compared to the same pharmaceutical formulation incubated at 5° C. for 1 week as measured by Differential Scanning Fluorometry.

38. 38. The pharmaceutical formulation of any one of claims 1 to 37, wherein after incubation of the pharmaceutical formulation at 5°C for 1 week, the pH of the pharmaceutical formulation does not change by more than a pH value of about 0.2 or about 0.

1.

39. 39. The pharmaceutical formulation of any one of claims 1 to 38, wherein after incubation of the pharmaceutical formulation at 50°C for 1 week, the pH of the pharmaceutical formulation does not change by more than a pH value of about 0.2 or about 0.

1.

40. 40. The pharmaceutical formulation of any one of claims 1-39, wherein the heterodimeric Fc-fusion protein in said pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 15 nm, less than about 14 nm, less than about 13 nm, or less than about 12 nm as measured by dynamic light scattering at 25°C.

41. 41. The pharmaceutical formulation of claim 40, wherein said heterodimeric Fc-fusion protein in said pharmaceutical formulation has a Z-average hydrodynamic diameter of about 11.6 nm.

42. 42. The pharmaceutical formulation of any one of claims 1-41, wherein the heterodimeric Fc-fusion protein in said pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 20 nm, less than about 19 nm, less than about 18 nm, less than about 17 nm, less than about 16 nm, or less than about 15 nm as measured by dynamic light scattering at 25°C after incubating the pharmaceutical formulation at 50°C for two weeks.

43. 43. The pharmaceutical formulation of claim 42, wherein said heterodimeric Fc-fusion protein in said pharmaceutical formulation has a Z-average hydrodynamic diameter of about 14.4 nm.

44. 44. The pharmaceutical formulation of any one of claims 1-43, wherein said heterodimeric Fc-fusion protein in said pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 20 nm, less than about 19 nm, less than about 18 nm, less than about 17 nm, or less than about 16 nm as measured by dynamic light scattering at 25°C after subjecting said pharmaceutical formulation to five freeze-thaw cycles.

45. 45. The pharmaceutical formulation of claim 44, wherein said heterodimeric Fc-fusion protein in said pharmaceutical formulation has a Z-average hydrodynamic diameter of about 15.3 nm.

46. 46. ​​The pharmaceutical formulation of any one of claims 1-45, wherein the polydispersity index of said heterodimeric Fc-fusion protein in said pharmaceutical formulation is less than about 0.30, less than about 0.29, less than about 0.28, or less than about 0.27 as measured by dynamic light scattering at 25°C.

47. 47. The pharmaceutical formulation of any one of claims 1 to 46, wherein the polydispersity index of said heterodimeric Fc-fusion protein in said pharmaceutical formulation is about 0.

26.

48. 48. The pharmaceutical formulation of any one of claims 1-47, wherein the polydispersity index of said heterodimeric Fc-fusion protein in said pharmaceutical formulation is less than about 0.30, less than about 0.29, less than about 0.28, less than about 0.27, or less than about 0.26, as measured by dynamic light scattering at 25°C after incubating said pharmaceutical formulation at 50°C for 2 weeks.

49. 49. The pharmaceutical formulation of any one of claims 1 to 48, wherein the polydispersity index of said heterodimeric Fc-fusion protein in said pharmaceutical formulation is about 0.

25.

50. 50. The pharmaceutical formulation of any one of claims 1-49, wherein the polydispersity index of said heterodimeric Fc-fusion protein in said pharmaceutical formulation is less than about 0.40, less than about 0.35, or less than about 0.34, as measured by dynamic light scattering at 25°C after subjecting said pharmaceutical formulation to five freeze-thaw cycles.

51. 51. The pharmaceutical formulation of any one of claims 1 to 50, wherein the polydispersity index of said heterodimeric Fc-fusion protein in said pharmaceutical formulation is about 0.

33.

52. 52. The pharmaceutical formulation of any one of claims 1-51, wherein the purity profile of the pharmaceutical formulation is greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis.

53. 53. The pharmaceutical formulation of claim 52, wherein the purity profile of the pharmaceutical formulation is about 99.0% as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis.

54. 54. The pharmaceutical formulation of any one of claims 1-53, wherein the purity profile of the pharmaceutical formulation, as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis, is greater than about 75%, greater than about 80%, greater than about 81%, greater than about 82%, greater than about 83%, greater than about 84%, or greater than about 85% after incubating the pharmaceutical formulation at 50°C for 2 weeks.

55. 55. The pharmaceutical formulation of claim 54, wherein the purity profile of the pharmaceutical formulation is about 85.2% as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis.

56. 56. The pharmaceutical formulation of any one of claims 1-55, wherein the purity profile of the pharmaceutical formulation is greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, or greater than about 98% as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis after subjecting the pharmaceutical formulation to five freeze-thaw cycles.

57. 57. The pharmaceutical formulation of claim 56, wherein the purity profile of the pharmaceutical formulation is about 98.9% as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis.

58. A method comprising administering to a subject in need thereof a pharmaceutical formulation according to any one of claims 1 to 57 as a single dose therapy.

59. 60. A method comprising administering to a subject in need thereof the pharmaceutical formulation of any one of claims 1 to 57 in a multiple dose therapy with an interval of at least 3 weeks between doses or at least 4 weeks between doses.

60. 60. The method of claim 59, wherein the pharmaceutical formulation is administered to the subject once every three weeks.

61. 60. The method of claim 59, wherein the pharmaceutical formulation is administered to the subject once every four weeks.

62. 60. The method of claim 59, wherein the pharmaceutical formulation is administered to the subject once every six weeks.

63. 63. The method of any one of claims 59-62, further comprising discontinuing said multiple dose therapy if the subject experiences disease progression, unacceptable toxicity, or if the subject meets a withdrawal criterion.

64. 64. The method of any one of claims 59-63, wherein if the subject experiences a complete response (CR) during said multiple dose therapy, said multiple dose therapy is further administered for at least 12 months after confirmation of said complete response.

65. 65. The method of claim 64, wherein the total duration of said multiple dose therapy is 24 months or less.

66. 65. The method of claim 64, wherein the total treatment duration is greater than 24 months.

67. The method of any one of claims 58 to 66, wherein the pharmaceutical formulation is administered by subcutaneous injection.

