Stable formulations of anti-CTLA4 antibodies alone and in combination with programmed death receptor 1 (PD-1) antibodies and methods of use thereof

Abstract

The present invention relates to a stable formulation comprising an antibody or an antigen-binding fragment thereof that binds to cytotoxic T lymphocyte-associated antigen 4 (CTLA4), optionally further comprising an anti-human programmed death receptor 1 (PD-1) antibody or an antigen-binding fragment thereof. Methods for treating various cancers and chronic infections using the formulations of the present invention are also provided.

Description

Technical Field

[0001] The present invention relates to preparations of therapeutic antibodies and their use in treating various diseases. In one aspect, the present invention relates to preparations comprising antibodies or antigen-binding fragments thereof that bind to cytotoxic T lymphocyte-associated antigen 4 (CTLA4). In another aspect, such preparations further comprise anti-human programmed death receptor 1 (PD-1) antibodies or antigen-binding fragments thereof. Methods of treating various cancers and chronic infections with the preparations described herein are also provided.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of USSN 62 / 500,268, filed May 2, 2017, the contents of which are incorporated herein by reference in their entirety.

[0004] References to electronically submitted sequence listings

[0005] The sequence table of the present application is electronically submitted in ASCII format via EFS-Web, with the file name "24449WOPCT-SEQTXT-30APR2018.TXT", the creation date is April 30, 2018, and the size is 96Kb. This sequence table submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Background Art

[0006] The constant domain amino acid sequence or framework sequence in the variable domain of antibody drugs for humans may be different, but the difference is usually the largest in the CDR sequence. Even antibodies that bind to the same protein, the same polypeptide, or even potentially the same epitope can contain completely different CDR sequences. Therapeutic antibodies for humans can also be obtained from human germline antibody sequences or non-human (e.g., rodent) germline antibody sequences (e.g., in humanized antibodies), resulting in further diversity of potential CDR sequences. These sequence differences lead to different stability in solution and different responsiveness to solution parameters. In addition, slight changes in the amino acid arrangement or changes in one or more amino acid residues can lead to significantly different antibody stability and sensitivity to sequence-specific degradation pathways. As a result, it is currently impossible to predict the solution conditions required for optimizing antibody stability. Each antibody must be studied separately to determine the best solution formulation. Bhambhani et al. (2012) J. Pharm. Sci. 101: 1120.

[0007] Antibodies are also relatively high molecular weight proteins (~150,000Da), for example, compared with other therapeutic proteins (such as hormones and cytokines). As a result, a relatively high weight content (weight amount) of antibody drug dosage is often required to obtain the desired drug molar concentration. In addition, it is generally desirable to administer antibody drugs subcutaneously because this enables self-administration. Self-administration avoids the time and cost associated with visiting a medical institution for administration (e.g., intravenous). Subcutaneous delivery is limited by the volume of solution that can be actually delivered at the injection site in a single injection, which is generally about 1 to 1.5ml. Subcutaneous self-administration is usually completed using a pre-filled syringe, or an automatic syringe filled with a liquid solution formulation of the drug rather than a lyophilized form, to avoid requiring the patient to resuspend the drug before injection. Antibody drugs must be stable during storage to ensure efficacy and consistent dosage, so it is crucial that no matter which formulation is selected, the desired properties, such as high concentration, transparency and acceptable viscosity, should be supported, and these properties and drug efficacy should also be maintained under typical storage conditions within an acceptable long shelf life.

[0008] CTLA4 has a very close relationship with CD28 molecules in terms of gene structure, chromosomal location, sequence homology and gene expression. They are both receptors for the co-stimulatory molecule B7 and are mainly expressed on the surface of activated T cells. After binding to B7, CTLA4 can inhibit the activation of mouse and human T cells and play a negative regulatory role in the activation of T cells.

[0009] CTLA4 mAb or CTLA4 ligand can prevent CTLA4 from binding to its natural ligand, thereby preventing CTLA4 from conducting T cell negative regulatory signals and enhancing the reactivity of T cells to various antigens. In this regard, the results of in vivo and in vitro studies are basically the same. At present, there are some CTLA4 mAbs being tested in clinical trials, which are used to treat prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso et al., CTLA-4blockade in tumor models: an overview of preclinical and translational research. Cancer Immun. 13: 5 (2013)).

[0010] As an important factor affecting T cell function, CTLA4 and CTLA4 mAb can produce specific therapeutic effects on diseases by interfering with the body's immune microenvironment. They have high efficacy and can make up for the shortcomings of traditional drugs, opening up a new path for gene therapy. CTLA4 and CTLA4 mAb are being tested at various stages of experimental and clinical trials. For example, in autoimmune diseases, they effectively inhibit airway hyperresponsiveness in asthma animal models, prevent the development of rheumatic diseases, mediate immune tolerance to allogeneic transplants in vivo, etc. On the other hand, although biological gene therapy has not shown any adverse reactions in short-term clinical trials, attention should be paid to the potential effects after long-term application. For example, excessive blocking of CTLA4-B7 signaling by CTLA4 mAb may lead to the development of autoimmune diseases. Since antibodies can specifically bind to their antigens and induce target cell lysis or prevent pathological progression, the development and utilization of antibody-based drugs, especially humanized antibodies, are of great significance in the clinical treatment of human malignancies and other immune diseases.

[0011] PD-1 is considered to be an important player in immunoregulation and maintenance of peripheral tolerance. PD-1 is moderately expressed on naive T, B and NKT cells and is upregulated by T / B cell receptor signaling on lymphocytes, monocytes and myeloid cells (Sharpe et al., The function of programmed cell death 1and its ligands inregulating autoimmunity and infection. Nature Immunology (2007); 8: 239-245). It has been proposed that the efficacy of anti-PD-1 antibodies may be enhanced if administered in combination with other approved or experimental cancer therapies (e.g., radiotherapy, surgery, chemotherapeutic agents, targeted therapies, agents that inhibit other signaling pathways that are dysregulated in tumors, and other immunopotentiators). One such drug that has been tested in combination with PD-1 antagonists is cytotoxic T lymphocyte-associated antigen 4 (abbreviated as CTLA4).

[0012] There is a need for stable formulations of anti-CTLA4 antibodies for pharmaceutical uses, such as for the treatment of various cancers and infectious diseases, and for stable formulations of anti-CTLA4 antibodies co-formulated with anti-human PD-1 antibodies. Preferably, such formulations will exhibit a long shelf life, be stable during storage and transportation, and will preferably exhibit stability under typical conditions of drug storage for self-administration, i.e., in a syringe at refrigerator temperature, for months to years, resulting in a long shelf life of the corresponding drug product. Summary of the invention

[0013] In one aspect, the present invention includes a formulation of an anti-CTLA4 antibody or an antigen-binding fragment thereof, comprising (i) an anti-CTLA4 antibody or an antigen-binding fragment thereof; (ii) a buffer, (iii) a non-reducing sugar; (iv) a non-ionic surfactant; and an antioxidant. In another embodiment, the formulation further comprises an anti-PD-1 antibody, e.g., pembrolizumab or nivolumab. In one embodiment, the formulation further comprises a chelating agent.

[0014] In one embodiment, the formulation comprises (i) about 10 mg / ml to about 200 mg / ml of an anti-CTLA4 antibody or antigen-binding fragment thereof; (ii) about 5 mM to about 20 mM of a buffer; (iii) about 6% to about 8% weight / volume (w / v) of a non-reducing sugar; (iv) about 0.01% to about 0.10% of a nonionic surfactant; and (v) about 1 mM to about 20 mM of an antioxidant. In another embodiment, the formulation further comprises an anti-PD-1 antibody, e.g., pembrolizumab or nivolumab. In another embodiment, the formulation further comprises a chelating agent. In one embodiment, the chelating agent is present in an amount of about 1 μM to about 50 μM. The chelating agent is DTPA. In one embodiment, the pH of the formulation is between 4.5-6.5. In a specific embodiment, the pH of the formulation is about pH 5.0 to about pH 6.0. In another embodiment, the pH of the formulation is about pH 5.3 to about pH 5.8. In another embodiment, the pH is 5.3. In another embodiment, the pH is 5.4. In one embodiment, the pH is 5.5. In one embodiment, the pH is 5.6. In another embodiment, the pH is 5.7. In one embodiment, the pH is 5.8.

[0015] In one embodiment of the formulation, the buffer is L-histidine buffer or sodium acetate buffer, the non-reducing sugar is sucrose, the nonionic surfactant is polysorbate 80, and the antioxidant is methionine or a pharmaceutically acceptable salt thereof. In one embodiment, the antioxidant is L-methionine. In another embodiment, the antioxidant is a pharmaceutically acceptable salt of L-methionine, for example, methionine HCl.

[0016] In another embodiment, the formulation comprises (i) about 10 mg / ml to about 200 mg / ml of an anti-CTLA4 antibody or antigen-binding fragment thereof; (ii) about 5 mM to about 20 mM L-histidine or about 5 mM to about 20 mM sodium acetate buffer; (iii) about 6% to about 8% w / v sucrose; (iv) about 0.01% to about 0.10% w / v polysorbate 80; and (v) about 1 mM to about 20 mM L-methionine. In another embodiment, the formulation further comprises an anti-PD-1 antibody, e.g., pembrolizumab or nivolumab. In one embodiment, the formulation further comprises a chelator. In one embodiment, the chelator is present in an amount of about 1 μM to about 50 μM. In one embodiment, the chelator is DTPA. In one embodiment, the buffer is an L-histidine buffer. In one embodiment, the formulation comprises about 8 mM to about 12 mM L-histidine. In another embodiment, the formulation comprises about 5 mM to about 10 mM L-methionine. In another embodiment, the formulation comprises about 0.02% w / v polysorbate 80. In one embodiment, the anti-CTLA4 formulation comprises about 7% (w / v) sucrose.

[0017] In an embodiment of the formulation, the concentration of the anti-CTLA4 antibody or its antigen-binding fragment is about 10 mg / ml to about 100 mg / ml. In another embodiment, the concentration of the anti-CTLA4 antibody or its antigen-binding fragment is about 10 mg / ml, 12.5 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 75 mg / ml or 100 mg / ml. In one embodiment, the concentration of the anti-CTLA4 antibody or its antigen-binding fragment is 25 mg / mL. In another embodiment, the concentration of the anti-CTLA4 antibody or its antigen-binding fragment is about 50 mg / ml. In another embodiment, the concentration of the anti-CTLA antibody or its antigen-binding fragment is about 75 mg / mL. In another embodiment, the concentration of the anti-CTLA4 antibody or its antigen-binding fragment is 100 mg / mL.

[0018] In one aspect, a formulation is provided comprising about 25 mg / mL of an anti-CTLA4 antibody or an antigen-binding fragment thereof, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

[0019] In one aspect, a formulation is provided comprising about 50 mg / mL of an anti-CTLA4 antibody or an antigen-binding fragment thereof, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

[0020] In one aspect, a formulation is provided comprising about 75 mg / mL of an anti-CTLA4 antibody or an antigen-binding fragment thereof, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

[0021] In one aspect, a formulation is provided comprising about 100 mg / mL of an anti-CTLA4 antibody or an antigen-binding fragment thereof, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

[0022] In one aspect of any of the above formulations, the pH of the formulation is about 5.3 to 5.8. In another aspect, the pH of the formulation is about 5.5 to about 5.6. In another aspect, the pH of the formulation is about 5.5. In another aspect, the pH of the formulation is about 5.6.

[0023] In one aspect of any of the above formulations, the formulation comprises an anti-PD1 antibody or an antigen-binding fragment thereof. In one aspect, the anti-PD1 antibody is pembrolizumab. In another aspect, the anti-PD1 antibody is nivolumab.

[0024] On the other hand, the formulation may further comprise a chelator. In one embodiment, the chelator is DTPA. In one embodiment, the chelator is EDTA. In one aspect, the chelator is present in an amount of about 1 μM to about 50 μM. In one embodiment, the formulation comprises about 5 μM of a chelator. In one embodiment, the formulation comprises about 10 μM of a chelator. In one embodiment, the formulation comprises about 15 μM of a chelator. In one embodiment, the formulation comprises about 20 μM of a chelator. In one embodiment, the formulation comprises about 25 μM of a chelator. In one embodiment, the formulation comprises about 30 μM of a chelator. In one embodiment, the formulation comprises about 35 μM of a chelator. In one embodiment, the formulation comprises about 40 μM of a chelator. In one embodiment, the formulation comprises about 45 μM of a chelator. In one embodiment, the formulation comprises about 50 μM of a chelator. In one embodiment, the chelator is DTPA, which is present in any amount as described above. In another embodiment, the chelating agent is EDTA, which is present in any of the amounts described above.

[0025] In one embodiment, the formulation is contained in a glass vial. In another embodiment, the formulation is contained in an injection device. In another embodiment, the formulation is a liquid formulation. In one aspect, the formulation is frozen to at least below -70°C. In another embodiment, the formulation is a reconstituted solution from a lyophilized formulation.

[0026] In certain embodiments, the formulation is stable at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. In one embodiment of the formulation, after 12 months at 5°C, the anti-CTLA4 antibody has a % monomer of ≥90% as determined by size exclusion chromatography. In another embodiment of the formulation, after 12 months at 5°C, the anti-CTLA4 antibody has a % monomer of ≥95% as determined by size exclusion chromatography. In another embodiment of the formulation, after 12 months at 5°C, the anti-CTLA4 antibody has a % heavy chain and light chain of ≥90% as determined by reduced CE-SDS. In another embodiment of the formulation, after 12 months at 5°C, the anti-CTLA4 antibody has a % heavy chain and light chain of ≥95% as determined by reduced CE-SDS. In another embodiment of the formulation, after 12 months at 5°C, the anti-CTLA4 antibody has a % intact IgG of ≥90% as determined by non-reduced CE-SDS. In another embodiment of the formulation, the % intact IgG of the anti-CTLA4 antibody is ≥ 95% as determined by non-reduced CE-SDS after 12 months at 5°C.

[0027] In one aspect of any of the above formulations, the formulation comprises an anti-CTLA4 antibody or antigen-binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 38, CDRL2 of SEQ ID NO: 39, CDRL3 of SEQ ID NO: 40 and the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 35, CDRH2 of SEQ ID NO: 36, and CDRH3 of SEQ ID NO: 37. In another aspect, the formulation comprises an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 88 and a light chain variable region comprising SEQ ID NO: 48. In another aspect, the formulation comprises an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a heavy chain comprising SEQ ID NO: 99 and a light chain comprising SEQ ID NO: 100.

[0028] In one aspect, the present invention provides a co-formulation of an anti-CTLA4 antibody or an antigen-binding fragment thereof and an anti-human PD-1 antibody or an antigen-binding fragment thereof, comprising (i) an anti-CTLA4 antibody or an antigen-binding fragment thereof; (ii) an anti-human PD-1 antibody or an antigen-binding fragment thereof, (iii) a buffer, and (iv) a non

[0029] reducing sugar; (iv) nonionic surfactant; and antioxidant. In one embodiment, the co-formulation further comprises a chelating agent. In one embodiment, the chelating agent is EDTA. In another embodiment, the chelating agent is DTPA. In one embodiment of the co-formulation, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 1:2. In another embodiment of the co-formulation, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 1:1. In another embodiment of the co-formulation, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 2:1. In another embodiment of the co-formulation, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 10:1. In another embodiment of the co-formulation, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 1:10. In another embodiment, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 8:1. In another embodiment, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 8:3.

[0030] In one embodiment of the present invention, the co-formulation comprises (i) about 1 mg / ml to about 100 mg / ml of an anti-CTLA4 antibody or an antigen-binding fragment thereof; (ii) about 1 mg / ml to about 100 mg / ml of an anti-human PD-1 antibody, (ii) about 5 mM to about 20 mM of a buffer; (iii) about 6% to about 8% weight / volume (w / v) of a non-reducing sugar; (iv) about 0.01% to about 0.10% of a nonionic surfactant; and (v) about 1 mM to about 20 mM of an antioxidant. In one embodiment, the co-formulation further comprises a chelating agent. In one embodiment, the chelating agent is DTPA. In one embodiment of the co-formulation, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 1:2. In another embodiment of the co-formulation, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 1:1. In another embodiment of the co-formulation, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 2:1. In another embodiment of the co-formulation, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 10:1. In another embodiment of the co-formulation, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 1:10. In another embodiment, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 8:1. In another embodiment, the ratio of the anti-human PD-1 antibody to the anti-CTLA4 antibody is 8:3. In one embodiment, the pH of the co-formulation is 4.5 to 6.5. In other embodiments, the pH of the formulation is about pH 5.0 to about pH 6.0. In another embodiment, the pH of the formulation is about pH 5.3 to about pH 5.8.

[0031] In one embodiment of the co-formulation, the buffer is a histidine buffer or a sodium acetate buffer, the non-reducing sugar is sucrose, the nonionic surfactant is polysorbate 80, and the antioxidant is methionine or a pharmaceutically acceptable salt thereof. In one embodiment, the antioxidant is L-methionine. In another embodiment, the antioxidant is a pharmaceutically acceptable salt of L-methionine, for example, methionine HCl.

[0032] In another aspect, the co-formulation comprises (i) about 1 mg / ml to about 100 mg / ml of an anti-CTLA4 antibody or an antigen-binding fragment thereof; (ii) about 1 mg / ml to about 100 mg / ml of an anti-human PD-1 antibody or an antigen-binding fragment thereof; (iii) about 5 mM to about 20 mM of L-histidine buffer or about 5 mM to about 20 mM of sodium acetate buffer; (iv) about 6% to about 8% w / v sucrose; (v) about 0.01% to about 0.10% w / v of polysorbate 80; and (vi) about 1 mM to about 20 mM of L-methionine. In one embodiment, the co-formulation further comprises a chelating agent. In one embodiment, the chelating agent is DTPA. In one embodiment, the buffer is an L-histidine buffer. In one embodiment, the co-formulation comprises about 8 mM to about 12 mM of L-histidine buffer. In another embodiment, the co-formulation comprises about 5 mM to about 10 mM of L-methionine. In another embodiment, the co-formulation comprises about 0.02% w / v polysorbate 80. In one embodiment, the co-formulation comprises about 7% (w / v) sucrose.

[0033] In an embodiment of the co-formulation, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is about 1 mg / mL to about 100 mg / mL. In another embodiment, the concentration of the anti-CTLA4 antibody is about 10 mg / ml to about 100 mg / ml. In another embodiment, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is about 10 mg / ml. In another embodiment, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 1.25 mg / ml. In another embodiment, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 2.5 mg / ml. In another embodiment, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 5 mg / ml. In another embodiment, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 12.5 mg / ml. In another embodiment, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 25 mg / ml. In another embodiment, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 50 mg / ml. In another embodiment, the anti-CTLA4 antibody or antigen binding fragment thereof is 75 mg / ml. In another embodiment, the concentration of the anti-CTLA4 antibody or antigen binding fragment thereof is 100 mg / ml. In another embodiment, the concentration of the anti-CTLA4 antibody or antigen binding fragment thereof is about 50 mg / ml. In another embodiment, the concentration of the anti-CTLA4 antibody or antigen binding fragment thereof is 2.9 mg / mL. In another embodiment, the concentration of the anti-CTLA4 antibody is 7.9 mg / mL.

[0034] In an embodiment of the co-formulation, the concentration of the anti-human PD-1 antibody is about 1 mg / mL to about 100 mg / mL. In another embodiment, the concentration of the anti-human PD-1 antibody is about 10 mg / ml to about 100 mg / ml. In another embodiment, the concentration of the anti-human PD-1 antibody is about 25 mg / ml. In another embodiment, the concentration of the anti-human PD-1 antibody is about 22.7 mg / ml. In another embodiment, the concentration of the anti-human PD-1 antibody is about 2.27 mg / ml. In another embodiment, the concentration of the anti-human PD-1 antibody is about 21.1 mg / ml. In another embodiment, the concentration of the anti-human PD-1 antibody is about 23.5 mg / ml.

[0035] In one embodiment, the co-formulation comprises about 25 mg / mL of anti-PD1 antibody, about 12.5 mg / mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

[0036] In one embodiment, the co-formulation comprises about 25 mg / mL of anti-PD1 antibody, about 25 mg / mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

[0037] In one embodiment, the co-formulation comprises about 25 mg / mL of anti-PD1 antibody, about 50 mg / mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

[0038] In one embodiment, the co-formulation comprises about 22.72 mg / mL of anti-PD1 antibody, about 2.3 mg / mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

[0039] In one embodiment, the co-formulation comprises about 2.27 mg / mL of anti-PD1 antibody, about 22.7 mg / mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

[0040] In one embodiment, the co-formulation comprises about 23.5 mg / mL of anti-PD1 antibody, about 2.9 mg / mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

[0041] In one embodiment, the co-formulation comprises about 21.1 mg / mL of anti-PD1 antibody, about 7.9 mg / mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and about 10 mM L-methionine.

[0042] In one aspect of any of the above formulations, the formulation comprises an anti-CTLA4 antibody or antigen-binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 38, CDRL2 of SEQ ID NO: 39, CDRL3 of SEQ ID NO: 40 and the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 35, CDRH2 of SEQ ID NO: 36, and CDRH3 of SEQ ID NO: 37. In another aspect, the formulation comprises an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 88 and a light chain variable region comprising SEQ ID NO: 48. In another aspect, the formulation comprises an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a heavy chain comprising SEQ ID NO: 99 and a light chain comprising SEQ ID NO: 100. In one aspect of any of the above formulations, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, CDRL3 of SEQ ID NO: 3 and the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDHR3 of SEQ ID NO: 8. In another aspect, the formulation comprises an anti-human PD1 antibody or antigen-binding fragment thereof comprising a light chain variable region comprising SEQ ID NO: 4 and a heavy chain variable region comprising SEQ ID NO: 9. In another aspect, the formulation comprises an anti-human PD1 antibody or antigen-binding fragment thereof comprising a light chain comprising SEQ ID NO: 5 and a heavy chain comprising SEQ ID NO: 10. In one aspect of any of the above formulations, the anti-human PD-1 antibody or antigen-binding fragment thereof is pembrolizumab. In another aspect, the anti-human PD-1 antibody or antigen-binding fragment thereof is nivolumab.

[0043] In one aspect of any of the above co-formulations, the formulation comprises (i) an anti-CTLA4 antibody or an antigen-binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 38, CDRL2 of SEQ ID NO: 39, CDRL3 of SEQ ID NO: 40 and the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 35, CDRH2 of SEQ ID NO: 36, and CDRH3 of SEQ ID NO: 37 and (ii) an anti-human PD-1 antibody or an antigen-binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, CDRL3 of SEQ ID NO: 3 and the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8.

[0044] In one aspect of any of the above co-formulations, the formulation comprises (i) an anti-CTLA4 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO: 88 and a light chain variable region comprising SEQ ID NO: 48 and (ii) an anti-human PD1 antibody or an antigen-binding fragment thereof comprising a light chain variable region comprising SEQ ID NO: 4 and a heavy chain variable region comprising SEQ ID NO: 9.

[0045] In another aspect of any of the above co-formulations, the formulation comprises (i) an anti-CTLA4 antibody or an antigen-binding fragment thereof comprising a heavy chain comprising SEQ ID NO: 99 and a light chain comprising SEQ ID NO: 100 and (ii) an anti-human PD1 antibody or an antigen-binding fragment thereof comprising a light chain comprising SEQ ID NO: 5 and a heavy chain comprising SEQ ID NO: 10.

[0046] In one embodiment, the formulation is contained in a glass vial. In another embodiment, the formulation is contained in an injection device. In another embodiment, the formulation is a liquid formulation. In one aspect, the formulation is frozen to at least below -70°C. In another embodiment, the formulation is a reconstituted solution from a lyophilized formulation.

[0047] In one aspect, a method of treating a chronic infection or cancer in a mammalian subject (eg, a human) in need thereof is provided, the method comprising administering an effective amount of an anti-CTLA4 formulation or co-formulation described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1AThe UV A350 absorption of Formulation A1 at 5°C, 25°C and 40°C over 8 weeks is shown. Figure 1B The UV A350 absorption of Formulation A2 at 5°C, 25°C and 40°C over 8 weeks is shown.

[0049] Figure 2 Shown are the UV A350 absorbance of formulations A1 and A2 used for freeze-thaw, agitation and light stress studies.

[0050] Figure 3A and 3B % HMW determined by UP-SEC versus time data are shown for Formulations A1 and A2 at 5°C, 25°C and 40°C storage conditions, respectively.

[0051] Figure 4A and 4B % Monomer versus time data determined by UP-SEC are shown for Formulations A1 and A2 at 5°C, 25°C and 40°C storage conditions, respectively.

[0052] Figure 5 Shown are the % HMW determined by UP-SEC for Formulations A1 and A2 used for freeze-thaw, agitation and light stress studies.

[0053] Figure 6 Shown are the % monomer determined by UP-SEC for Formulations A1 and A2 used for freeze-thaw, agitation, and light stress studies.

[0054] Fig. 7A and 7B % Acidity determined by HP-IEX versus time data for Formulations A1 and A2 at 5°C, 25°C and 40°C storage conditions are shown, respectively.

[0055] Fig. 8A and 8B % Basicity determined by HP-IEX versus time data are shown for Formulations A1 and A2 at 5°C, 25°C and 40°C storage conditions, respectively.

[0056] Fig.9A and 9B The % Main component (% Main) versus time data determined by HP-IEX for Formulations A1 and A2 at 5°C, 25°C, and 40°C storage conditions are shown, respectively.

[0057] Fig.10 Shown are the % acidity determined by HP-IEX for Formulations A1 and A2 used for freeze-thaw, agitation, and light stress studies.

[0058] Fig.11 Shown are the % basicity determined by HP-IEX for Formulations A1 and A2 used for freeze-thaw, agitation, and light stress studies.

[0059] Fig.12 Shown are the % principal components determined by HP-IEX for formulations A1 and A2 used for freeze-thaw, agitation and light stress studies.

[0060] Fig.13 The percent oxidation of LC-M4 (methionine oxidation) determined by peptide mapping of formulations A1 and A2 is shown.

[0061] Fig.14 Shown are the percent oxidation of HC-M34 (methionine oxidation) determined by peptide mapping of formulations A1 and A2.

[0062] Fig.15 Shown are the percent oxidation of HC-M250 (methionine oxidation) determined by peptide mapping of formulations A1 and A2.

[0063] Fig.16 Shown are the percent oxidation of HC-M426 (methionine oxidation) determined by peptide mapping of formulations A1 and A2.

[0064] Fig.17A The UV A350 absorption of Formulation Bl at 5°C, 25°C and 40°C over 8 weeks is shown.

[0065] Fig. 17B The UV A350 absorption of Formulation B2 at 5°C, 25°C and 40°C over 8 weeks is shown.

[0066] Fig.18 Shown are the UV A350 absorbances of formulations Bl and B2 used for freeze-thaw, agitation, and light stress studies.

[0067] Fig.19A and 19B % HMW determined by UP-SEC versus time data are shown for Formulations Bl and B2 at 5°C, 25°C and 40°C storage conditions, respectively.

[0068] Fig. 20A and 20B % Monomer versus time data determined by UP-SEC are shown for Formulations Bl and B2 at 5°C, 25°C, and 40°C storage conditions, respectively.

[0069] Fig.21 Shown are the % HMW determined by UP-SEC for formulations Bl and B2 used for freeze-thaw, agitation, and light stress studies.

[0070] Fig. 22 Shown are the % monomer determined by UP-SEC for formulations Bl and B2 used for freeze-thaw, agitation, and light stress studies.

[0071] Fig.23A and 23B% Acidity determined by HP-IEX versus time data are shown for Formulations Bl and B2 at 5°C, 25°C, and 40°C storage conditions, respectively.

[0072] Fig.24A and 24B % Basicity determined by HP-IEX versus time data are shown for Formulations Bl and B2 at 5°C, 25°C, and 40°C storage conditions, respectively.

[0073] Fig.25A and 25B The % principal component versus time data determined by HP-IEX are shown for formulations Bl and B2 at 5°C, 25°C, and 40°C storage conditions, respectively.

[0074] Fig.26 Shown are the % acidity determined by HP-IEX for formulations Bl and B2 used for freeze-thaw, agitation, and light stress studies.

[0075] Fig. 27 Shown are the % basicity determined by HP-IEX for Formulations Bl and B2 used for freeze-thaw, agitation, and light stress studies.

[0076] Fig.28 Shown are the % principal components determined by HP-IEX for formulations Bl and B2 used for freeze-thaw, agitation, and light stress studies.

[0077] Fig.29 The percent oxidation of LC-M4 (methionine oxidation) determined by peptide mapping of formulations B1 and B2 is shown.

[0078] Fig.30 The percent oxidation of HC-M34 (methionine oxidation) determined by peptide mapping of formulations B1 and B2 is shown.

[0079] Fig.31 The percent oxidation of HC-M250 (methionine oxidation) determined by peptide mapping of formulations B1 and B2 is shown.

[0080] Fig.32 Shown are the percent oxidation of HC-M426 (methionine oxidation) determined by peptide mapping of formulations B1 and B2.

[0081] Fig.33 The KD data for the co-formulations are shown, indicating that the formulations are stable at three different pH values ​​(5.0, 5.5, and 6.0).

[0082] Fig.34 The amino acid sequences of the heavy and light chains of ipilimumab are shown (SEQ ID NOs: 84 and 85, respectively). DETAILED DESCRIPTION

[0083] In one aspect, the present invention provides a formulation comprising an anti-CTLA4 antibody or an antigen-binding fragment thereof, which comprises methionine. Also provided is a co-formulation of an anti-CTLA4 antibody or an antigen-binding fragment thereof and an anti-human PD-1 antibody or an antigen-binding fragment thereof, which comprises methionine. In each case, the formulation and co-formulation optionally comprise a chelating agent.

[0084] I. Definitions and Abbreviations

[0085] As used throughout the specification and appended claims, the following abbreviations apply:

[0086] API Active Pharmaceutical Ingredient

[0087] CDR Unless otherwise indicated, the complementarity determining regions in immunoglobulin variable regions are defined using the Kabat numbering system.

[0088] CHO Chinese Hamster Ovary

[0089] CI confidence interval

[0090] CTLA4 Cytotoxic T lymphocyte-associated antigen 4

[0091] DTPA Diethylenetriaminepentaacetic acid

[0092] EC50 Concentration resulting in 50% efficacy or binding

[0093] ELISA enzyme-linked immunosorbent assay

[0094] FFPE formalin fixed, paraffin embedded

[0095] FR Framework Region

[0096] HRP Horseradish Peroxidase

[0097] HNSCC Head and neck squamous cell carcinoma

[0098] IC50 concentration causing 50% inhibition

[0099] IgG Immunoglobulin G

[0100] ICH International Conference on Harmonization

[0101] IHC Immunohistochemistry or immunohistochemical

[0102] mAb monoclonal antibody

[0103] MES 2-(N-morpholino)ethanesulfonic acid

[0104] NCBI National Center for Biotechnology Information

[0105] NSCLC Non-small cell lung cancer

[0106] PCR polymerase chain reaction

[0107] PD-1 Programmed death 1 (also known as programmed cell death-1 and programmed death receptor 1)

[0108] PD-L1 Programmed cell death 1 ligand 1

[0109] PD-L2 Programmed cell death 1 ligand 2

[0110] PS80 Polysorbate 80

[0111] TNBC Triple-negative breast cancer

[0112] V H Immunoglobulin heavy chain variable region

[0113] VK immunoglobulin kappa light chain variable region

[0114] V L Immunoglobulin light chain variable region

[0115] v / v volume / volume

[0116] WFI Water for Injection

[0117] w / v weight / volume

[0118] In order to more easily understand the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by ordinary technicians in the field to which the present invention belongs.

[0119] As used throughout the specification and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0120] Unless the context clearly indicates one of the indicated possibilities, references to "or" indicate one or both possibilities. In some cases, "and / or" is used to highlight one or both possibilities.

[0121] As used herein, "treating" or "treating" cancer means administering the formulation of the invention to a subject suffering from an immune condition or cancerous condition or diagnosed with cancer or pathogenic infection (e.g., viral, bacterial, fungal) to achieve at least one positive therapeutic effect, for example, reducing the number of cancer cells, reducing the size of a tumor, reducing the rate of cancer cells infiltrating surrounding organs, or reducing the rate of tumor metastasis or tumor growth. "Treatment" may include one or more of the following: inducing / increasing an anti-tumor immune response, stimulating an immune response to pathogens, toxins and / or self-antigens, stimulating an immune response to viral infection, reducing the number of one or more tumor markers, inhibiting the growth or survival of tumor cells, eliminating or reducing the size of one or more cancerous lesions or tumors, reducing the level of one or more tumor markers, improving, reducing the severity or duration of cancer, and prolonging the patient's survival relative to the expected survival of a similar patient who has not been treated.

[0122] "Immune conditions" or "immune disorders" encompass, for example, pathological inflammation, inflammatory disorders, and autoimmune disorders or diseases. "Immune conditions" also refer to infections, persistent infections, and proliferative conditions, such as cancer, tumors, and angiogenesis, including infections, tumors, and cancers that resist eradication by the immune system. "Cancerous conditions" include, for example, cancer, cancer cells, tumors, angiogenesis, and precancerous conditions such as dysplasia.

[0123] Positive therapeutic effects in cancer can be measured in many ways (see, WA Weber, J. Nucl. Med. 50: 1S-10S (2009)). For example, with respect to tumor growth inhibition, according to NCI standards, T / C ≤ 42% is the minimum level of anti-tumor activity. T / C < 10% is considered a high level of anti-tumor activity, where T / C (%) = median tumor volume treated / median tumor volume of control × 100. In some embodiments, the treatment achieved by administering the formulation of the present invention is any of progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). PFS, also known as "tumor progression time", refers to the length of time that cancer does not grow during and after treatment, and includes the amount of time that the patient experiences a complete response or partial response, and the amount of time that the patient experiences stable disease. DFS refers to the length of time that a patient remains disease-free during and after treatment. OS refers to the extension of life expectancy compared to an initial (naive) or untreated individual or patient. Although embodiments of the formulations, methods of treatment, and uses of the present invention may not be effective in achieving a positive therapeutic effect in every patient, it should be achieved in a statistically significant number of subjects, as determined by any statistical test known in the art, such as Student's t-test, chi-square test, or similar test. 2test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0124] The term "patient" (alternatively referred to herein as "subject" or "individual") refers to a mammal (e.g., rat, mouse, dog, cat, rabbit), most preferably a human, that can be treated with the formulations of the invention. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a pediatric patient.