68. 68. The method of any one of claims 58-67, wherein said pharmaceutical formulation is administered to said subject in an amount sufficient to provide said heterodimeric Fc-fusion protein at a dosage of about 0.05 μg / kg to about 1.75 μg / kg of the subject's body weight.

69. 69. The method of any one of claims 58-68, wherein said pharmaceutical formulation is administered to said subject in an amount sufficient to provide said heterodimeric Fc-fusion protein at a dosage of about 0.05 μg / kg, about 0.10 μg / kg, about 0.20 μg / kg, about 0.40 μg / kg, about 0.60 μg / kg, about 0.80 μg / kg, about 1.00 μg / kg, about 1.20 μg / kg, about 1.40 μg / kg, or about 1.75 μg / kg of the subject's body weight.

70. 68. The method of any one of claims 58-67, wherein said pharmaceutical formulation is administered to said subject in an amount sufficient to provide said heterodimeric Fc-fusion protein at a dosage of greater than 0.00 μg / kg and less than about 0.05 μg / kg of the subject's body weight.

71. 68. The method of any one of claims 58-67, wherein said pharmaceutical formulation is administered to said subject in an amount sufficient to provide said heterodimeric Fc-fusion protein at a dosage of greater than about 1.75 μg / kg of the subject's body weight.

72. The method of any one of claims 58 to 71, wherein the subject has cancer.

73. 73. The method of claim 72, wherein the subject has a locally advanced or metastatic solid tumor.

74. 74. The method of claim 72 or 73, wherein the presence of cancer in the subject is confirmed using the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.

1.

75. 75. The method of any one of claims 72 to 74, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, bladder cancer, urothelial carcinoma, high microsatellite instability carcinoma, colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma (RCC), endometrial carcinoma, cutaneous T-cell lymphoma, and triple-negative breast cancer.

76. The method of any one of claims 72 to 75, wherein the subject is anti-PD-1 refractory.

77. 76. The method of claim 75, wherein the subject has melanoma.

78. 78. The method of claim 77, wherein the subject has previously been treated with an anti-PD-1 antibody for at least 6 weeks.

79. 79. The method of claim 78, wherein the subject has confirmed disease progression at least 4 weeks after initial diagnosis of disease progression while receiving the anti-PD-1 antibody.

80. 80. The method of claim 79, wherein disease progression is confirmed by radiological or clinical findings.

81. 78. The method of claim 77, wherein the subject has a tumor containing a BRAF activating mutation, and the subject has previously been treated with a BRAF inhibitor.

82. 76. The method of claim 75, wherein the subject has RCC.

83. 83. The method of claim 82, wherein the RCC has clear cells.

84. 83. The method of claim 82, wherein the patient has previously been treated with an anti-PD-1 / PD-L1 antibody and / or an anti-vascular endothelial growth factor therapy.

85. 83. The method of claim 82, wherein the subject has received no more than three lines of prior therapy.

86. 76. The method of claim 75, wherein the subject has urothelial carcinoma.

87. 87. The method of claim 86, wherein the subject has locally advanced or metastatic transitional cell carcinoma of the urothelium.

88. 87. The method of claim 86, wherein the subject has previously been treated with a single therapy comprising a platinum-containing regimen and has demonstrated radiographic recurrence of disease progression within six months of the last dose of the platinum-containing regimen.

89. 87. The method of claim 86, wherein the subject has received no more than two lines of prior therapy.

90. 87. The method of claim 86, wherein the subject has not previously received checkpoint inhibitor (e.g., anti-PD-1 or anti-PD-L1 antibody) therapy as monotherapy or in combination with platinum-based chemotherapy.

91. The method of any one of claims 58 to 90, wherein the pharmaceutical formulation is administered to the subject as a monotherapy.

92. The method of any one of claims 58 to 90, wherein the pharmaceutical formulation is administered to the subject as a combination therapy.

93. 93. The method of claim 92, further comprising administering to the subject an anti-PD-1 antibody.

94. 94. The method of claim 93, wherein the anti-PD-1 antibody is pembrolizumab.

95. 95. The method of claim 94, wherein pembrolizumab is administered intravenously.

96. 96. The method of claim 94 or 95, wherein pembrolizumab is administered at a dose of 200 mg.

97. 97. The method of any one of claims 94-96, wherein administration of pembrolizumab occurs prior to each administration of the pharmaceutical formulation.

98. 98. The method of claim 97, wherein the pharmaceutical formulation is administered within 1 hour after completion of administration of pembrolizumab.

99. 93. The method of claim 92, wherein the anti-PD-1 antibody is nivolumab.

100. 100. The method of claim 99, wherein nivolumab is administered intravenously.

101. 101. The method of claim 99 or 100, wherein nivolumab is administered at a dose of 480 mg.

102. The method of any one of claims 99-101, wherein administration of nivolumab precedes each administration of the pharmaceutical formulation.

103. 103. The method of claim 102, wherein the pharmaceutical formulation is administered within 1 hour after completion of administration of nivolumab.

104. 104. The method of any one of claims 99 to 103, wherein the cancer is selected from the group consisting of melanoma, NSCLC, SCLC, RCC, classical Hodgkin's lymphoma, HNSCC, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, bladder cancer, and esophageal cancer.

105. 105. The method of claim 104, wherein the cancer is melanoma.

106. 106. The method of claim 105, wherein the melanoma is unresectable.

107. 105. The method of claim 104, wherein the cancer is colorectal cancer.

108. 108. The method of claim 107, wherein the colorectal cancer is microsatellite instability high (MSI-H) or mismatch repair deficient metastatic (dMMR) colorectal cancer.

109. 99. The method of any one of claims 92 to 98, further comprising performing a surgical intervention to lyse cancer cells, remove tumors, or debulk tumors in the subject.

110. 110. The method of claim 109, wherein the surgical intervention comprises cryotherapy.

111. 110. The method of claim 109, wherein the surgical intervention comprises thermotherapy.

112. 110. The method of claim 109, wherein the surgical intervention comprises administering radiation therapy to the subject.

113. 113. The method of claim 112, wherein the radiation therapy is stereotactic body radiation therapy (SBRT).

114. 114. The method of any one of claims 92-113, further comprising administering to the subject an NK cell targeted therapy.

115. 115. The method of claim 114, wherein the subject is administered a multispecific binding protein.

116. 116. The method of any one of claims 92-115, further comprising administering to the subject a chimeric antigen receptor therapy.

117. 117. The method of any one of claims 92-116, further comprising administering to the subject a cytokine therapy.

118. 118. The method of any one of claims 92-117, further comprising administering to the subject an innate immune system agonist therapy.

119. 119. The method of any one of claims 92-118, further comprising administering chemotherapy to the subject.

120. 120. The method of any one of claims 92-119, further comprising administering to the subject a targeted antigen therapy.

121. 121. The method of any one of claims 92-120, further comprising administering to the subject an oncolytic virus therapy.

122. 1. A method of detecting toxicity in a subject receiving a pharmaceutical formulation comprising measuring a C-reactive protein (CRP) concentration in the blood of the subject, wherein the pharmaceutical formulation comprises a heterodimeric Fc fusion protein and a pharma- ceutically acceptable carrier, and wherein the heterodimeric Fc fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and a p40 and p35 subunit of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or are linked to the second Fc region and the first Fc region, wherein the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and wherein the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization.

123. (1) identifying the subject as being at risk for developing an adverse drug reaction if the CRP concentration in the subject's blood is greater than a threshold CRP concentration; and (2) if the subject's blood CRP concentration is about the same as or lower than the threshold C-reactive protein concentration, the subject is identified as not at risk for developing an adverse drug reaction; The method of claim 122.

124. The method of claim 122, wherein if the subject's blood CRP concentration is higher than the threshold CRP concentration, (1) the administration of the pharmaceutical formulation is discontinued; (2) the heterodimeric Fc-fusion protein is administered at a reduced dose; or (3) therapeutic measures are taken to reduce or ameliorate the toxic effects of the formulation in the subject.

125. The pharmaceutical formulation of any one of claims 1 to 57 or the method of any one of claims 58 to 124, wherein said first and second antibody Fc domain polypeptides are human IgG1 Fc domain polypeptides.

126. 126. The pharmaceutical preparation or method of claim 125, wherein the multi-subunit cytokine is human IL12.

127. 127. The pharmaceutical preparation or method of claim 126, wherein said human IgGl Fc domain polypeptide comprises one or more mutations that reduce an Fc effector function.

128. 128. The pharmaceutical formulation or method of claim 127, wherein the first and second antibody Fc domain polypeptides comprise a mutation selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, numbered according to the EU numbering system.

129. 129. The pharmaceutical preparation or method of claim 128, wherein the first and second antibody Fc domain polypeptides each comprise the mutations L234A, L235A, and P329A.

130. 130. The pharmaceutical formulation or method of claim 129, wherein the first subunit of a multisubunit cytokine is the p40 subunit of IL12 and the second subunit of a multisubunit cytokine is the p35 subunit of IL12.

131. 131. The pharmaceutical formulation or method of claim 130, wherein the first subunit of the multi-subunit cytokine comprises the amino acid sequence of SEQ ID NO: 127 and the second subunit of the multi-subunit cytokine comprises the amino acid sequence of SEQ ID NO:

128.

132. 132. The pharmaceutical formulation or method of claim 131 , wherein the second subunit of a multisubunit cytokine is fused to the second antibody Fc domain by a linker comprising the amino acid sequence of SEQ ID NO:

108.

133. (a) the first antibody Fc domain comprises the mutations L234A, L235A, P329A, Y349C, K360E, and K409W; (b) the second antibody Fc domain comprises the mutations L234A, L235A, P329A, Q347R, S354C, D399V, and F405T; 133. The pharmaceutical formulation or method of claim 132.

134. (a) the first antibody Fc domain comprises the amino acid sequence of SEQ ID NO:215; (b) the second antibody Fc domain comprises the amino acid sequence of SEQ ID NO: 216; 134. The pharmaceutical formulation or method of claim 133.

135. 135. The pharmaceutical formulation or method of claim 134, wherein the first antibody Fc domain peptide comprises the amino acid sequence of SEQ ID NO:290 and the second antibody Fc domain peptide comprises the amino acid sequence of SEQ ID NO:

291.

136. 1. A kit comprising one or more containers containing a pharmaceutical formulation, said pharmaceutical formulation comprising: (a) a heterodimeric Fc fusion protein comprising a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or to the second Fc region and the first Fc region, the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization; and (b) a pharma- ceutically acceptable carrier. Including, and the one or more containers contain a total of about 0.1 mg to about 2 mg of the heterodimeric Fc-fusion protein.

137. 137. The kit of claim 136, wherein the one or more containers contain a total of about 0.5 mg to about 2 mg of heterodimeric Fc-fusion protein.

138. The kit of claim 137, wherein the one or more containers contain a total of about 1 mg of heterodimeric Fc-fusion protein.

139. 139. The kit of claim 138, comprising one container containing about 1 mg of the heterodimeric Fc-fusion protein.

140. The kit according to any one of claims 136 to 139, wherein the pharmaceutical formulation is a lyophilized formulation or a liquid formulation.

141. 141. The kit of claim 140, wherein the pharmaceutical formulation is a liquid formulation supplied in a volume of 1 mL.

142. 1. Use of a heterodimeric Fc-fusion protein in the manufacture of a medicament for the treatment of cancer, said medicament being manufactured as a liquid pharmaceutical formulation comprising about 0.5 g / L to about 1.5 g / L of said heterodimeric Fc-fusion protein in one or more containers; 13. Use of a heterodimeric Fc fusion protein comprising a first and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first and second Fc region, respectively, or to the second Fc region and the first Fc region, and the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and the CH3 domains of the first and second Fc regions each comprise one or more mutations that promote heterodimerization.

143. The use of a heterodimeric Fc-fusion protein according to claim 142, wherein the liquid pharmaceutical formulation comprises about 1.0 g / L of the heterodimeric Fc-fusion protein.

144. 1. Use of a heterodimeric Fc-fusion protein in the manufacture of a medicament for the treatment of cancer, said medicament being formulated as a liquid pharmaceutical formulation comprising about 0.1 mg to about 2 mg of the heterodimeric Fc-fusion protein in one or more containers; 13. Use of a heterodimeric Fc fusion protein comprising a first and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first and second Fc region, respectively, or to the second Fc region and the first Fc region, and the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and the CH3 domains of the first and second Fc regions each comprise one or more mutations that promote heterodimerization.