[0125] The term "antibody" refers to any form of antibody that exhibits a desired biological activity. Therefore, it is used in the broadest sense and specifically encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized, fully human antibodies, and chimeric antibodies. A "parent antibody" is an antibody obtained by exposing the immune system to an antigen before modifying the antibody for an intended use, such as humanization of an antibody for use as a human therapeutic antibody.

[0126] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The variable region of each light / heavy chain pair forms an antibody binding site. Therefore, typically, a complete antibody has two binding sites. The carboxyl-terminal portion of the heavy chain can define a constant region that is primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. In addition, human heavy chains are typically classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D" region of about 10 or more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W. ed., 2nd ed. Raven Press, NY (1989).

[0127] Typically, the variable domains of both the heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). The CDRs are typically aligned by the framework regions to enable binding to specific epitopes. Typically, from N-terminus to C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Typically, according to Sequences of Proteins of Immunological Interest, Kabat et al.; National Institutes of Health, Bethesda, Md.; 5th Edition; NIH Publication No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat et al., (1977) J. Biol. Chem. 252: 6609-6616; Chothia et al., (1987) J Mol. Biol. 196: 901-917 or Chothia et al., (1989) Nature 342: 878-883, and the amino acids are assigned to each domain.

[0128] An antibody that "specifically binds" a particular target protein is an antibody that exhibits preferential binding to the target compared to other proteins, but the specificity does not require absolute binding specificity. If antibody binding determines the presence of the target protein in the sample, for example, without producing undesirable results such as false positives, the antibody is considered to be "specific" for its intended target. The antibodies or binding fragments thereof that can be used in the present invention will bind to the target protein with an affinity that is at least 2 times greater, preferably at least 10 times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than the affinity for non-target proteins. As used herein, an antibody is said to specifically bind to a polypeptide comprising a given amino acid sequence (e.g., the amino acid sequence of a mature human CTLA4 or human PD-1 molecule) if the antibody binds to a polypeptide comprising the sequence but does not bind to a protein lacking the sequence.

[0129] "Chimeric antibodies" refer to antibodies in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species (e.g., human) or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species (e.g., mouse) or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity.

[0130] As used herein, "co-formulated" or "co-formulation" or "coformulation" or "coformulation" refers to at least two different antibodies or antigen-binding fragments thereof that are formulated together and stored in a single vial or container (e.g., injection device) as a combination product, rather than being formulated and stored separately and then mixed or administered separately prior to administration. In one embodiment, the co-formulation contains two different antibodies or antigen-binding fragments thereof.

[0131] The term "pharmaceutically effective amount" or "effective amount" means an amount of a therapeutic composition or formulation sufficient to introduce into a patient to treat a disease or condition. Those skilled in the art recognize that this level may vary depending on patient characteristics such as age, weight, etc.

[0132] The term "about", when modifying the amount of a substance or composition (e.g., mM or M), the percentage of a formulation component (v / v or w / v), the pH of a solution / formulation, or the value of a parameter of a step in a characterization method, etc., refers to variations in the numerical amount that may occur, for example, by typical measuring, handling, and sampling procedures involved in the preparation, characterization, and / or use of the substance or composition; by instrumental errors in these procedures; by differences in the manufacture, source, or purity of ingredients used to prepare or use the composition or perform the procedure; etc. In certain embodiments, "about" may mean a variation of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%.

[0133] As used herein, "x% (w / v)" equals x g / 100 ml (eg, 5% w / v equals 50 mg / ml).

[0134] The formulations of the present invention include antibodies and fragments thereof that are biologically active when reconstituted or in liquid form.

[0135] The terms "cancer," "cancerous," or "malignant" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer.

[0136] "Chothia" refers to the antibody numbering system described in Al-Lazikani et al., JMB 273:927-948 (1997).

[0137] As used herein, "Kabat" means the immunoglobulin alignment and numbering system originated by Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md.).

[0138] "Growth inhibitors" when used herein refer to compounds or compositions that inhibit cells, especially cancer cells that overexpress any gene identified herein, in vitro or in vivo. Therefore, growth inhibitors are inhibitors that significantly reduce the percentage of cells that overexpress such genes in the S phase. Examples of growth inhibitors include agents that prevent cell cycle progression (outside the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vincas (vincristine and vinblastine), taxanes, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, and etoposide. Those agents that block G1 also overflow into S phase arrest, such as DNA alkylating agents such as dacarbazine, nitrogen mustard, and cisplatin. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogens, and antineoplastic drugs" (Murakami et al.) (WB Saunders: Philadelphia, 1995).

[0139] The terms "CTLA4 binding fragment", "antigen binding fragment thereof", "binding fragment thereof" or "fragment thereof" include fragments or derivatives of antibodies that still substantially retain their biological activity of binding to the antigen (human CTLA4) and inhibiting its activity (e.g., blocking the binding of human CTLA4 to its natural ligand). Therefore, the term "antibody fragment" or CTLA4 binding fragment refers to a portion of a full-length antibody, typically its antigen binding region or variable region. Examples of CTLA4 antibody fragments include Fab, Fab', F(ab') 2and Fv fragments. Typically, the binding fragment or derivative retains at least 10% of its CTLA4 inhibitory activity. In some embodiments, the binding fragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its CTLA4 inhibitory activity, although any binding fragment with an affinity sufficient to exert the desired biological effect will be useful. In some embodiments, the antigen binding fragment binds to its antigen with an affinity that is at least two times greater, preferably at least ten times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than that with an unrelated antigen. In one embodiment, the affinity of the antibody is greater than about 10 9 Liter / mol, as determined, for example, by Scatchard analysis. Munsen et al. (1980) Analyt. Biochem. 107:220-239. It is also contemplated that CTLA4 binding fragments may include variants having conservative amino acid substitutions that do not substantially alter their biological activity.

[0140] The terms "PD-1 binding fragment", "antigen binding fragment thereof", "binding fragment thereof" or "fragment thereof" include fragments or derivatives of antibodies that still substantially retain their biological activity of binding to the antigen (human PD-1) and inhibiting its activity (e.g., blocking the binding of PD-1 to PDL1 and PDL2). Therefore, the term "antibody fragment" or PD-1 binding fragment refers to a portion of a full-length antibody, usually its antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments. Typically, the binding fragment or derivative retains at least 10% of its PD-1 inhibitory activity. In some embodiments, the binding fragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its PD-1 inhibitory activity, although any binding fragment with an affinity sufficient to exert the desired biological effect will be useful. In some embodiments, the antigen binding fragment binds to its antigen with an affinity that is at least two times greater, preferably at least ten times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than that with an unrelated antigen. In one embodiment, the affinity of the antibody is greater than about 10 9 Liter / mol, for example, as determined by Scatchard analysis. Munsen et al. (1980) Analyt. Biochem. 107: 220-239. It is also intended that the PD-1 binding fragment may include variants with conservative amino acid substitutions that do not substantially alter its biological activity.

[0141] "Human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. Human antibodies may contain murine carbohydrate chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, "mouse antibody" or "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively.

[0142] "Humanized antibody" refers to an antibody form containing sequences from non-human (e.g., mouse) antibodies and human antibodies. Such antibodies contain the minimum sequence derived from non-human immunoglobulins. Typically, a humanized antibody will comprise substantially all at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. Humanized antibodies also optionally comprise at least a portion of an immunoglobulin constant region (Fc) (typically a constant region of a human immunoglobulin). The humanized form of a rodent antibody typically comprises the same CDR sequence of a parent rodent antibody, although certain amino acid substitutions may be included in order to increase affinity, increase the stability of the humanized antibody, or for other reasons.

[0143] The antibodies of the present invention also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, for example, U.S. Pat. No. 5,624,821; WO2003 / 086310; WO2005 / 120571; WO2006 / 0057702; Presta (2006) Adv. Drug Delivery Rev. 58: 640-656. Such modifications can be used to enhance or inhibit various responses of the immune system and may have beneficial effects in diagnosis and treatment. Changes in the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and the addition of multiple Fcs. Changes in the Fc region can also change the half-life of the antibody in the therapeutic antibody, and a longer half-life will lead to a reduced frequency of dosing, while increasing convenience and reducing the use of materials. See Presta (2005) J. Allergy Clin. Immunol. 116: 731 (734-35).

[0144] A "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in mice, in mouse cells, or in a hybridoma derived from mouse cells, a fully human antibody may contain murine carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Fully human antibodies can be produced in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods.

[0145] "Hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining region" or "CDR" (e.g., as numbered by the Kabat numbering system (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health). Health, Bethesda, Md.), and / or those residues from the "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain) (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917). As used herein, the term "framework" or "FR" residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues. The CDR and FR residues are defined according to the standard sequence definition of Kabat. Kabat et al. (1987) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda Md.

[0146] "Conservatively modified variants" or "conservative substitutions" refer to amino acid substitutions that are known to those skilled in the art and can be made even in essential regions of a polypeptide without generally changing the biological activity of the resulting molecule. Such exemplary substitutions are preferably made according to those shown in Table 1, as shown below:

[0147] Table 1. Exemplary conservative amino acid substitutions

[0148]

[0149]

[0150] In addition, those skilled in the art recognize that, generally, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. See, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed.).

[0151] As used throughout the specification and claims, the phrase "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of" indicates that any recited element or set of elements is included, and optionally includes other elements having properties similar or different from the recited elements that do not substantially change the basic or novel properties of the specified dosage regimen, method, or composition. As a non-limiting example, a binding compound consisting essentially of a recited amino acid sequence may also include one or more amino acids that do not substantially affect the properties of the binding compound, including substitutions of one or more amino acid residues.

[0152] “Comprising” or variations such as “comprise,” “comprises,” or comprised of are used throughout the specification and claims in an inclusive sense, i.e., specifying the presence of recited features but not excluding the presence or addition of other features that may materially enhance the operation or utility of any embodiment of the invention, unless otherwise required by express language or necessary implicit context.

[0153] "Isolated antibodies" and "isolated antibody fragments" refer to a purified state, and in such a context mean that the named molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other substances such as cell debris and growth medium. Generally, the term "isolated" is not intended to imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that substantially interfere with the experimental or therapeutic use of the binding compounds as described herein.

[0154] As used herein, "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically include a number of different antibodies having different amino acid sequences in their variable domains (particularly their CDRs), which are often specific for different epitopes. The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homologous antibody population, and should not be construed as requiring the production of the antibody by any ad hoc method. For example, the monoclonal antibodies to be used according to the present invention can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0155] "Tumor" as it applies to a subject diagnosed with cancer or suspected of having cancer, refers to a tumor or mass of tissue of any size that is malignant or potentially malignant, and includes primary tumors and secondary tumors. Solid tumors are abnormal growths or masses of tissue that usually do not contain cysts or fluid areas. Different types of solid tumors are named after the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) do not usually form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

[0156] The term "tumor size" refers to the overall size of a tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by a variety of methods known in the art, for example, by measuring the size of the tumor after removal from a subject (e.g., using calipers), or using imaging techniques while in vivo, e.g., bone scans, ultrasound, CT or MRI scans.

[0157] As used herein, "variable region" or "V region" means a fragment of an IgG chain whose sequence is variable between different antibodies. It extends to Kabat residues 109 in the light chain and 113 in the heavy chain.

[0158] The term "buffer" encompasses those agents that maintain the solution pH of the formulations of the invention within an acceptable range or, for lyophilized formulations of the invention, provide an acceptable solution pH prior to lyophilization.

[0159] The terms "lyophilization", "lyophilized" and "freeze-dried" refer to a process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. Excipients may be included in the formulation prior to lyophilization to enhance the stability of the lyophilized product during storage.

[0160] The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the active ingredients to be effective and that contains no other components that are toxic to the subject to which the preparation is to be administered. The terms "formulation" and "pharmaceutical formulation" are used interchangeably throughout.

[0161] "Pharmaceutically acceptable" refers to excipients (vehicles, additives) and compositions that can be reasonably administered to a subject to provide an effective dose of the active ingredient employed, and which are "generally regarded as safe", e.g., they are physiologically tolerable and do not generally produce allergic reactions or similar unpleasant reactions, such as stomach upset, etc., when administered to humans. In another embodiment, the term refers to molecular entities and compositions approved by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or another generally recognized pharmacopeia for use in animals, particularly humans.

[0162] A "reconstituted" formulation is one that is prepared by dissolving a lyophilized protein formulation in a diluent so that the protein is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration, such as parenteral administration, and may optionally be suitable for subcutaneous administration.

[0163] The "reconstitution time" is the time required to rehydrate a lyophilized formulation with a solution into a clear solution free of particles.

[0164] A "stable" formulation is one in which the protein substantially maintains its physical stability and / or chemical stability and / or biological activity during storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected time period at a selected temperature. For example, in one embodiment, a stable formulation is a formulation that has not changed significantly for at least 12 months at a refrigerated temperature (2-8°C). In another embodiment, a stable formulation is a formulation that has not changed significantly for at least 18 months at a refrigerated temperature (2-8°C). In another embodiment, a stable formulation is a formulation that has not changed significantly for at least 3 months at room temperature (23-27°C). In another embodiment, a stable formulation is a formulation that has not changed significantly for at least 6 months at room temperature (23-27°C). In another embodiment, a stable formulation is a formulation that has not changed significantly for at least 12 months at room temperature (23-27°C). In another embodiment, a stable formulation is a formulation that has not changed significantly for at least 18 months at room temperature (23-27°C). The stability criteria for antibody formulations are as follows. Typically, no more than 10%, preferably no more than 5%, of the antibody monomers are degraded as measured by SEC-HPLC. Typically, the formulation is colorless or is clear to slightly milky white by visual analysis. Typically, the concentration, pH, and weight molar osmotic pressure concentration of the formulation do not vary by more than + / -10%. The potency is typically within 60-140% of the control or reference, preferably within 80-120%. Typically, no more than 10%, preferably no more than 5% of antibody truncation is observed, i.e., % low molecular weight species, such as determined by HP-SEC. Typically, no more than 10%, preferably no more than 5% of antibody aggregation is observed, i.e., % high molecular weight species, such as determined by HP-SEC.

[0165] An antibody "maintains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering. Changes in protein conformation can be assessed by fluorescence spectroscopy to determine protein tertiary structure and FTIR spectroscopy to determine protein secondary structure.

[0166] An antibody "maintains its chemical stability" in a pharmaceutical formulation if it does not show significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping coupled to mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0167] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody is within a predetermined range of the biological activity exhibited at a given time when the pharmaceutical formulation is prepared. The biological activity of an antibody can be determined, for example, by antigen binding assays.

[0168] The term "isotonic" means that the target formulation has substantially the same osmotic pressure as human blood. Isotonic formulations typically have an osmotic pressure of about 270-328 mOsm. Slightly hypotonic pressures are 250-269 mOsm, and slightly hypertonic pressures are 328-350 mOsm. Osmotic pressure can be measured, for example, using a vapor pressure or ice-type osmometer.

[0169] II. Formulations and Co-formulations of the Invention.

[0170] In one aspect, the present invention provides a stable biologic formulation comprising an anti-CTLA4 antibody or an antigen-binding fragment thereof that specifically binds to human CTLA4 as an active pharmaceutical ingredient. Including methionine in such a formulation reduces oxidation of methionine residues present in the Fc region of the anti-CTLA4 antibody.

[0171] In one aspect, the present invention also provides a co-formulation of an anti-CTLA4 antibody and an anti-PD-1 antibody. The main degradation pathways of pembrolizumab include oxidation of methionine 105 (Met105) (e.g., M105 of SEQ ID NO: 10) in the heavy chain CDR3 under peroxide stress and oxidation of Met105 and Fc methionine residues when exposed to light. For the degradation levels tested, pembrolizumab maintained its biological activity under most stress conditions. However, a reduced affinity of peroxide stressed samples for PD-1 was observed by surface plasmon resonance (SPR). Exposed methionine residues or methionine residues in the CDR of an antibody may affect the biological activity of the antibody through oxidation. The addition of methionine can reduce the oxidation of Met105 within the CDR of the heavy chain of pembrolizumab.

[0172] Anti-PD-1 antibodies and antigen-binding fragments thereof

[0173] In one aspect, the present invention provides a stable biologic comprising an anti-CTLA4 antibody or antigen-binding fragment thereof co-formulated with an anti-human PD-1 antibody or antigen-binding fragment thereof (e.g., a human or humanized anti-PD-1 antibody) as an active pharmaceutical ingredient (PD-1 API) that specifically binds to human PD-1, and a method of using the formulation of the present invention. Any anti-PD-1 antibody or antigen-binding fragment thereof can be used in the co-formulations and methods of the present invention. In a specific embodiment, the PD-1 API is an anti-PD-1 antibody selected from pembrolizumab and nivolumab. In a specific embodiment, the anti-PD-1 antibody is pembrolizumab. In an alternative embodiment, the anti-PD-1 antibody is nivolumab. Table 2 provides the amino acid sequences of exemplary anti-human PD-1 antibodies pembrolizumab and nivolumab. Table 3 shows alternative PD-1 antibodies and antigen-binding fragments that can be used in the co-formulations and methods of the present invention.

[0174] As used herein, "pembrolizumab" (formerly known as MK-3475, SCH 900475, and lambrolizumab), or "pembro" herein, is a humanized IgG4 mAb whose structure is described in WHO Drug Information, Vol. 27, No. 2, pp. 161-162 (2013), and which comprises the heavy and light chain amino acid sequences and CDRs described in Table 2. Pembrolizumab has been approved by the U.S. FDA for the treatment of patients with unresectable or metastatic melanoma and for the treatment of certain patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), urothelial carcinoma, gastric cancer, microsatellite instability-high (MSI-H) cancer, and non-small cell lung cancer, as KEYTRUDA TM (Merck & Co., Inc., Whitehouse Station, NJ USA; initially approved in the U.S. in 2014, updated September 2017) as described in the prescribing information.

[0175] In some embodiments, the anti-human PD-1 antibody or antigen-binding fragment thereof used in the co-formulation of the present invention comprises the three light chain CDRs of CDRL1, CDRL2 and CDRL3 and / or the three heavy chain CDRs of CDRH1, CDRH2 and CDRH3.

[0176] In one embodiment of the invention, CDRL1 is SEQ ID NO: 1 or a variant of SEQ ID NO: 1, CDRL2 is SEQ ID NO: 2 or a variant of SEQ ID NO: 2, and CDRL3 is SEQ ID NO: 3 or a variant of SEQ ID NO: 3.

[0177] In one embodiment, CDRH1 is SEQ ID NO:6 or a variant of SEQ ID NO:6, CDRH2 is SEQ ID NO:7 or a variant of SEQ ID NO:7, and CDRH3 is SEQ ID NO:8 or a variant of SEQ ID NO:8.

[0178] In one embodiment, the three light chain CDRs are SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the three heavy chain CDRs are SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0179] In an alternative embodiment of the invention, CDRL1 is SEQ ID NO:11 or a variant of SEQ ID NO:11, CDRL2 is SEQ ID NO:12 or a variant of SEQ ID NO:12, and CDRL3 is SEQ ID NO:13 or a variant of SEQ ID NO:13.

[0180] In one embodiment, CDRH1 is SEQ ID NO: 16 or a variant of SEQ ID NO: 16, CDRH2 is SEQ ID NO: 17 or a variant of SEQ ID NO: 17, and CDRH3 is SEQ ID NO: 18 or a variant of SEQ ID NO: 18.

[0181] In one embodiment, the three light chain CDRs are SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the three heavy chain CDRs are SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0182] In an alternative embodiment, the three light chain CDRs are SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and the three heavy chain CDRs are SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0183] In another embodiment of the invention, CDRL1 is SEQ ID NO:21 or a variant of SEQ ID NO:21, CDRL2 is SEQ ID NO:22 or a variant of SEQ ID NO:22, and CDRL3 is SEQ ID NO:23 or a variant of SEQ ID NO:23.

[0184] In yet another embodiment, CDRH1 is SEQ ID NO:24 or a variant of SEQ ID NO:24, CDRH2 is SEQ ID NO:25 or a variant of SEQ ID NO:25, and CDRH3 is SEQ ID NO:26 or a variant of SEQ ID NO:26.

[0185] In another embodiment, the three light chain CDRs are SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, and the three heavy chain CDRs are SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26.

[0186] Some anti-human PD-1 antibodies and antigen-binding fragments of the present invention comprise a light chain variable region and a heavy chain variable region. In some embodiments, the light chain variable region comprises SEQ ID NO: 4 or a variant of SEQ ID NO: 4, and the heavy chain variable region comprises SEQ ID NO: 9 or a variant of SEQ ID NO: 9. In further embodiments, the light chain variable region comprises SEQ ID NO: 14 or a variant of SEQ ID NO: 14, and the heavy chain variable region comprises SEQ ID NO: 19 or a variant of SEQ ID NO: 19. In further embodiments, the heavy chain variable region comprises SEQ ID NO: 27 or a variant of SEQ ID NO: 27, and the light chain variable region comprises SEQ ID NO: 28 or a variant of SEQ ID NO: 28, SEQ ID NO: 29 or a variant of SEQ ID NO: 29, or SEQ ID NO: 30 or a variant of SEQ ID NO: 30. In such embodiments, the variant light chain or heavy chain variable region sequence is identical to the reference sequence except for one, two, three, four or five amino acid substitutions. In some embodiments, the substitutions are in the framework regions (ie, outside of the CDRs). In some embodiments, one, two, three, four or five amino acid substitutions are conservative substitutions.

[0187] In one embodiment of the co-formulation of the present invention, the anti-human PD-1 antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO: 4 and a heavy chain variable region comprising or consisting of SEQ ID NO: 9. In another embodiment, the anti-human PD-1 antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO: 14 and a heavy chain variable region comprising or consisting of SEQ ID NO: 19. In one embodiment of the formulation of the present invention, the anti-human PD-1 antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO: 28 and a heavy chain variable region comprising or consisting of SEQ ID NO: 27. In another embodiment, the anti-human PD-1 antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO: 29 and a heavy chain variable region comprising or consisting of SEQ ID NO: 27. In another embodiment, the antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO: 30 and a heavy chain variable region comprising or consisting of SEQ ID NO: 27.

[0188] In another embodiment, the co-formulation of the present invention comprises an anti-human PD-1 antibody or antigen binding protein having the same V L Domain or V H V or V having at least 95%, 90%, 85%, 80%, 75% or 50% sequence homology to one of the structural domains. L Domain and / or V H In another embodiment, the anti-human PD-1 antibody or antigen-binding protein of the co-formulation of the present invention comprises a V having at most 1, 2, 3, 4 or 5 or more amino acid substitutions. L and V H domain and exhibited specific binding to PD-1.

[0189] In any of the above embodiments, the PD-1 API can be a full-length anti-PD-1 antibody or an antigen-binding fragment thereof that specifically binds to human PD-1. In certain embodiments, the PD-1 API is a full-length anti-PD-1 antibody selected from any class of immunoglobulins (including IgM, IgG, IgD, IgA, and IgE). Preferably, the antibody is an IgG antibody. Any isotype of IgG can be used, including IgG 1 IgG 2 IgG 3 and IgG 4 Different constant domains can be appended to the V L and V HRegion. For example, if the specific intended use of the antibody (or fragment) of the present invention requires a change in effector function, a heavy chain constant domain other than IgG1 can be used. Although IgG1 antibodies provide a longer half-life and effector functions, such as complement activation and antibody-dependent cellular cytotoxicity, such activities may not be ideal for all uses of the antibody. In this case, for example, an IgG4 constant domain can be used.

[0190] In an embodiment of the present invention, the PD-1 API is an anti-PD-1 antibody comprising a light chain and a heavy chain, the light chain comprising or consisting of the amino acid residue sequence shown in SEQ ID NO: 5, and the heavy chain comprising or consisting of the amino acid residue sequence shown in SEQ ID NO: 10. In an alternative embodiment, the PD-1 API is an anti-PD-1 antibody comprising a light chain and a heavy chain, the light chain comprising or consisting of the amino acid residue sequence shown in SEQ ID NO: 15, and the heavy chain comprising or consisting of the amino acid residue sequence shown in SEQ ID NO: 20. In a further embodiment, the PD-1 API is an anti-PD-1 antibody comprising a light chain and a heavy chain, the light chain comprising or consisting of the amino acid residue sequence shown in SEQ ID NO: 32, and the heavy chain comprising or consisting of the amino acid residue sequence shown in SEQ ID NO: 31. In another embodiment, the PD-1 API is an anti-PD-1 antibody comprising a light chain and a heavy chain, the light chain comprising or consisting of the amino acid residue sequence shown in SEQ ID NO: 33, and the heavy chain comprising or consisting of the amino acid residue sequence shown in SEQ ID NO: 31. In another embodiment, the PD-1 API is an anti-PD-1 antibody comprising a light chain and a heavy chain, wherein the light chain comprises or consists of the amino acid residue sequence shown in SEQ ID NO: 34, and the heavy chain comprises or consists of the amino acid residue sequence shown in SEQ ID NO: 31. In some co-formulations of the present invention, the PD-1 API is pembrolizumab or a pembrolizumab biosimilar. In some co-formulations of the present invention, the PD-1 API is nivolumab or a nivolumab biosimilar.

[0191] Typically, the amino acid sequence variants of the anti-PD-1 antibodies and antigen-binding fragments of the invention or the anti-CTLA4 antibodies and antigen-binding fragments of the invention will have amino acid sequences similar to those of the reference antibody or antigen-binding fragment (e.g., heavy chain, light chain, V H 、V Lor humanized sequence) with at least 75% amino acid sequence identity, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, 98% or 99% amino acid sequence. Identity or homology relative to a sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the anti-PD-1 residues after alignment of the sequences and introduction of gaps if necessary to achieve the maximum percentage of sequence identity, without considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions into the antibody sequence shall not be construed as affecting sequence identity or homology.

[0192] Sequence identity refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. Sequence identity can be determined using the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence. The following references relate to BLAST algorithms that are frequently used for sequence analysis: BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W. et al., (1993) Nature Genet. 3: 266-272; Madden, TL et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al., (1997) Genome Res.7:649-656; Wootton, JC et al., (1993) Comput. Chem.17:149-163; Hancock, JM et al., (1994) Comput. Appl. Biosci.10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, MO et al., "A model of evolutionary change in proteins." Atlas of Protein Sequence and Structure, (1978) Vol. 5, Supplement 3. MO Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, RM et al., "Matrices for detecting distant relationships." Atlas of Protein Sequence and Structure, (1978) Vol. 5, Supplement 3. MO Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, RM et al., "Matrices for detecting distant relationships." Atlas of Protein Sequence and Structure, (1978) Vol. 5, Supplement 3." MO Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, (1991) J. Mol. Biol. 219: 555-565; States, DJ et al., (1991) Methods 3: 66-70; Henikoff, S. et al., (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919; Altschul, SF et al., (1993) J. Mol. Evol.36:290-300; ALIGNMENT STATISTICS:Karlin, S. et al., (1990) Proc.Natl.Acad.Sci.USA 87:2264-2268; Karlin, S. et al., (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877; Dembo, A. et al., (1994) Ann. Prob. 22: 2022-2039; and Altschul, SF "Evaluating the statistical significance of multiple distinct local alignments." Theoretical and Computational Methods in Genome Research (edited by S. Suhai), (1997) pp. 1-14, Plenum, New York. .

[0193] Likewise, any class of light chains can be used in the compositions and methods herein. Specifically, kappa, lambda, or variants thereof can be used in the compositions and methods of the present invention.

[0194] Table 2. Exemplary PD-1 antibody sequences

[0195]

[0196]

[0197]

[0198] Table 3. Additional PD-1 antibodies and antigen-binding fragments useful in the co-formulations, methods and uses of the invention.

[0199]

[0200] In some embodiments of the co-formulations of the present invention, the PD-1 API (i.e., anti-PD-1 antibody or antigen-binding fragment thereof) is present at a concentration of about 25 mg / mL to about 100 mg / mL. In alternative embodiments, the API is present at a concentration of about 10 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, or about 100 mg / mL.

[0201] Anti-CTLA4 antibodies and antigen-binding fragments thereof

[0202] The present invention provides stable biologics comprising an anti-CTLA4 antibody or an antigen-binding fragment thereof that specifically binds to human CTLA4 (e.g., a human or humanized anti-CTLA4 antibody) as an active pharmaceutical ingredient (CTLA4 API), and methods of using the formulations of the present invention.

[0203] The present invention also provides a stable biological co-formulation comprising (i) an anti-CTLA4 antibody or an antigen-binding fragment thereof (e.g., a human or humanized anti-CTLA4 antibody) that specifically binds to human CTLA4 and (ii) an anti-human PD-1 antibody or an antigen-binding fragment thereof that specifically binds to human PD-1. Any anti-CTLA4 antibody or antigen-binding fragment thereof can be used in the formulations and methods of the present invention including the co-formulation. Tables 4-8 and Fig.34 Amino acid sequences of exemplary anti-CTLA4 antibodies and antigen-binding fragments useful in the formulations, including co-formulations, and methods of the invention are provided.

[0204] In one embodiment of the formulation comprising the co-formulation, the anti-CTLA-4 antibody is the human monoclonal antibody 10D1, currently known as ipilimumab and marketed as Yervoy TM In another embodiment, the anti-CTLA-4 antibody is tremelimumab, also known as CP-675,206, which is an IgG2 monoclonal antibody described in U.S. Patent Application Publication No. 2012 / 263677 or PCT International Application Publication No. WO 2012 / 122444 or 2007 / 113648A2.

[0205] In a formulation comprising the co-formulation, the anti-CTLA-4 antibody is a monoclonal antibody comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO: 84 and a light chain comprising an amino acid sequence as shown in SEQ ID NO: 85. In some embodiments, the CTLA4 antibody is an antigen-binding fragment of SEQ ID NO: 84 and / or SEQ ID NO: 85, wherein the antigen-binding fragment specifically binds to CTLA4.

[0206] In one embodiment of the formulation of the present invention including the co-formulation, the anti-CTLA-4 antibody is any anti-CTLA-4 antibody or antigen-binding fragment thereof disclosed in International Application Publication No. WO 2016 / 015675A1. In one embodiment, the anti-CTLA4 antibody is a monoclonal antibody comprising the following CDRs:

[0207] HCDR1 comprising the amino acid sequence GFTFSDNW (SEQ ID NO: 35)

[0208] HCDR2 comprising the amino acid sequence IRNKPYNYET (SEQ ID NO: 36)

[0209] HCDR3 comprising the amino acid sequence TAQFAY (SEQ ID NO: 37)

[0210] and / or

[0211] LCDR1 comprising the amino acid sequence ENIYGG (SEQ ID NO: 38)

[0212] LCDR2 comprising the amino acid sequence GAT (SEQ ID NO: 39)

[0213] LCDR3 comprising an amino acid sequence selected from the group consisting of: QNVLRSPFT (SEQ ID NO: 40); QNVLSRHPG (SEQ ID NO: 41); or QNVLSSRPG (SEQ ID NO: 42)

[0214] In one embodiment of the preparation of the present invention including the co-formulation, the anti-CTLA4 antibody or its antigen-binding fragment comprises a variable heavy chain and a variable light chain. In one embodiment, the variable heavy chain and the variable light chain comprise the VH and VL sequences of 8D2 / 8D2(RE) or its variants. In another embodiment, the variable heavy chain and the variable light chain comprise the VH and VL sequences of 8D2H1L1 or its variants. In another embodiment, the variable heavy chain and the variable light chain comprise the VH and VL sequences of 8D2H2L2 or its variants. In another embodiment, the variable heavy chain and the variable light chain comprise the VH and VL sequences of 8D3H3L3 or its variants. In another embodiment, the variable heavy chain and the variable light chain comprise the VH and VL sequences of 8D2H2l17 or its variants. In one embodiment, the methionine at position 18 of any variant of 8D2 / 8D2(RE), 8D2H1L1, 8D2H2L2, 8D2H2L15 or 8D2H2L17 is independently substituted with an amino acid selected from the group consisting of leucine, valine, isoleucine and alanine. In another embodiment of the variant, the methionine at position 18 of any variant of 8D2 / 8D2(RE), 8D2H1L1, 8D2H2L2, 8D2H2L15 or 8D2H2L17 is substituted with leucine.

[0215] In one embodiment of the formulation including the co-formulation, the anti-CTLA4 antibody or antigen-binding fragment thereof is 8D2H2L2 or a variant thereof, wherein the methionine at position 18 in the variable heavy (VH) chain amino acid sequence of the 8D2H2L2 variant is independently substituted with an amino acid selected from the group consisting of leucine, valine, isoleucine and alanine.

[0216] Table 4: Exemplary sequences of anti-CTLA4 antibodies

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] In another embodiment of the formulation of the present invention including the co-formulation, the anti-CTLA4 antibody or antigen-binding fragment thereof comprises the VH and VL chain sequences of 8D2 / 8D2(RE) variant 1. In another embodiment, the anti-CTLA4 antibody or antigen-binding fragment thereof comprises the VH and VL chain sequences of 8D2H1L1 variant 1. In another embodiment, the anti-CTLA4 antibody or antigen-binding fragment thereof comprises the VH and VL chain sequences of 8D2H2L2 variant 1. In another embodiment, the anti-CTLA4 antibody or antigen-binding fragment thereof comprises the VH and VL chain sequences of 8D2H2L15 variant. In another embodiment, the anti-CTLA4 antibody or antigen-binding fragment thereof comprises the VH and VL chain sequences of 8D2H2l17 variant.