145. The use of a heterodimeric Fc-fusion protein according to claim 144, wherein the liquid pharmaceutical formulation comprises about 1 mg of the heterodimeric Fc-fusion protein.

146. 146. The use of the heterodimeric Fc-fusion protein according to any one of claims 142 to 145, wherein said medicament is contained in one container.

147. 147. Use of a heterodimeric Fc-fusion protein according to any one of claims 142 to 146, wherein each container contains 1 mg of the heterodimeric Fc-fusion protein.

148. 148. The use according to any one of claims 146 to 147, wherein the medicament is administered to the subject on the first day of every three weeks.

149. 148. The use according to any one of claims 146 to 147, wherein the medicament is administered to the subject on the first day of every four weeks.

150. The use according to any one of claims 146 to 148, wherein the medicament is administered subcutaneously.

151. 151. The use of any one of claims 146 to 150, wherein the medicament is administered in a volume of from about 0.1 mL to about 1 mL.

152. 152. The use of claim 151, wherein the medicament is administered in a volume of about 1 mL.

153. 153. The use according to any one of claims 146 to 152, wherein the medicament is administered at up to two injection sites.

154. 154. The use of claim 153, wherein the second injection is completed within 10 minutes after the first injection.

155. 155. The use of any one of claims 146 to 154, wherein the medicament is administered at a dose of from about 0.05 mg / kg to about 1.75 mg / kg.

156. 156. The use of claim 155, wherein the medicament is administered at a dose of about 1 mg / kg.

157. The use according to any one of claims 146 to 156, wherein the medicament is diluted in a solution of 0.9% saline (sodium chloride for injection) and 0.01% polysorbate 80 prior to administration.

158. A method for producing a heterodimeric Fc-fusion protein for preparation of a pharmaceutical formulation thereof, comprising adding acetic acid to a solution containing said heterodimeric Fc-fusion protein obtained from a Chinese Hamster Ovary (CHO) cell culture expressing said heterodimeric Fc-fusion protein for 30 minutes to 90 minutes, wherein said acetate adjusts and maintains the pH of said solution at pH 3.55 to 3.75; and wherein the heterodimeric Fc fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or to the second Fc region and the first Fc region, wherein the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and wherein the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization.

159. 159. The method of claim 158, wherein said acetic acid is added to said solution containing said heterodimeric Fc-fusion protein for about 60 minutes.

160. 160. The method of claim 158 or 159, wherein the acetic acid adjusts and maintains the pH of the solution at about 3.

65.

161. 159. The method of claim 158, wherein the CHO cell culture expressing the heterodimeric Fc-fusion protein is maintained in suspension.

162. 162. The method of claim 161, wherein the CHO cell culture expressing the heterodimeric Fc-fusion protein is cultured in a bioreactor for 7 to 21 days.

163. 163. The method of claim 161 or 162, wherein the CHO cell culture expressing the heterodimeric Fc-fusion protein is cultured in a bioreactor for 14 days.

164. 164. The method of any one of claims 158-163, wherein the CHO cell culture expressing the heterodimeric Fc-fusion protein is harvested by depth filtration to yield CHO harvest medium.

165. 165. The method of claim 164, wherein the depth filtration is a two-stage disposable depth filtration consisting of a DOHC and an XOHC filter.

166. 166. The method of claim 164 or 165, wherein the heterodimeric Fc-fusion protein is purified from the CHO harvest medium using Protein A capture chromatography, mixed mode chromatography, and cation exchange chromatography to produce the solution comprising the heterodimeric Fc-fusion protein.

167. Protein A capture chromatography Equilibrate Protein A resin with 20 mM Tris, 150 mM NaCl, pH 7.5; loading CHO harvest media onto said Protein A resin; washing the loaded Protein A resin with 20 mM Tris, 150 mM NaCl, pH 7.5; washing the loaded Protein A resin with 50 mM acetate, pH 5.4; and Eluting the heterodimeric Fc-fusion protein from the Protein A resin with 50 mM acetate, 100 mM arginine, pH 3.7, and collecting by 280 nm UV starting at 1.25 AU / cm up and ending at 1.25 AU / cm down.

167. The method of claim 166, comprising:

168. 168. The method of claim 167, wherein acetic acid is added to the solution containing the heterodimeric Fc-fusion protein eluted from the Protein A resin at a concentration of 0.5 M, the acetic acid acidifies the pH of the solution to pH 3.65 for 60 minutes, and then the solution is neutralized to pH 5.2 by adding 2 M Tris.

169. 169. The method of claim 168, wherein following acidification and neutralization of the solution, the solution containing the heterodimeric Fc-fusion protein is passed through a 0.2 μm filter.

170. 170. The method of claim 169, wherein the filtered solution containing the heterodimeric Fc-fusion protein eluted from the Protein A resin is passed through XOSP depth filtration.

171. Mixed mode chromatography Equilibrating a mixed-mode chromatography column with 50 mM acetate, pH 5.2; loading the X0SP filtered solution onto the mixed-mode chromatography column; washing the loaded mixed-mode chromatography column with 50 mM acetate, pH 5.2; and Eluting the heterodimeric Fc-fusion protein from the mixed-mode chromatography column with 50 mM acetate, 250 mM NaCl, pH 5.2, and collecting by 280 nm UV starting at 0.625 AU / cm up and ending at 1.50 AU / cm down.

171. The method of claim 170, comprising:

172. 172. The method of claim 171, wherein the solution containing the heterodimeric Fc-fusion protein eluted from the mixed-mode chromatography column is passed through a 0.2 μm filter.

173. Cation exchange chromatography Equilibrating the cation exchange chromatography resin with 50 mM Tris, pH 7.4; loading the filtered solution eluted from the mixed-mode chromatography column onto the cation exchange chromatography resin; washing the loaded cation exchange chromatography resin with 50 mM Tris, pH 7.4; and Eluting the heterodimeric Fc-fusion protein from the cation exchange chromatography resin with a gradient of 50 mM Tris pH 7.4 and 50 mM Tris pH 7.4, 0.5 M NaCl, and collecting by 280 nm UV starting at a 2.5 AU / cm rise and ending at a 4.5 AU / cm fall.