[0223] In one embodiment of the formulation of the present invention including the co-formulation, the anti-CTLA4 antibody is any anti-CTLA4 antibody or antigen-binding fragment thereof described in PCT International Application No. PCT / CN2016 / 096357 filed on August 23, 2016. In one embodiment, the anti-CTLA4 antibody is mouse antibody 4G10, which comprises the following VH chain and VL chain amino acid sequences and a humanized form of the antibody.

[0224] Table 5: Mouse anti-CTLA4 antibodies

[0225]

[0226] In one embodiment of the formulation of the present invention comprising the co-formulation, the anti-CTLA4 antibody is a monoclonal antibody comprising the following CDRs:

[0227] HCDR1 comprising a moiety selected from GYSFTGYT (SEQ ID NO: 57) or GYTX 1 N (SEQ ID NO: 58), wherein X 1 For M, V, L, I, G, A, S, T.

[0228] HCDR2 comprising a member selected from INPYNX 1 IX 2 (SEQ ID NO: 59), wherein X 1 is N, D, or E, and X 2 is T, D, E, G, or A; or LINPYNX 1 IX 2 NYX 3 QKFX 4 G (SEQ ID NO: 60), wherein X 1 N, D; X 2 is T, D, E, G or A; X 3 is A or N; and X 4 It is Q or M.

[0229] HCDR3 comprising an amino acid sequence selected from LDIRSY (SEQ ID NO: 61) or ARLDYRSY (SEQ ID NO: 62)

[0230] and / or

[0231] LCDR1 comprising a member selected from TGAVTTSNF (SEQ ID NO: 63) or GSTSGAVTTSNFX 1 N (SEQ ID NO: 64), wherein X 1 is P or A;

[0232] LCDR2, which comprises a compound selected from GTN or GTNNX 1 AX 2 (SEQ ID NO: 65), wherein X 1 is K, R, or any amino acid other than M or C; and X 2 is S or P;

[0233] LCDR3, which comprises ALX 1 YS 2 (SEQ ID NO: 66), wherein X 1 is W or any amino acid except M or C, and X 2 is W or any amino acid except M or C; or ALX1 YS 2 V (SEQ ID NO: 67), wherein X 1 is W or any amino acid except M or C, and X 2 is W or any amino acid except M or C.

[0234] In another embodiment, the humanized VH sequence of the 4G10 antibody comprises any of the following VH sequences:

[0235] Table 6: Exemplary anti-CTLA4 antibody sequences

[0236]

[0237]

[0238] In other embodiments of the formulations of the present invention including the co-formulations, the humanized VL sequence of the 4G10 antibody comprises any of the following VL sequences:

[0239] Table 7: Exemplary anti-CTLA4 antibody sequences

[0240]

[0241]

[0242] In some embodiments, the anti-CTLA4 antibody comprises a variable heavy chain and a variable light chain sequence corresponding to the VH and VL sequences of 4G10H1L1. In another embodiment, the anti-CTLA4 antibody comprises a variable heavy chain and a variable light chain sequence corresponding to the VH and VL sequences of 4G10H3L3. In one embodiment, the anti-CTLA4 antibody comprises a variable heavy chain and a variable light chain sequence corresponding to the VH and VL sequences of 4G10H3L3. In another embodiment, the anti-CTLA4 antibody comprises a variable heavy chain and a variable light chain sequence corresponding to the VH and VL sequences of 4G10H5L3.

[0243] Table 8: Exemplary anti-CTLA4 antibody sequences

[0244]

[0245]

[0246]

[0247]

[0248] In another embodiment of the formulation of the present invention including the co-formulation, the anti-CTLA-4 antibody is an antibody or an antigen-binding fragment thereof that cross-competes with ipilimumab, tremelimumab or any of the above antibodies for binding to human CTLA-4, or is an antibody or an antigen-binding fragment thereof that binds to the same epitope region of human CTLA-4 as ipilimumab, tremelimumab or any of the above antibodies, including 8D2 / 8D2(RE) or 8D2 / 8D2(RE) variant 1, 8D2H1L1 or 8D2H1L1 variant 1, 8D2H2L2 or 8D2H2L2 variant 1, 8D3H3L3, 8D2H2L15 or 8D2H2L15 variant 1, 8D2H2L17 or 8D2H2L17 variant 1, 4G10H1L1 or variants thereof, 4G10H3L3 or variants thereof, 4G10H3L3 or variants thereof, and 4G10H5L3 or variants thereof.

[0249] preparation

[0250] In some aspects of the invention, the formulations described herein minimize the formation of antibody aggregates (high molecular weight species) and microparticles, both high and low molecular weight species, minimize oxidation of methionine residues, and ensure that the antibody retains biological activity over time.

[0251] In one aspect, the present invention includes various preparations of anti-CTLA4 antibodies or antigen-binding fragments thereof. For example, the present invention includes preparations comprising the following ingredients: (i) anti-CTLA4 antibodies or antigen-binding fragments thereof, (ii) buffer (e.g., L-histidine or acetate), (iii) non-reducing sugars (e.g., sucrose); (iv) non-ionic surfactants (e.g., polysorbate 80); and (v) antioxidants (e.g., L-methionine). In one aspect, the preparation further comprises an anti-PD-1 antibody. In one aspect, the preparation may further comprise a chelating agent. In one embodiment, the chelating agent is diethylenetriaminepentaacetic acid (DTPA).

[0252] In one aspect, the present invention also includes various co-formulations of anti-CTLA4 antibodies or antigen-binding fragments thereof and anti-human PD-1 antibodies or antigen-binding fragments thereof. In one embodiment, the present invention includes a formulation comprising the following ingredients: (i) anti-CTLA4 antibodies or antigen-binding fragments thereof, (ii) anti-human PD-1 antibodies or antigen-binding fragments thereof, (iii) buffer (e.g., L-histidine or acetate), (iv) non-reducing sugars (e.g., sucrose), (v) non-ionic surfactants (e.g., polysorbate 80) and (vi) antioxidants (e.g., L-methionine). In one embodiment, the formulation may further include a chelating agent (e.g., DTPA).

[0253] The pharmaceutical formulations described herein may include a buffer. The term "buffer" encompasses those agents that maintain the solution pH of the liquid formulations described herein within an acceptable range, or for the lyophilized formulations described herein, those agents that provide an acceptable solution pH before lyophilization and / or after reconstitution.

[0254] Buffers that can be used in the pharmaceutical preparations and methods of the present invention include succinate (sodium or potassium), L-histidine, phosphate (sodium or potassium), Tris (tris (hydroxymethyl) aminomethane), diethanolamine, citrate (sodium), acetate (sodium), etc. In one embodiment of the invention, the buffer is present in the preparation at a concentration of about 1-20 mM (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 mM). In a specific embodiment of the present invention, the buffer is a histidine buffer. In another embodiment, the buffer is an L-histidine buffer.

[0255] In one embodiment, the pH of the buffer is in the range of about 4.5 to about 6.5. In another embodiment, the pH is in the range of about 5.0-6.0. In another embodiment, the pH range is about 5.3-5.8. In another embodiment, the pH is about 5.5. In order to obtain exemplary formulations, the applicability of histidine and acetate buffers with a pH range of 5.0-6.0 was explored. When describing the range of pH values, such as "pH between pH 5.5 and 6.0", the range is intended to include the described values. For example, the range of about 5.0 to about 6.0 includes 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0. For lyophilized formulations, unless otherwise stated, pH refers to the pH after reconstitution. pH is usually measured at 25°C using a standard glass bulb pH meter. As used herein, a solution comprising "a histidine buffer at pH X" refers to a solution having a pH of X and comprising a histidine buffer, i.e., the pH is intended to refer to the pH of the solution. In some embodiments of the co-formulation wherein the co-formulation contains a higher concentration of anti-human PD-1 antibody than anti-CTLA4 antibody, the pH of the co-formulation is about 5.0.

[0256] In one embodiment of the present invention, the anti-CTLA4 preparation and the co-formulation of anti-CTLA4 and anti-human PD-1 contain non-reducing sugars. As used herein, "non-reducing sugars" are sugars that cannot be used as reducing agents because they do not contain or cannot be converted to contain free aldehyde groups or free ketone groups. Examples of non-reducing sugars include, but are not limited to, disaccharides, such as sucrose and trehalose. In one embodiment, the non-reducing sugar is present in an amount of about 1-10% (w / v) (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%). In another embodiment, the non-reducing sugar is present in an amount of about 6% to about 8% (w / v) (6, 7 or 8%). In another embodiment, the non-reducing sugar is present in an amount of about 6% (w / v). In another embodiment, the non-reducing sugar is present in an amount of about 7% (w / v). In another embodiment, the non-reducing sugar is present in an amount of about 8% (w / v). In one embodiment, the non-reducing sugar is sucrose, trehalose or raffinose. In another embodiment, the non-reducing sugar is sucrose. In another embodiment, the sucrose is present at 6-8% w / v. In one embodiment, the sucrose is present at 6% (w / v). In one embodiment, the sucrose is present at 7% (w / v). In one embodiment, the sucrose is present at 8% (w / v).

[0257] The formulations described herein also include surfactants. As used herein, surfactants are essentially amphipathic surfactants. Surfactants can be added to the formulations herein to provide stability during processing conditions such as purification, filtration, freeze drying, transportation, storage and delivery, reduce and / or prevent aggregation or prevent and / or inhibit protein damage. In one aspect of the invention, surfactants can be used to provide additional stability for one or more active ingredients.

[0258] Nonionic surfactants that can be used in the formulations and co-formulations described herein include, but are not limited to, polyoxyethylene sorbitan fatty acid esters (polysorbates, marketed under the trade name (sold by Uniquema Americas LLC, Wilmington, DE), including polysorbate-20 (polyoxyethylene sorbitan monolaurate), polysorbate-40 (polyoxyethylene sorbitan monopalmitate), polysorbate-60 (polyoxyethylene sorbitan monostearate), and polysorbate-80 (polyoxyethylene sorbitan monooleate); polyoxyethylene alkyl ethers, such as 58 (Uniquema Americas LLC, Wilmington, DE) and 35; poloxamer (eg, poloxamer 188); X-100 (Union Carbide Corp., Houston, TX) and X-114; NP40; Span 20, Span 40, Span 60, Span 65, Span 80 and Span 85; copolymers of ethylene glycol and propylene glycol (e.g., A series of nonionic surfactants, such as F68, 10R5, F108, F127, F38, L44, L62 (BASF Corp., Ludwigshafen, Germany); and sodium dodecyl sulfate (SDS). In one embodiment, the nonionic surfactant is polysorbate 80 or polysorbate 20. In one embodiment, the nonionic surfactant is polysorbate 20. In another embodiment, the surfactant is polysorbate 80.

[0259] The amount of nonionic surfactant included in the formulation of the present invention is an amount sufficient to perform the desired function, i.e., the minimum amount required to stabilize the active pharmaceutical ingredient (i.e., anti-CTLA4 antibody or antigen-binding fragment thereof, or anti-CTLA4 antibody or antigen-binding fragment thereof and anti-human PD-1 antibody or antigen-binding fragment thereof) in the formulation. All percentages of polysorbate 80 listed are % w / v. Typically, the surfactant is present at a concentration of about 0.008% to about 0.1% w / v. In some embodiments of this aspect of the invention, the surfactant is present in the formulation in an amount of about 0.01% to about 0.1%; about 0.01% to about 0.09%; about 0.01% to about 0.08%; about 0.01% to about 0.07%; about 0.01% to about 0.06%; about 0.01% to about 0.05%; about 0.01% to about 0.04%; about 0.01% to about 0.03%, about 0.01% to about 0.02%, about 0.015% to about 0.04%; about 0.015% to about 0.03%, about 0.015% to about 0.02%, about 0.02% to about 0.04%, about 0.02% to about 0.035% or about 0.02% to about 0.03%. In a specific embodiment, the surfactant is present in an amount of about 0.02%. In alternative embodiments, the surfactant is present in an amount of about 0.01%, about 0.015%, about 0.025%, about 0.03%, about 0.035%, or about 0.04%.

[0260] In an exemplary embodiment of the present invention, the surfactant is a nonionic surfactant selected from the group consisting of polysorbate 20 and polysorbate 80. In a preferred embodiment, the surfactant is polysorbate 80.

[0261] In a specific embodiment, the formulation of the present invention including the co-formulation comprises about 0.01% to about 0.04% w / v of polysorbate 80. In a further embodiment, the formulation described herein comprises polysorbate 80 in an amount of about 0.008% w / v, about 0.01% w / v. In one embodiment, the amount of polysorbate 80 is about 0.015 w / v%. In another embodiment, the amount of polysorbate 80 is about 0.02% w / v. In another embodiment, the amount of polysorbate 80 is about 0.025% w / v. In another embodiment, the amount of polysorbate 80 is about 0.03% w / v. In another embodiment, the amount of polysorbate 80 is about 0.035% w / v. In another embodiment, the amount of polysorbate 80 is about 0.04% w / v. In another embodiment, the amount of polysorbate 80 is about 0.045% w / v. In a specific embodiment, the formulations of the invention comprise about 0.02% w / v polysorbate 80.

[0262] The preparation of the present invention including the co-formulation also includes methionine or a pharmaceutically acceptable salt thereof. In one embodiment, the methionine is L-methionine. In another embodiment, the methionine is a pharmaceutically acceptable salt of L-methionine, for example, methionine HCl. In one embodiment, methionine is present in the preparation at a concentration of about 1-20 mM (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19 and 20 mM). In another embodiment, the methionine is present at about 5 mM to about 10 mM (5, 6, 7, 8, 9 and 10 mM). In another embodiment, the methionine is present at about 10 mM.

[0263] The formulations and co-formulations described herein may further comprise a chelating agent. In one embodiment of the invention, the chelating agent is present in the formulation at a concentration of about 1-50 μM (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μM). In one embodiment, the chelating agent is DTPA. In another embodiment, the chelating agent is EDTA. In some other embodiments, the DTPA is an antioxidant, which may be present in any of the formulations described herein in any of the following amounts: 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μM.

[0264] Freeze-dried composition

[0265] Lyophilized formulations of therapeutic proteins provide several advantages. Lyophilized formulations generally have better chemical stability than solution formulations, thus increasing the half-life. Lyophilized formulations can also be reconstituted at different concentrations according to clinical factors, such as route of administration or dosage. For example, if subcutaneous administration is required, the lyophilized formulation can be reconstituted at a high concentration (i.e., in a small volume), or if it is intravenous administration, it can be reconstituted at a lower concentration. If a high dose is required for a particular subject, particularly if the injection volume must be reduced to a minimum for subcutaneous administration, a high concentration may also be required. One such lyophilized antibody formulation is disclosed in U.S. Patent No. 6,267,958, which is incorporated herein by reference in its entirety. Another lyophilized formulation of a therapeutic protein is disclosed in U.S. Patent No. 7,247,707, which is incorporated herein by reference in its entirety.

[0266] Typically, a lyophilized formulation is prepared in anticipation of reconstitution of a high concentration of a drug product (DP, in one exemplary embodiment, a humanized anti-PD-1 antibody pembrolizumab or an antigen-binding fragment thereof), i.e., in anticipation of reconstitution in a small volume of water. Subsequent dilution with water or an isotonic buffer can then be easily used to dilute the DP to a lower concentration. Typically, the content of excipients included in the lyophilized formulation of the present invention will result in a substantially isotonic formulation when reconstituted at a high DP concentration, such as for subcutaneous administration. Reconstitution in a larger volume of water to produce a lower DP concentration will necessarily reduce the tonicity of the reconstituted solution, but such a reduction is of little significance for non-subcutaneous (e.g., intravenous) administration. If isotonicity is desired at a lower DP concentration, the lyophilized powder can be reconstituted in a standard small volume of water and then further diluted with an isotonic diluent (e.g., 0.9% sodium chloride).

[0267] The lyophilized preparation of the present invention is formed by freeze drying (freeze drying) of the pre-freeze drying solution. Freeze drying is accomplished by freezing the preparation and then sublimating water at a temperature suitable for primary drying. Under such conditions, the product temperature is lower than the eutectic point or collapse temperature of the preparation. Typically, the storage temperature (shelf temperature) of the primary drying will be in the range of about -30 to 25°C (assuming that the product remains frozen during the primary drying period) at a suitable pressure of usually about 50 to 250mTorr. The size and type of the preparation, the container (e.g., glass vial) holding the sample, and the volume of the liquid determine the time required for drying, which can range from several hours to several days (e.g., 40-60hrs). The secondary drying stage can be carried out at about 0-40°C, which depends primarily on the type and size of the container and the type of protein used. The secondary drying time depends on the desired residual humidity level in the product, and typically requires at least about 5 hours. Typically, the moisture content of the lyophilized preparation is less than about 5%, and preferably less than about 3%. The pressure can be the same as that used in the primary drying step. Freeze drying conditions may vary depending on the formulation and vial size.

[0268] In some cases, it may be desirable to lyophilize the protein formulation in the container in which the protein is to be reconstituted to avoid a transfer step. In this case, the container may be, for example, a 3, 5, 10, 20, 50 or 100 cc vial.

[0269] The lyophilized formulation of the present invention is reconstructed before administration. The protein can be reconstructed at a concentration of about 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90 or 100 mg / mL or higher concentrations such as 150 mg / mL, 200 mg / mL, 250 mg / mL or 300 mg / mL to about 500 mg / mL. In one embodiment, the protein concentration after reconstruction is about 10-300 mg / ml. In one embodiment, the protein concentration after reconstruction is about 20-250 mg / ml. In one embodiment, the protein concentration after reconstruction is about 150-250 mg / ml. In one embodiment, the protein concentration after reconstruction is about 180-220 mg / ml. In one embodiment, the protein concentration after reconstruction is about 50-150 mg / ml. In one embodiment, the protein concentration after reconstruction is about 100 mg / ml. In one embodiment, the protein concentration after reconstruction is about 75 mg / ml. In one embodiment, the protein concentration after reconstruction is about 50 mg / ml. In one embodiment, the protein concentration after reconstitution is about 25 mg / ml. In the case of a reconstituted formulation intended for subcutaneous delivery, a high protein concentration is particularly useful. However, for other routes of administration, such as intravenous administration, a lower concentration of protein (e.g., about 5-50 mg / mL) may be desirable.

[0270] Reconstitution is typically performed at a temperature of about 25°C to ensure complete hydration, although other temperatures may be employed as desired. The time required for reconstitution will depend, for example, on the type of diluent, the amount of excipients and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

[0271] Liquid composition

[0272] Liquid antibody formulations can be prepared by taking a liquid form of a pharmaceutical substance (e.g., anti-humanized PD-1) (e.g., pembrolizumab in an aqueous formulation) and exchanging it with a buffer into a desired buffer as the last step of the purification process. There is no lyophilization step in this embodiment. The pharmaceutical substance in the final buffer is concentrated to the desired concentration. Excipients such as sucrose and polysorbate 80 are added to the pharmaceutical substance and diluted to the final protein concentration using an appropriate buffer. The final formulated pharmaceutical substance is filtered using a 0.22 μm filter and filled into a final container (e.g., a glass vial).

[0273] III. How to use

[0274] The present invention also relates to a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of any formulation of the present invention, i.e., any formulation described herein. In some specific embodiments of the method, the formulation is administered to the subject by intravenous administration. In other embodiments, the formulation is administered to the subject by subcutaneous administration. In one embodiment, the present invention includes a method for treating cancer in a human patient, the method comprising administering to the patient any formulation of the present invention.

[0275] In any of the methods of the invention, the cancer can be selected from melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, lymphoma, kidney cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary cancer, prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), pancreatic cancer, colon cancer, esophageal cancer, liver cancer, thyroid cancer, glioblastoma, glioma and other neoplastic malignancies.

[0276] In some embodiments, the lung cancer is non-small cell lung cancer.

[0277] In an alternative embodiment, the lung cancer is small cell lung cancer.

[0278] In some embodiments, the lymphoma is Hodgkin lymphoma.

[0279] In other embodiments, the lymphoma is non-Hodgkin's lymphoma. In a specific embodiment, the lymphoma is mediastinal large B-cell lymphoma.

[0280] In some embodiments, the breast cancer is triple-negative breast cancer.

[0281] In a further embodiment, the breast cancer is ER+ / HER2- breast cancer.

[0282] In some embodiments, the bladder cancer is urothelial carcinoma.

[0283] In some embodiments, the head and neck cancer is nasopharyngeal cancer. In some embodiments, the cancer is thyroid cancer. In other embodiments, the cancer is salivary cancer. In other embodiments, the cancer is squamous cell carcinoma of the head and neck.

[0284] In one embodiment, the present invention includes a method for treating metastatic non-small cell lung cancer (NSCLC) in a human patient, the method comprising administering a formulation of the present invention to the patient. In a specific embodiment, the patient has a tumor with high PD-L1 expression [(tumor proportion score (TPS) ≥ 50%)] and has not been previously treated with platinum-containing chemotherapy. In other embodiments, the patient has a tumor with PD-L1 expression (TPS ≥ 1%) and has been previously treated with platinum-containing chemotherapy. In other embodiments, the patient has a tumor with PD-L1 expression (TPS ≥ 1%) and has not been previously treated with platinum-containing chemotherapy. In a specific embodiment, the patient develops the disease while or after receiving platinum-containing chemotherapy. In certain embodiments, the PD-L1 TPS is determined by an FDA-approved test. In certain embodiments, the patient's tumor has no EGFR or ALK genomic aberrations. In certain embodiments, the patient's tumor has EGFR or ALK genomic aberrations, and the disease develops while or after receiving treatment for EGFR or ALK aberrations (one or more) before receiving an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0285] In some embodiments, the cancer is metastatic colorectal cancer with high levels of microsatellite instability (MSI-H).

[0286] In some embodiments, the cancer is metastatic colorectal cancer with high levels of microsatellite instability (MSI-H).

[0287] In some embodiments, the cancer is a solid tumor with high levels of microsatellite instability (MSI-H).

[0288] In some embodiments, the cancer is a solid tumor with a high mutational burden.

[0289] In some embodiments, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, relapsed or refractory classical Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal cancer, gastric cancer, and hepatocellular carcinoma.

[0290] In other embodiments of the above-mentioned treatment methods, the cancer is a heme malignancy. In certain embodiments, the heme malignancy is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T cell / histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia 1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL) or small lymphocytic lymphoma (SLL).

[0291] Malignancies that exhibit improved disease-free and overall survival (which correlate with the presence of tumor-infiltrating lymphocytes in biopsies or surgical material) are encompassed in the methods and treatments described herein, such as melanoma, colorectal cancer, liver cancer, kidney cancer, gastric / esophageal cancer, breast cancer, pancreatic cancer, and ovarian cancer. Such cancer subtypes are known to be susceptible to immune control by T lymphocytes. In addition, refractory or recurrent malignancies whose growth can be inhibited using the antibodies described herein are included.

[0292] Other cancers that may benefit from treatment with the formulations described herein include those associated with persistent infection with viruses, such as human immunodeficiency virus, hepatitis A, B and C viruses, Epstein-Barr virus, human papillomavirus known to be causally associated with, for example, Kaposi's sarcoma, liver cancer, nasopharyngeal cancer, lymphoma, cervical cancer, vulvar cancer, anal cancer, penile cancer, and oral cancer.

[0293] The preparation can also be used to prevent or treat infection and infectious diseases. Therefore, the present invention provides a method for treating chronic infection in a mammalian subject, the method comprising administering an effective amount of the preparation of the present invention to the subject. In some specific embodiments of the method, the preparation is administered to the subject by intravenous administration. In other embodiments, the preparation is administered to the subject by subcutaneous administration.

[0294] These agents can be used alone or in combination with vaccines to stimulate immune responses to pathogens, toxins, and self-antigens. The antibodies or antigen-binding fragments thereof can be used to stimulate immune responses to viruses that infect humans, including but not limited to: human immunodeficiency virus, hepatitis A, B, and C viruses, Epstein-Barr virus, human cytomegalovirus, human papillomavirus, and herpes virus. Antagonist anti-PD-1 antibodies or antibody fragments can be used to stimulate immune responses to infections with bacteria or fungal parasites and other pathogens. Viral infections with hepatitis B and C and HIV are among those infections that are considered chronic viral infections.

[0295] The formulations of the present invention can be administered to a patient in combination with one or more "additional therapeutic agents". Additional therapeutic agents can be biological therapeutic agents (including but not limited to antibodies to the following substances: VEGF, EGFR, Her2 / neu, VEGF receptors, other growth factor receptors, CD20, CD40, CD-40L, OX-40, 4-1BB and ICOS), immunogenic agents (e.g., attenuated cancer cells, tumor antigens, antigen presenting cells (such as dendritic cells stimulated with tumor-derived antigens or nucleic acid pulses), immunostimulatory cytokines (e.g., IL-2, IFNα2, GM-CSF), and cells transfected with genes encoding immunostimulatory cytokines (such as but not limited to GM-CSF).

[0296] As described above, in some embodiments of the method of the present invention, the method further comprises administering an additional therapeutic agent. In a specific embodiment, the additional therapeutic agent is an anti-LAG3 antibody or an antigen binding fragment thereof, an anti-GITR antibody or an antigen binding fragment thereof, an anti-TIGIT antibody or an antigen binding fragment thereof, an anti-CD27 antibody or an antigen binding fragment thereof. In one embodiment, the additional therapeutic agent is a Newcastle disease virus vector expressing IL-12. In another embodiment, the additional therapeutic agent is dinaciclib. In a further embodiment, the additional therapeutic agent is a STING agonist.

[0297] Suitable routes of administration may include, for example, parenteral delivery, including intramuscular, subcutaneous, and intrathecal, direct intraventricular, intravenous, intraperitoneal. The drug may be administered in a variety of conventional ways, such as intraperitoneal, parenteral, intraarterial or intravenous injection. Modes of administration that must limit solution volume (e.g., subcutaneous administration) require lyophilized formulations to be reconstituted at high concentrations.

[0298] The dosage of the additional therapeutic agent selected depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissues, or organs in the individual being treated. The dosage of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Therefore, the dosage and frequency of administration of each additional therapeutic agent (e.g., a biotherapeutic or chemotherapeutic agent) will depend in part on the specific therapeutic agent, the severity of the cancer being treated, and the patient's characteristics. Guidance for selecting appropriate dosages of antibodies, cytokines, and small molecules is available. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 345:783-792. Engl. J. Med. 342: 613-619; Ghosh et al. (2003) New Engl. J. Med. 348: 24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th edition); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). The clinician can determine the appropriate dosage regimen, for example, using parameters or factors known or suspected in the art to affect treatment or expected to affect treatment, and will depend on, for example, the patient's clinical history (e.g., previous therapy), the type and stage of cancer to be treated, and biomarkers that respond to one or more therapeutic agents in combination therapy.

[0299] Various literature references are available to facilitate the selection of pharmaceutically acceptable carriers or excipients for other therapeutic agents. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984); Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York. York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parental Medications, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker,NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0300] The pharmaceutical antibody formulation can be administered by continuous infusion or at intervals of, for example, one day, 1-7 times per week, one week, two weeks, three weeks, monthly, every two months, etc. The preferred dosage regimen is a dosage regimen involving the maximum dose or dosage frequency that avoids significant undesirable side effects. The total weekly dose is generally at least 0.05 μg / kg, 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.2 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg body weight or higher. See, e.g., Yang et al. (2003) New Engl. J. Med. 349:427-434; Herold et al. (2002) New Engl. J. Med. 346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji et al. (20003) Cancer Immunol. Immunother. 52:133-144. The expected dosage of small molecule therapeutics, such as peptide mimetics, natural products or organic chemical agents, is substantially the same as that of antibodies or polypeptides on a moles / kg basis.

[0301] Embodiments of the invention also include one or more of the biologics described herein (i) for use, (ii) as a medicament or composition for use, or (iii) for the preparation of a medicament for use in: (a) therapy (e.g., human therapy); (b) a medicament; (c) inducing or increasing an anti-tumor immune response; (d) reducing the amount of one or more tumor markers in a patient; (e) stopping or slowing the growth of a tumor or blood cancer; (f) stopping or slowing the progression of a CTLA4 or PD-1 related disease; (g) preventing or delaying cancer progression; (h) stabilizing CTLA4 or PD-1. 1 related disease; (i) inhibiting the growth or survival of tumor cells; (j) eliminating or reducing the size of one or more cancerous lesions or tumors; (k) reducing the progression, onset or severity of CTLA4 or PD-1 related diseases; (l) reducing the severity or duration of clinical symptoms of CTLA4 or PD-1 related diseases such as cancer; (m) prolonging patient survival relative to the expected survival of similar untreated patients; n) inducing complete or partial remission of cancer conditions or other CTLA4 or PD-1 related diseases; o) treating cancer; or p) treating chronic infections.

[0302] General approach

[0303] Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 1989 2nd edition, 2001 3rd edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0304] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described (Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modifications, post-translational modifications, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) Bio Directory, Piscataway, NJ, pp. 384-391). The production, purification and fragmentation of polyclonal and monoclonal antibodies are described (Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0305] Monoclonal antibodies, polyclonal antibodies, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Harbor, NY, pp. 139-243; Carpenter et al. (2000) J. Immunol. 165:6205; He et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

[0306] An alternative to humanization is to use human antibody libraries displayed on phage or in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).

[0307] Purification of the antigen is not necessary for the production of antibodies. Animals can be immunized with cells bearing the target antigen. Splenocytes can then be isolated from the immunized animal and fused with a myeloma cell line to produce hybridomas (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithamana et al. (1999) J. Immunol. 163:5157-5164).

[0308] The antibody can be conjugated to, for example, a small drug molecule, an enzyme, a liposome, polyethylene glycol (PEG). The antibody can be used for treatment, diagnosis, kits, or other purposes, and includes, for example, antibodies coupled to dyes, radioisotopes, enzymes, or metals (e.g., colloidal gold) (see, e.g., Le Doussal et al. (1991) J. Immunol. 146: 169-175; Gibellini et al. (1998) J. Immunol. 160: 3891-3898; Hsing and Bishop (1999) J. Immunol. 162: 2804-2811; Everts et al. (2002) J. Immunol. 168: 883-889).

[0309] Methods for flow cytometry, including fluorescence activated cell sorting (FACS), are available (see, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids (including nucleic acid primers and probes, polypeptides and antibodies useful as, e.g., diagnostic reagents) are available (Molecular Probesy (2003) Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO).

[0310] Standard methods for the histology of the immune system are described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Philadelphia, PA; Louis et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

[0311] Software packages and databases are available for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments (see, e.g., GenBank, Vector Suite (Informax, Inc., Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); (TimeLogic Corp., Crystal Bay, Nevada); Menne et al. (2000) Bioinformatics 16:741-742; Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren et al. (2002) Comput. Methods Programs Biomed. 68: 177-181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0312] Analytical methods

[0313] Analytical methods suitable for evaluating product stability include size exclusion chromatography (SEC), dynamic light scattering test (DLS), differential scanning calorimetry (DSC), iso-aspartic acid (iso-asp) quantification, potency, UV at 340 nm, UV spectroscopy and FTIR.SEC (J. Pharm. Scien., 83: 1645-1650, (1994); Pharm. Res., 11: 485 (1994); J. Pharm. Bio. Anal., 15: 1928 (1997); J. Pharm. Bio. Anal., 14: 1133-1140 (1986)) measures the percentage of monomers in the product and gives information on the amount of soluble aggregates.DSC (Pharm. Res., 15: 200 (1998); Pharm. Res., 9: 109 (1982)) gives information on protein denaturation temperature and glass transition temperature. DLS (American Lab., November (1991)) measures the average diffusion coefficient and gives information on the amount of soluble and insoluble aggregates. UV at 340 nm measures the intensity of scattered light at 340 nm and gives information on the amount of soluble and insoluble aggregates. UV spectroscopy measures the absorbance at 278 nm and gives information on protein concentration. FTIR (Eur. J. Pharm. Biopharm., 45:231 (1998); Pharm. Res., 12:1250 (1995); J. Pharm. Scien., 85:1290 (1996); J. Pharm. Scien., 87:1069 (1998)) measures the IR spectrum of the amide one region and gives information on the secondary structure of the protein.

[0314] The isoaspartate content in the sample is measured using the Isoquant isoaspartate detection system (IsoquantIsoaspartate Detection System, Promega). The kit uses the enzyme protein isoaspartyl methyltransferase (PIMT) to specifically detect the presence of isoaspartate residues in the target protein. PIMT catalyzes the transfer of the methyl group at the α-carboxyl position from S-adenosyl-L-methionine to isoaspartate, producing S-adenosyl-L-homocysteine ​​(SAH) in the process. This is a relatively small molecule and can usually be separated and quantified by reversed-phase HPLC using the SAH HPLC standards provided in the kit.

[0315] The potency or bioidentity of an antibody can be measured by its ability to bind to an antigen. Specific binding of an antibody to its antigen can be quantified by any method known to those skilled in the art, for example, an immunoassay such as ELISA (enzyme-linked immunosorbent assay).

[0316] All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing methodologies and materials that might be used in connection with the present invention.

[0317] While different embodiments of the present invention have been described herein with reference to the accompanying drawings, it should be understood that the invention is not limited to those precise embodiments and that various changes and modifications may be made therein by those skilled in the art without departing from the scope or spirit of the invention as defined by the appended claims.

[0318] EXAMPLES Example 1

[0319] Stability of anti-CTLA4 antibody formulations with or without methionine

[0320] This study was conducted to investigate the effect of 10 mM L-methionine on the stability of anti-CTLA4 antibody formulations. The effects of the following stresses on anti-CTLA4 formulations with and without L-methionine were evaluated:

[0321] (1) Heat stress for up to 3 months at 5±3°C (ambient humidity), 25°C (60% relative humidity), and 40°C (75% relative humidity).

[0322] (2) Stirring stress in horizontal position (300 rpm, 3 days)

[0323] (3) Freeze-thaw stress (five freeze-thaw cycles from -80°C to 18-22°C (4 h thawing at room temperature)).