173. The method of claim 172, comprising:

174. 174. The method of claim 173, wherein the solution containing the heterodimeric Fc-fusion protein eluted from the cation exchange chromatography resin is passed through a 0.2 μm filter.

175. 175. The method of claim 174, wherein the filtered solution comprising the heterodimeric Fc-fusion protein eluted from the cation exchange chromatography resin is nanofiltered with a prefilter, a 20 nm nominal filter, and a 0.2 μm membrane.

176. 176. The method of claim 175, wherein the nanofiltered solution containing the heterodimeric Fc-fusion protein is ultrafiltered and diafiltered, wherein the ultrafiltration and diafiltration comprises: Equilibrate the ultrafiltration system with 50 mM Tris, 265 mM NaCl, pH 7.4; concentrating the nanofiltered solution containing the heterodimeric Fc-fusion protein to a concentration of about 5.0 g / L; exchanging the buffer using 7 diavolumes of 20 mM citrate, pH 6.5; concentrating the diafiltered solution containing the heterodimeric Fc-fusion protein to a concentration of about 11.0 g / L; diluting the concentrated solution containing the heterodimeric Fc-fusion protein with 20 mM citrate at pH 6.5 to a concentration of about 5 g / L to about 10 g / L; and Adding 20 mM citrate, 18% (w / v) sucrose, 3% (w / v) mannitol, 0.03% (w / v) polysorbate 80 at pH 6.5 to achieve a final concentration of the ultrafiltration / diafiltration retentate solution containing the heterodimeric Fc-fusion protein of 20 mM citrate, 6% (w / v) sucrose, 1% (w / v) mannitol, 0.01% (w / v) polysorbate 80. A method comprising:

177. The method of claim 176, wherein the ultrafiltered / diafiltered solution containing the heterodimeric Fc-fusion protein is passed through a 0.2 μm membrane to produce a bulk drug substance.

178. The method of claim 177, wherein the bulk drug substance is diluted to an 80% drug product solution in a 0.2 μm filtered buffer containing 20 mM citrate, 6% (w / v) sucrose, 1% (w / v) mannitol, and 0.01% (w / v) polysorbate 80 at pH 6.

5.

179. 179. The method of claim 177 or 178, wherein the bulk drug product or 80% drug product is diluted in a 0.2 μm filtered buffer containing 20 mM citrate, 6% (w / v) sucrose, 1% (w / v) mannitol, and 0.01% (w / v) polysorbate 80 at pH 6.5 to a dosing concentration of 1 mg / mL of the heterodimeric Fc-fusion protein.

180. 1. A method of treating cancer in a subject who has received at least six weeks of treatment with a checkpoint inhibitor antibody, comprising administering to the subject a pharmaceutical formulation comprising a heterodimeric Fc-fusion protein and a pharma- ceutical acceptable carrier, wherein the heterodimeric Fc-fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or are linked to the second Fc region and the first Fc region, wherein the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and wherein the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization.

181. 181. The method of claim 180, wherein the checkpoint inhibitor antibody is an anti-programmed cell death protein 1 (PD-1) antibody.

182. 182. The method of claim 180 or 181, wherein the cancer is melanoma.

183. 183. The method of claim 182, wherein the melanoma is unresectable or metastatic.

184. 184. The method of claim 182 or 183, wherein the subject is confirmed to have disease progression at least 4 weeks after initial diagnosis of disease progression while receiving the anti-PD-1 antibody.

185. 185. The method of any one of claims 182-184, wherein the subject is confirmed to have disease progression at least 4 weeks after initial diagnosis of disease progression while receiving the anti-PD-1 antibody.

186. 186. The method of claim 184 or 185, wherein disease progression is confirmed by radiological or clinical findings.

187. 1. A method of treating cancer in a subject who has previously been treated with a checkpoint inhibitor antibody or an anti-vascular endothelial growth factor therapy, either as monotherapy or in combination, comprising administering to the subject a pharmaceutical formulation comprising a heterodimeric Fc fusion protein and a pharma- ceutical acceptable carrier, wherein the heterodimeric Fc fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or are linked to the second Fc region and the first Fc region, wherein the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and wherein the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization.

188. 188. The method of claim 187, wherein the checkpoint inhibitor antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

189. The method of claim 187 or 188, wherein the cancer is advanced renal cell carcinoma (RCC).

190. 190. The method of claim 189, wherein the RCC is unresectable or metastatic.

191. 191. The method of claim 189 or 190, wherein the RCC has a clear cell component.

192. 192. The method of any one of claims 189-191, wherein the subject has received no more than three lines of prior therapy.

193. 193. The method of any one of claims 189-192, wherein the subject has not been treated with a checkpoint inhibitor.

194. 200. The method of claim 193, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody or an anti-PD-L1 antibody as monotherapy or in combination with platinum-based chemotherapy.

195. 1. A method of treating cancer in a subject who has been treated with only one platinum-containing regimen, comprising administering to the subject a pharmaceutical formulation comprising a heterodimeric Fc-fusion protein and a pharma- ceutical acceptable carrier, wherein the heterodimeric Fc-fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or are linked to the second Fc region and the first Fc region, wherein the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and wherein the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization.

196. 196. The method of claim 195, wherein the platinum-containing regimen is platinum in combination with an agent selected from gemcitabine, methotrexate, vinblastine, and doxorubicin.

197. 197. The method of claim 195 or 196, wherein the cancer is locally advanced or metastatic urothelial transitional cell carcinoma.

198. 200. The method of claim 197, wherein the urothelial cancer comprises one or more of the group consisting of renal pelvis, ureter, urinary urothelium, and urethra.

199. 199. The method of claim 197 or 198, wherein the urothelial carcinoma is inoperable.

200. 200. The method of any one of claims 197-199, wherein the urothelial cancer is characterized by radiographic progression or recurrence within six months of the last dose of an adjuvant platinum-containing regimen.

201. The method of any one of claims 197 to 200, wherein the urothelial cancer is considered a failure of a first-line platinum-containing regimen.

202. The method of any one of claims 197-201, wherein the subject has received no more than two lines of therapy (including said platinum-containing regimen) for the treatment of said urothelial carcinoma prior to administration of said pharmaceutical formulation.