[0324] (4) Light stress (ICH conditions at 0.2x ICH, 0.5x ICH, 1x ICH).

[0325] Based on the data, formulations containing L-methionine are more stable than corresponding formulations without L-methionine.

[0326] Materials and methods

[0327] The following liquid formulations were prepared using an anti-CTLA4 antibody having the following CDRs: CDRH1 of SEQ ID NO: 35, CDRH2 of SEQ ID NO: 36, CDRH3 of SEQ ID NO: 37, CDRL1 of SEQ ID NO: 38, CDRL2 of SEQ ID NO: 39, and CDRL3 of SEQ ID NO: 40 on an IgG1 backbone. The variable heavy chain and variable light chain sequences of the anti-CTLA4 antibody are listed in SEQ ID NOs: 88 and 48, respectively. Each formulation was filled at 1 mL into a 2-mL Type 1 glass vial. A total of 36 vials for formulation A1 and 19 vials for formulation A2 were filled. The target pH for each formulation was 5.5.

[0328] Table 10: Anti-CTLA4 Antibody Preparations

[0329]

[0330]

[0331] The vials were then incubated under three different storage conditions: 5°C (ambient humidity), 25°C (60% relative humidity), and 40°C (75% relative humidity). Data were collected as follows:

[0332] Photostability studies were conducted using 1 mL of A1 and A2 liquid formulations in glass vials at 0.2X ICH; 0.5X ICH; and IX ICH at room temperature.

[0333] Protein concentration was measured by using UV absorption at 280 nm.

[0334] The samples were equilibrated to room temperature and their turbidity was studied spectrophotometrically at absorbance at 350 nm (A350).

[0335] The purity of the samples was assessed by size exclusion chromatography (SEC), where the percentage of monomers was determined, as well as the percentage of high molecular weight species (HMW) and late eluting peaks (LMW species). Ultra-high performance-size exclusion chromatography (UP-SEC) was performed by diluting the samples to 5.0 mg / mL in a mobile phase (50 mM sodium phosphate, 450 mM arginine monohydrochloride, pH 7.0). The diluted samples were injected (6 μL) into a UPLC equipped with a Waters BEH200 column and a UV detector. The proteins in the samples were separated by size and detected by UV absorption at 280 nm.

[0336] Ion exchange chromatography was performed to evaluate chemical stability and monitor the charge change curve over time. The ion exchange HPLC method was performed using a Dionex ProPac WCX-10 column and a UV detector at 280nm. The samples were diluted in purified water and 80μg was injected for analysis. The mobile phase for IEX analysis of thermal stability samples was a gradient of the following mobile phases (mobile phase A: 20mM MOPS, pH 7.2; mobile phase B: 50mM sodium phosphate, 60mM sodium chloride pH 8.0). The determination was performed using a mobile phase gradient from 20mM MOPS (pH 7.2) to 50mM sodium phosphate, 60mM NaCl, pH 8.0. UV detection was performed at 280nm. These methods are considered equivalent and the results are presented as relative percentages based on the total area of ​​the chromatogram.

[0337] Peptide mapping was performed by Lys-C digestion. Samples were injected into Q Exactive at 30ul / sample. Data analysis was performed by PinPoint software.

[0338] The results of the study are listed in the table below:

[0339] Table 11

[0340]

[0341] Table 12

[0342]

[0343]

[0344] Table 13:

[0345]

[0346]

[0347] Table 14

[0348]

[0349]

[0350] Table 15

[0351]

[0352]

[0353] Table 16

[0354]

[0355] Table 17

[0356]

[0357] Table 18

[0358]

[0359] result

[0360] For the conditions and durations studied, there was no measurable variation in protein concentration between formulations as measured by UV absorbance at 280 nm.

[0361] For the conditions tested and the duration of the study, there were no measurable changes in pH between formulations.

[0362] Turbidity (A350) data such as Figure 1A and Figure 1B At 40°C, both formulations showed a trend of increasing turbidity at the 8-week time point. For both formulations, at 25°C and 5°C, there was no substantial change in turbidity at the 8-week time point. Figure 2 As shown in , compared to Formulation A1, under all light stress conditions, Formulation A2 showed slightly more (but consistent) turbidity increase. Compared to the control (T0) sample, freeze-thaw (up to 5X) or stirring stress (300rpm, up to 3 days) treatment of Formulation A1 samples did not change the sample turbidity. Formulation A2 was not subjected to freeze-thaw or stirring stress. Therefore, based on the turbidity data, Formulation A1 containing L-methionine is slightly preferred.

[0363] like Figure 3A , 3B , 4A and 4B, UP-SEC analysis of samples to determine HMW percentage and monomer percentage showed that at 40°C, both formulations showed a trend of increasing %HMW peak and %LMW peak (and a corresponding decrease in %monomer peak) at time points up to 12 weeks. At 25°C, both formulations showed similar trends, but with smaller changes compared to 40°C. At 5°C, no substantial changes were observed. Figure 5 As shown, Formulation A2 showed a slightly greater but consistent increase in %HMW (and correspondingly a slightly greater but consistent decrease in %Monomer) compared to Formulation A1 under all light stress conditions studied. Subjecting Formulation A1 samples to freeze-thaw (up to 5X) or agitation stress (300 rpm for up to 3 days) did not alter %HMW or %Monomer compared to the control (T0) samples (see Figure 5 and Figure 6 ). Formulation A2 was not subjected to freeze-thaw or stirring stress. Therefore, based on the UP-SEC data, Formulation A1 containing L-methionine is slightly preferred.

[0364] like Fig. 7A , 7B , 8A, 8B, 9A, and 9B, the HP-IEX data indicate that at 40°C, both formulations showed trends of increasing % Acidic Peak and % Basic Peak, and corresponding trends of decreasing % Main Peak, at time points up to 12 weeks. At 25°C, both formulations showed similar trends, but with smaller changes compared to 40°C. At 5°C, no substantial changes were observed for Formulation 1 or Formulation 2, except for the 8-week time point for Formulation A2, where a small increase in % Basic Peak and a corresponding small decrease in % Main Peak were observed. This trend could not be confirmed at the 12-week / 5°C time point because the Formulation A2 sample was not tested. Figure 10-12 As shown, under all light stress conditions, Formulation A2 showed a slightly greater but consistent increase in % Acidic Peak and % Basic Peak (and a correspondingly slightly greater but consistent decrease in % Main Peak), as well as a corresponding decrease in % Main Peak (compared to Formulation A1). Figure 10-12 As shown, subjecting Formulation A1 samples to freeze-thaw (up to 5X) or agitation stress (300 rpm, up to 3 days) did not change the % acidic peak, % basic peak, or % main peak compared to the control (T0) samples (Formulation 2 was not subjected to freeze-thaw or agitation stress). Therefore, based on the HP-IEX data, Formulation A1 containing L-methionine is slightly preferred.

[0365] Monoclonal antibodies often have methionine residues in the CDR region and Fc region, which may be susceptible to oxidation under light stress. For anti-CTLA4 antibodies, LC-M4, HC-M34, HC-M250, and HC-M426 may be susceptible to oxidation under light stress. Peptide mapping studies were performed to determine changes in oxidation levels of these residues after exposure to 40°C or .2X / 0.5X / 1X ICH light stress treatment for 8 weeks.

[0366] The results of the peptide mapping study show the oxidation percentages of residues LC-M4, HC-M34, HC-M250, and HC-M426, respectively. Fig.13 , Fig.14 , Fig.15and Fig.16 middle.

[0367] like Figure 13-16 As shown, oxidation of certain methionine residues increased under light stress conditions. Notably, residues HC-M250 and HC-M426 in both formulations showed significant increases in oxidation levels after 0.5X and 1X ICH light stress treatment. However, the presence of 10 mM L-methionine in formulation A1 resulted in smaller increases in oxidation levels for M250 and M426 compared to formulation A2, which did not contain L-methionine. Therefore, based on the peptide mapping data, formulation 1 (containing L-Met) appears to be slightly preferred.

[0368] Based on comparison of analytical data from the above studies, compared to Formulation A1, Formulation A1 exhibited (i) increased turbidity under all light stress conditions, (ii) a lower increase in aggregate levels (% HMW) under all light stress conditions, (iii) a slightly smaller but consistent decrease in % Main Peak under all light stress conditions, and (iv) a lower increase in oxidation levels of residues HC M250 and HC M426 after light stress.

[0369] Example 2

[0370] Anti-CTLA4 Antibody Preparation Buffer Screening

[0371] This study compared the stability of an anti-CLTA4 antibody comprising a heavy chain variable region comprising SEQ ID NO: 88 and a light chain variable region comprising SEQ ID NO: 48 in two different viable formulation buffers (L-histidine and acetate) in the presence of sucrose, polysorbate 80, and L-methionine. Protein-protein interactions (indicative of colloidal and thermal stability) were measured for both formulations (in L-histidine and acetate buffers, as shown below). The positive diffusion interaction parameter (K D ) value (K D >0), indicating a repulsive protein-protein interaction indicating a stable formulation with a low tendency to aggregate. The K values ​​of the two formulations at three different pH values ​​(pH 5, 5.5, and 6) were D At least three measurements (N=3) were performed to obtain standard deviations. Based on protein-protein interactions (data not shown or Figure Y), anti-CTLA4 antibodies are stable in both L-histidine and acetate buffers in the pH range of 5.0-6.0. Therefore, these two formulations were placed for additional thermal stability at 5°C, 25°C, and 40°C, respectively, at pH 5.5.

[0372] To evaluate the stability of the formulations, the effects of the following pressures on two anti-CTLA4 formulations (L-histidine buffer and acetate buffer) were evaluated:

[0373] (1) Heat stress for up to 3 months at 5±3°C (ambient humidity), 25°C (60% relative humidity), and 40°C (75% relative humidity).

[0374] (2) Stirring stress in horizontal position (300 rpm, 3 days)

[0375] (3) Freeze-thaw stress (five freeze-thaw cycles from -80°C to 18-22°C (4 h thawing at room temperature)).

[0376] (4) Light stress (ICH conditions at 0.2x ICH, 0.5x ICH, 1x ICH).

[0377] Based on the data, anti-CTLA4 antibodies are stable in both L-histidine and acetate buffers.

[0378] Materials and methods

[0379] The following liquid formulations were prepared using an anti-CTLA4 antibody having the following CDRs: HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, HCDR3 of SEQ ID NO: 37, LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 40 on the IgG1 backbone. Each formulation was filled at 1 mL into a 2 mL Type 1 glass vial. A total of 72 batches were produced for each of the two formulations. The following table lists the composition of the two formulations. 7% (w / v) sucrose was added as a stabilizer; PS-80 is a surfactant with stability against stress caused by stirring; and L-methionine is an antioxidant because it reduces methionine oxidation under light stress conditions (see Example 1 above).

[0380] Table 19: Formulation

[0381]

[0382]

[0383] The vials were then incubated under three different storage conditions: 5°C (ambient humidity), 25°C (60% relative humidity), and 40°C (75% relative humidity). Data were collected as follows:

[0384] Table 20: Schedule of Formulation B1

[0385]

[0386] Table 21: Schedule of Formulation B2

[0387]

[0388] Photostability studies were conducted using 1 mL of B1 and B2 liquid formulations in glass vials at 0.2X ICH; 0.5X ICH; and IX ICH at room temperature.

[0389] Protein concentration was measured by using UV absorption at 280 nm.

[0390] The samples were equilibrated to room temperature and their turbidity was studied spectrophotometrically at absorbance at 350 nm (A350).

[0391] The purity of the samples was assessed by size exclusion chromatography (SEC), where the percentage of monomers was determined, as well as the percentage of high molecular weight species (HMW) and late eluting peaks (LMW species). Ultra-high performance-size exclusion chromatography (UP-SEC) was performed by diluting the samples to 5.0 mg / mL in a mobile phase (50 mM sodium phosphate, 450 mM arginine monohydrochloride, pH 7.0). The diluted samples were injected (6 μL) into a UPLC equipped with a Waters BEH200 column and a UV detector. The proteins in the samples were separated by size and detected by UV absorption at 280 nm.

[0392] Ion exchange chromatography was performed to evaluate chemical stability and monitor the charge change curve over time. The ion exchange HPLC method was performed using a Dionex ProPac WCX-10 column and a UV detector at 280nm. The samples were diluted in purified water and 80μg was injected for analysis. The mobile phase for IEX analysis of thermal stability samples was a gradient of the following mobile phases (mobile phase A: 20mM MOPS, pH 7.2; mobile phase B: 50mM sodium phosphate, 60mM sodium chloride pH 8.0). The determination was performed using a mobile phase gradient from 20mMMOPS, pH 7.2 to 50mM sodium phosphate, 60mM NaCl, pH 8.0. UV detection was performed at 280nm. These methods are considered equivalent and the results are presented as relative percentages based on the total area of ​​the chromatogram.

[0393] Peptide mapping was performed by Lys-C digestion. Samples were injected into Q Exactive at 30ul / sample. Data analysis was performed by PinPoint software.

[0394] The results of the study are listed in the table below:

[0395] Table 22

[0396]

[0397]

[0398] Table 23

[0399]

[0400]

[0401] Table 24

[0402]

[0403]

[0404] Table 25

[0405]

[0406]

[0407] Table 26

[0408]

[0409]

[0410] For the conditions and durations studied, there was no measurable variation in protein concentration between formulations as measured by UV absorbance at 280 nm.

[0411] For the conditions tested and the duration of the study, there were no measurable changes in pH between formulations.

[0412] Turbidity (A350) data is shown in Fig.17A , Fig. 17B and Fig.18 After comparing the data, it was found that at 40°C, both formulations showed a trend of increasing turbidity at the 8-week time point. At 25°C and 5°C, both formulations showed no substantial change in turbidity at the 8-week time point. Fig.14 As shown, it was also observed that Formulation B1 showed slightly more but consistent turbidity increase after light stress conditions (0.5X ICH and 1X ICH) compared to Formulation B2. Subjecting the samples to freeze-thaw (up to 5X) or agitation stress (300 rpm, up to 3 days) did not change the sample turbidity compared to the control (T0) sample.

[0413] like Fig.19A , 19B, 20A and 20B, UP-SEC analysis of samples to determine HMW percentage and monomer percentage showed that at 40°C, both formulations showed a trend of increasing %HMW peak and %LMW peak (and a corresponding decrease in %monomer peak) at time points up to 8 weeks. At 25°C, both formulations showed similar trends, but with smaller changes compared to 40°C. At 5°C, no substantial changes were observed. Fig.21 and 22 As shown, Formulation B2 showed a slightly greater (but consistent) increase in %HMW (and correspondingly a slightly greater but consistent decrease in %Monomer) under all light stress conditions studied compared to Formulation B1. Subjecting the samples to freeze-thaw (up to 5X) or agitation stress (300 rpm for up to 3 days) did not alter %HMW or %Monomer compared to the control (T0) samples. Fig.21 and 22 ).

[0414] like Fig.23A , 23B As shown in Figures 24A, 24B, 25A and 25B, the HP-IEX data indicate that at 40°C, both formulations showed a trend of increasing % acidic peak and % basic peak, and a corresponding trend of decreasing % main peak at time points up to 8 weeks. However, formulation B2 showed a slightly greater but consistent decrease in % main peak compared to formulation B1. At 25°C, both formulations showed similar trends, but with less change compared to 40°C. Fig.26 , 27 As shown in Figures 28 and 29, after light stress conditions (0.5X ICH and 1X ICH), Formulation B1 showed a slightly greater but consistent increase in % Acidic Peak, while Formulation B2 showed a slightly greater (but consistent) increase in % Basic Peak. However, the corresponding decreases in % Main Peak were comparable for both formulations. Subjecting the samples to freeze-thaw (up to 5X) or agitation stress (300 rpm for up to 3 days) did not change % Acidic Peak, % Basic Peak, or % Main Peak (Figure 2A). Figure 26-28 ).

[0415] Peptide mapping studies were performed to determine changes in oxidation levels of these residues after exposure to 40°C or .2X / 0.5X / 1X ICH light stress treatments for 8 weeks.

[0416] The results of the peptide mapping study show the oxidation percentages of residues LC-M4, HC-M34, HC-M250, and HC-M426, respectively, presented in Fig.29 , Fig.30 , Fig.31 and Fig.32 middle.

[0417] like Figure 29-32As shown, oxidation of certain methionine residues increased under light stress conditions. Notably, residues HC-M250 and HC-M426 in both formulations showed significant increases in oxidation levels after 0.5X and 1X ICH light stress treatments. However, formulation B1 resulted in smaller increases in oxidation levels of M250 and M426 compared to formulation B2.

[0418] Based on comparison of analytical data from the above studies, Formulation B1 (L-histidine buffer) exhibited (i) lower increases in aggregate levels (% HMW) for all light stress conditions, (ii) slightly smaller but consistent decreases in % main peak at 40°C, and (iii) lower increases in oxidation levels of residues HC M250 and HC M426 after light stress compared to Formulation B2 (acetate buffer). Thus, based on protein-protein interaction data and stability data, the anti-CTLA4 antibody was shown to be stable in both L-histidine buffer and acetate buffer.