203. The method of any one of claims 197 to 202, wherein the subject has not received treatment with a checkpoint inhibitor (CPI) as first line therapy.

204. The method of claim 203, wherein the checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

205. The method of claim 203 or 204, wherein the checkpoint inhibitor is a monotherapy or in combination with platinum-based chemotherapy.

206. The method of any one of claims 195-205, wherein the pharmaceutical preparation is administered in combination with pembrolizumab.

207. The method of claim 206, wherein pembrolizumab is administered once every three weeks.

208. The method of claim 206 or 207, wherein pembrolizumab is administered prior to administration of the pharmaceutical formulation.

209. The method of claim 208, wherein the pharmaceutical formulation is administered within 1 hour after completion of administration of pembrolizumab.

210. The method of any one of claims 206-209, wherein pembrolizumab is administered at a dose of 200 mg.

211. The method of any one of claims 206-210, wherein pembrolizumab is administered intravenously.

212. The method of any one of claims 206 to 211, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma, primary mediastinal large B-cell lymphoma, urothelial carcinoma, high microsatellite instability carcinoma, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma, and endometrial carcinoma.

213. The method of any one of claims 195-205, wherein the pharmaceutical formulation is administered in combination with nivolumab.

214. 214. The method of claim 213, wherein nivolumab is administered prior to administration of the pharmaceutical formulation.

215. 215. The method of claim 214, wherein the pharmaceutical formulation is administered within 1 hour after completion of administration of nivolumab.

216. The method of any one of claims 213-215, wherein nivolumab is administered at a dose of about 480 mg.

217. The method of any one of claims 213 to 216, wherein nivolumab is administered intravenously.

218. 218. The method of any one of claims 213 to 217, wherein the cancer is selected from the group consisting of melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), renal cell carcinoma, classical Hodgkin's lymphoma, head and neck squamous cell carcinoma (HNSCC), colorectal cancer, hepatocellular carcinoma, bladder cancer, and esophageal cancer.

219. 219. The method of claim 218, wherein the cancer is melanoma.

220. 220. The method of claim 219, wherein the melanoma is unresectable or metastatic.

221. 219. The method of claim 218, wherein the cancer is colorectal cancer.

222. 222. The method of claim 221, wherein the colorectal cancer is microsatellite instability high (MSI-H) or mismatch repair deficient (dMMR) metastatic colorectal cancer.

223. The method of any one of claims 180 to 211, wherein the pharmaceutical formulation is administered to the subject on the first day of every three weeks.

224. 223. The method of any one of claims 180-222, wherein the pharmaceutical formulation is administered to the subject on the first day of every four weeks.

225. The method of any one of claims 180 to 223, wherein the pharmaceutical formulation is administered subcutaneously.

226. 226. The method of any one of claims 180-225, wherein the pharmaceutical formulation is administered in a volume of about 0.1 mL to about 1 mL.

227. 227. The method of claim 226, wherein the pharmaceutical formulation is administered in a volume of about 1 mL.

228. 228. The method of any one of claims 180 to 227, wherein the pharmaceutical formulation is administered at up to two injection sites.

229. 229. The method of claim 228, wherein the second injection is completed within 10 minutes after the first injection.

230. 230. The method of any one of claims 180-229, wherein the pharmaceutical formulation is administered at a dose of about 0.05 mg / kg to about 1.75 mg / kg.

231. 231. The method of claim 230, wherein the pharmaceutical formulation is administered at a dose of about 1 mg / kg.

232. 232. The method of any one of claims 180-231, wherein the pharmaceutical formulation is diluted in a solution of 0.9% saline (sodium chloride for injection) and 0.01% polysorbate 80 prior to administration.

233. 233. The method of any one of claims 180 to 232, wherein the presence of cancer is determined using the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.

1.

234. The method of any one of claims 180-233, wherein a subject with confirmed complete response is treated with the pharmaceutical preparation for at least 12 months after confirmation, unless censoring criteria are met.

235. 1. A method of treating a subject having a blood level of C-reactive protein (CRP) monitored comprising administering to the subject a pharmaceutical formulation comprising a heterodimeric Fc fusion protein and a pharma- ceutical acceptable carrier, wherein the heterodimeric Fc fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and the p40 and p35 subunits of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or are linked to the second Fc region and the first Fc region, wherein the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and wherein the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization.

236. (1) identifying the subject as being at risk for developing an adverse drug reaction if the CRP concentration in the subject's blood is greater than a threshold CRP concentration; and (2) If the subject's blood CRP concentration is about the same as or lower than the threshold C-reactive protein concentration, the subject is identified as not at risk of developing an adverse drug reaction.

237. The method of claim 235, wherein if the subject's blood CRP concentration is higher than the threshold CRP concentration, (1) the administration of the pharmaceutical formulation is discontinued; (2) the heterodimeric Fc-fusion protein is administered at a reduced dose; or (3) therapeutic measures are taken to reduce or ameliorate the toxic effects of the formulation in the subject.

238. 1. A method of treating cancer in a subject in need thereof comprising subcutaneously administering to said subject a pharmaceutical formulation comprising a heterodimeric Fc-fusion protein and a pharma- ceutical acceptable carrier, wherein the heterodimeric Fc fusion protein comprises a first Fc region and a second Fc region of an immunoglobulin Fc (fragment crystallizable) pair and a p40 and p35 subunit of IL-12, wherein the p40 and p35 subunits of IL-12 are separately linked to the first Fc region and the second Fc region, respectively, or are linked to the second Fc region and the first Fc region, wherein the p40 and p35 subunits are each linked to the N-terminus or C-terminus of the Fc region, and wherein the CH3 domains of the first Fc region and the second Fc region each comprise one or more mutations that promote heterodimerization; and wherein the pharmaceutical formulation comprises citrate; a sugar; a sugar alcohol; and a non-ionic surfactant, and wherein the pH of the formulation is 5.5 to 7.

0.

239. The kit of any one of claims 136 to 141, the use of any one of claims 142 to 157, or the method of any one of claims 158 to 238, wherein the first and second Fc regions are human IgG1 Fc regions.

240. 240. The kit, use, or method of claim 239, wherein the human IgGl Fc region comprises one or more mutations that reduce an Fc effector function.

241. 241. The kit, use, or method of claim 240, wherein the first Fc region and the second Fc region comprise one or more mutations selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, numbered according to the EU numbering system.

242. 242. The kit, use, or method of claim 241, wherein the first Fc region and the second Fc region each comprise the mutations L234A, L235A, and P329A.

243. 243. The kit, use, or method of claim 242, wherein the p40 subunit of IL-12 comprises the amino acid sequence of SEQ ID NO: 127 and the p35 subunit of IL-12 comprises the amino acid sequence of SEQ ID NO:

128.

244. 244. The kit, use, or method of claim 243, wherein the p35 subunit of IL-12 is fused to the second Fc region by a linker comprising the amino acid sequence of SEQ ID NO:

108.

245. (a) the first Fc region comprises the mutations L234A, L235A, P329A, Y349C, K360E, and K409W; (b) the second Fc region comprises the mutations L234A, L235A, P329A, Q347R, S354C, D399V, and F405T; 245. The kit, use, or method of claim 244.

246. (a) the first Fc region comprises the amino acid sequence of SEQ ID NO: 215; (b) the second Fc region comprises the amino acid sequence of SEQ ID NO: 216; 246. The kit, use, or method of claim 245.

247. 247. The kit, use, or method of claim 246, wherein the first Fc region linked to the p40 subunit of IL-12 comprises the amino acid sequence of SEQ ID NO:290 and the second Fc region linked to the p35 subunit of IL-12 comprises the amino acid sequence of SEQ ID NO:

291.

248. 248. The kit, use, or method of any one of claims 239-247, wherein the pharmaceutical formulation comprises: (a) citrate; (b) a sugar; (c) a sugar alcohol; and (d) a non-ionic surfactant, and further wherein the pH of the formulation is from about 6.0 to about 7.

0.

249. 249. The kit, use, or method of claim 248, wherein the concentration of citrate in the pharmaceutical formulation is from about 10 mM to about 30 mM.

250. 250. The kit, use, or method of claim 249, wherein the concentration of citrate in the pharmaceutical formulation is about 20 mM.

251. 251. The kit, use, or method of any one of claims 248 to 250, wherein the concentration of said sugar in said pharmaceutical formulation is from about 3% to about 12% (w / v).

252. 252. The kit, use, or method of claim 251, wherein the concentration of the sugar in the pharmaceutical formulation is about 6% (w / v).

253. 253. The kit, use, or method of claim 251 or 252, wherein the sugar is a disaccharide.

254. 254. The kit, use, or method of claim 253, wherein the disaccharide is sucrose.

255. 255. The kit, use, or method of any one of claims 248 to 254, wherein the concentration of the sugar alcohol in the pharmaceutical formulation is from about 0.5% to about 6% (w / v).

256. 256. The kit, use, or method of claim 255, wherein the concentration of the sugar alcohol in the pharmaceutical formulation is about 1% (w / v).

257. 257. The kit, use, or method of any one of claims 248 to 256, wherein the sugar alcohol is derived from a monosaccharide.

258. 258. The kit, use, or method of claim 257, wherein the sugar alcohol is mannitol.

259. 259. The kit, use, or method of any one of claims 248 to 258, wherein the concentration of said non-ionic surfactant in said pharmaceutical formulation is from about 0.005% to about 0.02% (w / v).

260. 260. The kit, use, or method of claim 259, wherein the concentration of polysorbate 80 in the pharmaceutical formulation is about 0.01% (w / v).

261. 261. The kit, use, or method of claim 259 or 260, wherein the non-ionic surfactant is a polysorbate.

262. 262. The kit, use, or method of claim 261, wherein the polysorbate is polysorbate 80.

263. 263. The kit, use, or method of any one of claims 248 to 262, wherein the pH is from about 6.1 to about 6.

9.

264. 264. The kit, use, or method of claim 263, wherein the pH is from about 6.2 to about 6.

8.

265. 265. The kit, use, or method of claim 264, wherein the pH is from about 6.3 to about 6.

7.

266. 266. The kit, use, or method of claim 265, wherein the pH is from about 6.4 to about 6.

6.

267. 267. The kit, use, or method of claim 266, wherein the pH is about 6.

5.

268. The pharmaceutical formulation, further comprising water, the kit, use, or method of any one of claims 248 to 267.

269. 269. The kit, use, or method of claim 268, wherein the water is USP water for injection.

270. 270. The kit, use or method of any one of claims 248 to 269, wherein said pharmaceutical formulation comprises a bulk concentration of the heterodimeric Fc-fusion protein of about 1 g / L to about 10 g / L.

271. 271. The kit, use, or method of claim 270, wherein said pharmaceutical formulation comprises a bulk concentration of the heterodimeric Fc-fusion protein of about 2 g / L to about 8 g / L.

272. 272. The kit, use, or method of claim 271, wherein said pharmaceutical formulation comprises a bulk concentration of the heterodimeric Fc-fusion protein of about 4 g / L to about 6 g / L.

273. 273. The kit, use, or method of claim 272, wherein said pharmaceutical formulation comprises a bulk concentration of the heterodimeric Fc-fusion protein of about 5 g / L.

274. 274. The kit, use, or method of any one of claims 248 to 273, wherein said pharmaceutical formulation comprises said protein in an administration concentration of about 0.5 g / L to about 1.5 g / L.

275. 275. The kit, use, or method of claim 274, wherein said pharmaceutical formulation comprises said protein in an administration concentration of about 0.75 g / L to about 1.25 g / L.

276. 276. The kit, use, or method of claim 275, wherein the pharmaceutical formulation comprises the protein at an administration concentration of about 1 g / L.

277. 277. The kit, use, or method of any one of 248-276, wherein the formulation is designed for storage at a temperature between 2°C and 8°C.

278. 278. The kit, use, or method of any one of claims 248 to 277, wherein the pharmaceutical formulation is a clear, colorless solution and is free of visible particulate matter.