[0419] Example 3

[0420] Co-formulation of anti-CTLA4 antibody and anti-PD-1 antibody.

[0421] Co-formulating the two antibodies into a single formulation is more convenient for patients and improves patient compliance. Based on protein-protein interactions (as shown below), all co-formulations (as shown below) were found to be stable in the pH 5.0-6.0 range. Therefore, three co-formulations (P1C1, P1C2, and P2C1) with a pH of 5.5 were selected and placed in additional thermal stability at 5°C, 25°C, and 40°C together with two controls (anti-PD1 and anti-CTLA4).

[0422] This study evaluated the stability of an anti-CLTA4 antibody with the following CDRs on an IgG1 backbone co-formulated with pembrolizumab at various concentrations as shown below: HCDR1 of SEQ ID NO:35, HCDR2 of SEQ ID NO:36, HCDR3 of SEQ ID NO:37, LCDR1 of SEQ ID NO:38, LCDR2 of SEQ ID NO:39, and LCDR3 of SEQ ID NO:40:

[0423] Table 27

[0424]

[0425]

[0426] The formulation was prepared as a liquid formulation as follows:

[0427] Table 28

[0428]

[0429]

[0430] Each formulation was filled into 2R vials at 1 mL. Stability was measured by visual inspection, turbidity of PD350, protein concentration, microflow imaging (MFI) (particle evaluation), mixed mode size exclusion chromatography (SEC) (aggregation evaluation), cIEF (charge variant evaluation), IEX (charge variant evaluation) and UP-SEC (aggregation evaluation). Since anti-CTLA4 and anti-PD1 were co-eluted in UP-SEC, anti-PD1 and anti-CTLA4 were separated by mixed mode SEC to evaluate the stability of the co-formulation for the co-formulation. The co-formulation at pH 5.5 was used for thermal stability studies. The thermal stability protocol is as follows:

[0431] Table 29

[0432]

[0433]

[0434] Protein-protein interactions indicating colloidal and thermal stability of different co-formulations (3 co-formulations and 2 controls) were measured. For example, the positive diffusion interaction parameter (K D ) value K D >0, indicating a repulsive protein-protein interaction indicating a stable formulation with a low tendency to aggregate. The Kd of all formulations at different pH values ​​(pH 5.0, 5.5 and 6.0) was measured at least three times to obtain the standard deviation. Fig.33 As shown, the combinations P1C1, P2C2, and P1C2 are stable at each of the three pH values ​​because the K D The values ​​are all positive. Compared with the CTLA4-rich combination (P1C2), pembrolizumab (PD1) and the pembrolizumab-rich combination (P2C1) are more stable at pH 5.0. The CTLA4-rich combination (P1C2) is equally stable at pH 5.0 and pH 5.5. That is, compared with the co-formulation with a greater proportion of CTLA4, the co-formulation with a greater proportion of pembrolizumab is more stable at pH 5.0, which is equally stable at pH 5.0 and 5.5.

[0435] 12-month stability results

[0436] At twelve months, not much change in total protein concentration (determined by UV 280) was observed under any condition (data not shown).

[0437] Turbidity Change: Turbidity changes were observed for each formulation for up to 12 months in the following order: 5°C < 25°C < 40°C. The rate of turbidity change for up to 6 months of data at 40°C appeared to be proportional to the total protein concentration in each formulation (data not shown).

[0438] Under all conditions, the number of particles in all formulations (measured by MFI) appeared quite low (data not shown). Microflow (MFI) imaging was used to characterize the co-formulated samples. One milliliter of sample was drawn into a pipette tip and gently pumped (0.17 mL / min) through the flow cell of the microscope system (depth of field was 150 microns) to perform particle counting and image capture by a digital camera. As the sample passed through the flow cell, bright field images were captured continuously in real time. The output at the end of the analysis was particle count and particle concentration data. MFI images can also be processed for different morphological parameters (such as size, intensity, transparency and shape) using system software.

[0439] For each anti-PD-1 and anti-CTLA4 antibody, an increase in the % acidic charge variants indicating deamidation was observed for each antibody at higher temperatures (25°C and 40°C). The ion exchange HPLC method was performed using a Dionex ProPac WCX-10 column and a UV detector at 280nm. The samples were diluted in purified water and 80μg was injected for analysis. The mobile phase for IEX analysis of thermal stability samples was a gradient of the following mobile phases (mobile phase A: 24mM MES pH 6, 4% acetonitrile; mobile phase B: 20mMNaPO4, 95mM NaCl pH 8, 4% acetonitrile). A summary of the standardized cIEF data (initial, 3 months and 6 months) is listed below:

[0440] Table 30

[0441]

[0442]

[0443] Aggregation was measured using UPSEC and mixed-mode SEC. Although anti-CTLA4 and anti-PD1 antibodies co-eluted in UP-SEC, they were separated and visualized by mixed-mode SEC. HMW aggregates and LMW aggregates increased with temperature. UP-SEC results showed that very low percentages of aggregate (%HMW) species were detected at 5°C and 25°C for up to 12 months, with no major changes compared to the initial time point.

[0444] Table 31

[0445]

[0446]

[0447] Mixed mode SEC was used to analyze aggregates and oxidative species of both antibodies in the co-formulation. The results are listed in the table below. Pembrolizumab showed an increase in oxidative species 1 and 2 at high temperature, reflecting oxidation of M105 on one and both arms, respectively. M105 is in CDR3 of pembrolizumab. Exposed methionine residues or methionine residues in the CDR of an antibody may affect the biological activity of the antibody through oxidation. Methionine reduces oxidation of Met105 within the CDR of the heavy chain of pembrolizumab. The minor changes in oxidative species compared to the initial ones indicate that the co-formulation is stable for up to 12 months at 5°C.

[0448]

[0449]

[0450] Based on twelve months of data, the antibodies performed well in solution when co-formulated, similar to single antibody formulations. The co-formulations were stable at pH 5.0-6.0 as shown by repulsive protein-protein interactions measured by the protein diffusion interaction parameter kD, an indicator of colloidal and thermal stability.

[0451] Example 4

[0452] Other co-formulations

[0453] This study evaluated the stability of an anti-CLTA4 antibody having the following CDRs on an IgG1 backbone: HCDR1 of SEQ ID NO:35, HCDR2 of SEQ ID NO:36, HCDR3 of SEQ ID NO:37, LCDR1 of SEQ ID NO:38, LCDR2 of SEQ ID NO:39, and LCDR3 of SEQ ID NO:40, co-formulated with pembrolizumab at various concentrations as shown below:

[0454] Table 32

[0455]

[0456] The formulation was prepared as a liquid formulation as follows:

[0457] Table 33

[0458]

[0459]

[0460] The formulations were placed under three different storage conditions: 5°C (ambient humidity), 25°C (60% relative humidity), and 40°C (75% relative humidity). The 75 / 200 formulation was also exposed to light stress (0 ICH, 0.5x ICH, or 1x ICH).

[0461] result

[0462] The results showed that increasing methionine concentration reduced subvisible particulates under all conditions based on MFI measurements (data not shown). Increasing methionine concentration at 40°C reduced the % HMW species as observed by UP-SEC. Increasing methionine concentration at 40°C slightly reduced turbidity.

[0463]

[0464]

[0465] Embodiment 5:

[0466] Long-term stability of CTLA4 monotherapy

[0467] The following liquid formulation was prepared using an anti-CTLA4 antibody with the following CDRs: 100 mg / mL anti-CTLA4 antibody, 10 mM L-histidine buffer, 7% w / v sucrose, 0.02% w / v polysorbate 80 at pH 5.5. The product was dispensed into 2R I-type glass with an elastomeric stopper and aluminum seal. The fill volume was 2.0 mL and the overfill was 0.2 mL. The samples were placed in stability under the following conditions:

[0468] (i) 5℃ / ambient humidity

[0469] (ii) 25°C / 60% relative humidity

[0470] (iii) 40℃ / 75% relative humidity

[0471] Samples were tested initially and at months 1, 3, 6, 9 and 12. The results are listed in the table below:

[0472] Table 34

[0473]

[0474]

[0475] Table 35

[0476]

[0477]

[0478] Table 36

[0479]

[0480]

[0481] result:

[0482] As shown in the above data, no significant changes were observed when the formulations were stored at 5°C. Such formulations are expected to be stable for 24 months at 5°C. The biological potency of the formulations determined by binding ELISA showed that no significant changes in potency were observed under all conditions. The pH did not change with storage time or conditions.

[0483] In all three conditions of stability testing up to 12 months, samples showed no significant changes in protein concentration under all storage conditions.

[0484] Charge variants (% acidic variants, % main component, and % basic variants) were determined by HP-IEX. For 5°C conditions, no significant changes were observed within 12 months. For 25°C conditions, % acidic variants increased, % total main peak decreased, and % basic variants remained unchanged within 12 months. These results are not unexpected considering the storage conditions. For 40°C conditions, % total main peak showed a significant decrease from the initial time point to the 6-month time point, % acidic variants increased significantly, and % basic variants remained unchanged. These results are not unexpected considering the storage conditions.

[0485] Purity was determined by UP-SEC (% HMW species, % monomer, and % LMW species). There was no change in % monomer or % HMW species at stability up to 12 months at 5°C. No peaks for LMW species were detected at 5°C over 12 months. For storage conditions at 25°C, there was no change in HMW species, a slight decrease in % monomer, and a slight increase in LMW species over 12 months. At 40°C for up to 6 months, there was a slight increase in % HMW species, while at the 6 month time point, the % monomer decreased by less than 85%. These results are not unexpected given the storage conditions.

[0486] The pH did not change with storage time or conditions.

[0487] Example 6

[0488] Addition of chelating agents as optional excipients

[0489] The stability of the formulations in the presence or absence of a chelating agent (DTPA) was evaluated. In order to evaluate the stability of the formulations, the two formulations were filled into vials and graded for stability at 5°C (ambient humidity), 25°C (60% relative humidity) and 40°C (75% relative humidity) - protected from light for twelve weeks. The two formulations are as follows:

[0490]

[0491]

[0492] The colloidal stability of the samples was assessed by size exclusion chromatography (SEC) for determination of purity of percent monomer, as well as the percentage of high molecular weight species (HMW) and late eluting peaks (LMW species). The table below lists the UPSEC data for assessing the levels of high molecular weight species (HMW or aggregates), % monomer and LMW (low molecular weight species).

[0493] Table 37

[0494]

[0495]

[0496] As shown in the table above, UP-SEC analysis of samples to determine the HMW percentage and monomer percentage showed that at 5°C, 25°C and 40°C, both formulations showed a trend of increasing %HMW peak and %LMW peak (and a concomitant decrease in %monomer peak) at time points up to 12 weeks. At 25°C, both formulations showed similar trends compared to 40°C, but with smaller changes. At 5°C, no substantial changes were observed. Based on the data, Formulation 1 (without DTPA) showed a slight increase in %HMW and %LMW compared to Formulation 2 (containing DTPA). In addition, Formulation 1 showed a greater decrease in %monomer compared to Formulation 2.

[0497] HP-IEX analysis of samples to determine chemical stability showed that at 5°C, 25°C, and 40°C, both formulations showed a trend of increasing % acidic peak and a concomitant decrease in % monomeric peak at time points up to 12 weeks. At 25°C, both formulations showed similar trends, but with less change compared to 40°C. At 5°C, no substantial changes were observed (data not shown).

[0498] The results in the table below show the trend of PS80 concentration decreasing over time at time points up to 8 weeks. At 25°C, both formulations showed similar trends, but with less change compared to 40°C. At 5°C, no substantial changes were observed. At 40°C, less PS80 degradation was observed in formulation 2 (anti-CTLA4 antibody with DTPA) compared to formulation 1 (anti-CTLA4 antibody without DTPA).

[0499]

[0500]

[0501] Thus, DTPA may also provide even greater stability to the formulations described herein.

Claims

1. A preparation comprising: (i) 1 mg / ml to 100 mg / ml of an anti-CTLA4 antibody or an antigen-binding fragment thereof; wherein the anti-CTLA4 antibody or the antigen-binding fragment thereof comprises three light chain CDRs and three heavy chain CDRs, the three light chain CDRs are CDRL1 of SEQ ID NO:38, CDRL2 of SEQ ID NO:39, and CDRL3 of SEQ ID NO:40, and the three heavy chain CDRs are CDRH1 of SEQ ID NO:35, CDRH2 of SEQ ID NO:36, and CDRH3 of SEQ ID NO:37; (ii) 1 mg / ml to 100 mg / ml of an anti-PD1 antibody or an antigen-binding fragment thereof; wherein the anti-PD1 antibody or the antigen-binding fragment thereof comprises three light chain CDRs and three heavy chain CDRs, the three light chain CDRs are CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the three heavy chain CDRs are CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8; (iii) 5 mM to 20 mM L-histidine buffer; (iv) 6% to 8% weight / volume (w / v) sucrose; (v) 0.01% to 0.10% w / v polysorbate 80; and (vi) 1 mM to 20 mM L-methionine, The pH of the formulation is between 5.0 and 6.

0.

2. The formulation of claim 1, wherein the anti-CTLA4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 88 and a light chain variable region comprising SEQ ID NO:

48.

3. The preparation of claim 1, comprising 8 mM to 12 mM L-histidine buffer. The preparation of claim 1 , comprising 5 mM to 10 mM L-methionine.

5. The formulation of claim 1, comprising polysorbate 80 in an amount of 0.02% w / v.

6. The formulation of any one of claims 1-5, wherein the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 1-50 mg / ml.

7. The formulation of any one of claims 1-5, comprising 25 mg / mL of anti-PD1 antibody and 50 mg / mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, 7% w / v sucrose, 0.02% polysorbate 80 and 10 mM L-methionine.

8. The formulation of any one of claims 1-5, comprising 23.5 mg / mL of anti-PD1 antibody and 2.9 mg / mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, 7% w / v sucrose, 0.02% polysorbate 80 and 10 mM L-methionine.

9. The formulation of any one of claims 1-5, wherein the pH of the formulation is 5.3 to 5.

8.

10. The formulation of claim 9, wherein the pH of the formulation is 5.5 to 5.

6.

11. The formulation of any one of claims 1-5, wherein after 12 months at 5°C: (i) the anti-CTLA4 antibody has a % monomer of ≥ 95% as determined by size exclusion chromatography; (ii) the % heavy and light chains of the anti-CTLA4 antibody are ≥ 90% as determined by reduced CE-SDS; (iii) the % heavy and light chains of the anti-CTLA4 antibody are ≥ 95% as determined by reduced CE-SDS; (iv) the % intact IgG of the anti-CTLA4 antibody is ≥ 90% as determined by non-reduced CE-SDS; and / or (v) the % intact IgG of the anti-CTLA4 antibody is ≥ 95% as determined by non-reduced CE-SDS.

12. The formulation of any one of claims 1-5, wherein the anti-PD1 antibody or antigen-binding fragment thereof comprises V L Area and V H Area, the V L The region comprises the amino acid sequence shown in SEQ ID NO: 4, and the V H The region comprises the amino acid sequence shown in SEQ ID NO:

9.

13. The formulation of any one of claims 1-5, wherein the anti-PD1 antibody is pembrolizumab.

14. The formulation of any one of claims 1-5, wherein the ratio of the anti-PD1 antibody to the anti-CTLA4 antibody is 1:2, 1:1, 2:1, 10:1, 1:10, 8:3 or 8:

1.

15. The formulation of claim 14, wherein the ratio of the anti-PD1 antibody to the anti-CTLA4 antibody is 8:

1.

16. The formulation of any one of claims 1-5, wherein the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 1.25 mg / ml, 2.5 mg / ml, 2.9 mg / ml, 5 mg / ml, 7.9 mg / ml, 10 mg / ml, 12.5 mg / ml, 25 mg / ml, 50 mg / ml, 75 mg / ml or 100 mg / ml.

17. The formulation of any one of claims 1-5, wherein the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 1-25 mg / ml.

18. The formulation of any one of claims 1-5, wherein the concentration of the anti-PD1 antibody or antigen-binding fragment thereof is 25 mg / mL, 22.7 mg / mL, 2.27 mg / mL, 21.1 mg / mL or 23.5 mg / mL.

19. The formulation of any one of claims 1-5, wherein the concentration of the anti-PD1 antibody or antigen-binding fragment thereof is 1-25 mg / mL.

20. The formulation of any one of claims 1-5, comprising 23.5 mg / mL of anti-PD1 antibody and 2.9 mg / mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, 7% w / v sucrose, 0.02% w / v polysorbate 80, and 10 mM L-methionine.

21. The formulation of any one of claims 1-5, further comprising a chelating agent, wherein the chelating agent is diethylenetriaminepentaacetic acid (DTPA).

22. The formulation of any one of claims 1-5, wherein the formulation is contained in a glass vial or an injection device.

23. The formulation of any one of claims 1-5, which is a liquid formulation, or which is frozen to at least below -70°C, or which is a reconstituted solution from a lyophilized formulation.

24. Use of the formulation of any one of claims 1 to 23 in the preparation of a medicament for treating cancer or for treating a chronic infection, wherein the cancer is a solid tumor with a high level of microsatellite instability (MSI-H) or a solid tumor with a high mutation burden.

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