279. The pharmaceutical formulation comprises: When measured by differential scanning fluorimetry, (a) a T of greater than about 60° C., greater than about 61° C., greater than about 62° C., greater than about 63° C., greater than about 64° C., greater than about 65° C., or greater than about 66° C. ｍ１ and / or (b) a T of greater than about 70° C., greater than about 71° C., greater than about 72° C., greater than about 73° C., greater than about 74° C., greater than about 75° C., greater than about 76° C., or greater than about 77° C. ｍ２ 279. The kit, use or method of any one of claims 248 to 278, having a thermostability profile as defined by:

280. The formulation has a T ｍ１ and a T of about 77.3° C. ｍ２ 280. The kit, use, or method of claim 279, having a thermostability profile as defined by:

281. T ｍ１ and / or T ｍ２ The kit, use, or method of claim 280, wherein the thermal stability profile of the pharmaceutical formulation, as defined by: changes by less than about 2°C or less than about 1°C when the pharmaceutical formulation is incubated at 50°C for 1 week compared to the same pharmaceutical formulation incubated at 5°C for 1 week as measured by differential scanning fluorimetry.

282. The formulation has a T of greater than 60° C., greater than about 61° C., greater than about 62° C., greater than about 63° C., greater than about 64° C., greater than about 65° C., greater than about 66° C., or greater than about 67° C. as measured by differential scanning fluorimetry. ａｇｇ 282. The kit, use, or method of any one of 248-281, having a thermostability profile as defined by:

283. T ａｇｇ The kit, use, or method of claim 282, wherein the thermal stability profile of the pharmaceutical formulation, as defined by: changes by less than about 2°C or less than about 1°C when the pharmaceutical formulation is incubated at 50°C for 1 week compared to the same pharmaceutical formulation incubated at 5°C for 1 week as measured by differential scanning fluorimetry.

284. 284. The kit, use or method of any one of claims 248 to 283, wherein after incubation of the pharmaceutical formulation at 5°C for 1 week, the pH of the pharmaceutical formulation does not change by more than a pH value of about 0.2 or about 0.

1.

285. 285. The kit, use or method of any one of claims 248 to 284, wherein after incubation of the pharmaceutical formulation at 50°C for 1 week, the pH of the pharmaceutical formulation does not change by more than a pH value of about 0.2 or about 0.

1.

286. 286. The kit, use, or method of any one of claims 248-285, wherein said heterodimeric Fc-fusion protein in said pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 15 nm, less than about 14 nm, less than about 13 nm, or less than about 12 nm as measured by dynamic light scattering at 25°C.

287. 287. The kit, use, or method of claim 286, wherein the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of about 11.6 nm.

288. 288. The kit, use, or method of any one of claims 248-287, wherein said heterodimeric Fc-fusion protein in said pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 20 nm, less than about 19 nm, less than about 18 nm, less than about 17 nm, less than about 16 nm, or less than about 15 nm as measured by dynamic light scattering at 25°C after incubating said pharmaceutical formulation at 50°C for two weeks.

289. 289. The kit, use, or method of claim 288, wherein the heterodimeric Fc-fusion protein in the pharmaceutical formulation has a Z-average hydrodynamic diameter of about 14.4 nm.

290. 290. The kit, use, or method of any one of claims 248-289, wherein said heterodimeric Fc-fusion protein in said pharmaceutical formulation has a Z-average hydrodynamic diameter of less than about 20 nm, less than about 19 nm, less than about 18 nm, less than about 17 nm, or less than about 16 nm, as measured by dynamic light scattering at 25°C after subjecting said pharmaceutical formulation to 5 freeze-thaw cycles.

291. 291. The kit, use, or method of claim 290, wherein said heterodimeric Fc-fusion protein in said pharmaceutical formulation has a Z-average hydrodynamic diameter of about 15.3 nm.

292. 292. The kit, use, or method of any one of claims 248-291, wherein said heterodimeric Fc-fusion protein in said pharmaceutical formulation has a polydispersity index of less than about 0.30, less than about 0.29, less than about 0.28, or less than about 0.27 as measured by dynamic light scattering at 25°C.

293. 293. The kit, use or method of any one of claims 248 to 292, wherein the polydispersity index of said heterodimeric Fc-fusion protein in said pharmaceutical formulation is about 0.

26.

294. 294. The kit, use, or method of any one of claims 248 to 293, wherein the polydispersity index of said heterodimeric Fc-fusion protein in said pharmaceutical formulation is less than about 0.30, less than about 0.29, less than about 0.28, less than about 0.27, or less than about 0.26 when measured by dynamic light scattering at 25°C after incubating said pharmaceutical formulation at 50°C for 2 weeks.

295. 295. The kit, use or method of any one of claims 248 to 294, wherein the polydispersity index of said heterodimeric Fc-fusion protein in said pharmaceutical formulation is about 0.

25.

296. 300. The kit, use, or method of any one of claims 248-295, wherein said heterodimeric Fc-fusion protein in said pharmaceutical formulation has a polydispersity index of less than about 0.40, less than about 0.35, or less than about 0.34, as measured by dynamic light scattering at 25°C after subjecting said pharmaceutical formulation to 5 freeze-thaw cycles.

297. 300. The kit, use or method of any one of claims 248 to 296, wherein the polydispersity index of said heterodimeric Fc-fusion protein in said pharmaceutical formulation is about 0.

33.

298. 300. The kit, use or method of any one of claims 248 to 297, wherein the purity profile of the pharmaceutical formulation is greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis.

299. 300. The kit, use or method of claim 298, wherein the purity profile of said pharmaceutical formulation is about 99.0% as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis.

300. 300. The kit, use or method of any one of claims 248 to 299, wherein the purity profile of the pharmaceutical formulation, as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis, is greater than about 75%, greater than about 80%, greater than about 81%, greater than about 82%, greater than about 83%, greater than about 84%, or greater than about 85% after incubating the pharmaceutical formulation at 50°C for 2 weeks.

301. 301. The kit, use or method of claim 300, wherein the purity profile of said pharmaceutical formulation is about 85.2% as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis.

302. 302. The kit, use or method of any one of claims 248-301, wherein the purity profile of said pharmaceutical formulation is greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, or greater than about 98% as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis after subjecting said pharmaceutical formulation to five freeze-thaw cycles.

303. 303. The kit, use or method of claim 302, wherein the purity profile of said pharmaceutical formulation is about 98.9% as measured by the area of ​​the main peak as a percentage of the total area detected in SEC-HPLC analysis.

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