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

Abstract

To provide stable formulations of anti-CTLA4 antibodies for treating various cancer and infectious diseases.SOLUTION: Disclosed is a formulation comprising: (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 buffer; (iii) about 6 to about 8% weight / volume (w / v) of a non-reducing sugar; (iv) about 0.01 to about 0.10 w / v% of a nonionic surfactant; and (v) about 1 mM to about 20 mM anti-oxidant. Preferably the buffer is an L-histidine buffer, the non-reducing sugar is sucrose, the nonionic surfactant is polysorbate 80, and the antioxidant is L-methionine. The formulation may further contain an anti-PD1 antibody or antigen binding fragment thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the formulation of therapeutic antibodies and their use in the treatment of various disorders. In an embodiment, the present invention provides an antibody that binds to cytotoxic T-lymphocyte-associated antigen 4 (CTLA4). In another embodiment, the preparation comprises an antibody or an antigen-binding fragment thereof. The present invention further includes a human programmed death receptor 1 (PD-1) antibody or an antigen-binding fragment thereof. Also provided are methods of treating various cancers and chronic infections with the formulations described herein. can be.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of USSN 62 / 500,268, filed May 2, 2017. The benefit of which is claimed, the contents of which are incorporated herein by reference in their entirety. .

[0003] Reference to Electronically Submitted Sequence Listing The sequence listing of the present application is incorporated herein by reference in its entirety under the file name “24449WOPCT-SEQTXT-30APR 2018.TXT", created on April 30, 2018 and 96Kb in size. The sequence listing in CII format is submitted electronically via EFS-Web. EFS-Web This sequence listing, submitted via the Web site, is a part of the specification and is incorporated herein by reference in its entirety. Be absorbed. [Background technology]

[0004] Antibody drugs for use in humans have amino acid sequences of their constant domains that are Although their framework sequences within the variable domains may differ somewhat, typically Typically, the CDR sequences differ most dramatically. Even antibodies that bind essentially the same epitope may contain completely different CDR sequences. Therapeutic antibodies for use in humans may also be derived from human germline antibody sequences or e.g. They may also be derived from non-human (e.g., rodent) germline antibody sequences, such as in humanized antibodies. This leads to even greater diversity of potential CDR sequences. The differences result in different stabilities in solution and different responses to solution parameters. In addition, small changes in the amino acid sequence or changes in one or more amino acid residues can have dramatic effects. These results suggest that different antibody stabilities and susceptibility to sequence-specific degradation pathways may be conferred on different antibody sequences. As a result, it is currently not possible to predict the solution conditions necessary for optimal antibody stability. Each antibody must be tested individually to determine the optimal solution formulation. mbhani et al. (2012) J.Pharm.Sci. 101:112 0.

[0005] Antibodies also have the potential to be used in therapeutic applications, as compared to other therapeutic proteins, such as hormones and cytokines. It is also a relatively high molecular weight protein (approximately 150,000 Da). In order to achieve a drug molar concentration of 100 mg / kg, a relatively large amount of antibody drug is often administered. In addition, it will be possible to administer the antibody drug subcutaneously, which will allow self-administration. Self-administration may sometimes be desirable, for example in conjunction with a medical visit for intravenous administration. Subcutaneous delivery avoids the time and expense of actually delivering the drug to the injection site in a single injection. Subcutaneous administration is limited by the amount of solution that can be administered, which is generally about 1 to 1.5 ml. Self-administration of the drug is typically freeze-dried to eliminate the need for the patient to resuspend the drug prior to injection. Prefilled syringes or auto-injectors that contain a liquid solution formulation of the drug rather than a syringe This is achieved using vectors. Antibody drugs are typically engineered to ensure efficacy and consistent dosing. Regardless of the formulation chosen, the desired Supporting desirable properties such as high density, clarity and acceptable viscosity, and Additionally, these properties and drug efficacy can be evaluated for acceptable long-term shelf life under typical storage conditions. It is crucial to maintain this over time.

[0006] CTLA4 is similar to CD2 in gene structure, chromosomal location, sequence homology, and gene expression. 8, which are both receptors for the costimulatory molecule B7, After binding to B7, CTLA4 is expressed on activated T cells in mice and humans. It can inhibit cell activation and plays a negative regulatory role in T cell activation .

[0007] CTLA4 mAb or CTLA4 ligand is a method to bind CTLA4 to its natural ligand. This prevents CTLA4 from binding to T cells, thereby negatively regulating T cells. It blocks the transmission of IL-1 and enhances the responsiveness of T cells to various antigens. Results from in vivo and in vitro studies are substantially consistent. CTLA4 mAb is effective in treating prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, and liver cancer. , currently being tested in clinical trials to treat malignant melanoma, etc. (Grosso et al. CTLA-4 blockade in tumor models: an overview of preclinical and translation al research. Cancer Immun. 13:5(2013)).

[0008] CTLA4 and CTLA4 mAbs as important factors influencing T cell function By interfering with the immune microenvironment in the body, it is possible to produce specific therapeutic effects against diseases. These drugs are highly effective and can improve the shortcomings of conventional drug treatments. Opening up new avenues for gene therapy. CTLA4 and CTLA4 mAb have been shown to be effective in experimental and It is currently in various stages of clinical trials. They found that it effectively inhibited airway hyperresponsiveness in animal models of asthma and reduced the development of rheumatic diseases. On the other hand, short-term clinical trials have shown that Even if biological gene therapy does not show any adverse effects in the current study, potential side effects after long-term application may be Attention should be paid to potential effects of CTLA4 mAb on CTLA4-B activation. 7 Excessive blockage of signal transduction can lead to the development of autoimmune diseases. It can specifically bind to the antigen and induce lysis of the target cell or inhibit the progression of the disease. Therefore, the development and use of drugs based on antibodies, especially humanized antibodies, is essential for the treatment of malignant tumors in humans. and other immune disorders.

[0009] PD-1 is recognized as a key player in immune regulation and the maintenance of peripheral tolerance. PD-1 is moderately expressed on naive T cells, B cells, 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 1 and its ligands in regulating autoimmunity and infection . Nature Immunolog (2007) 8:239-245). anti-PD The efficacy of the -1 antibody will be compared with other approved or experimental cancer treatments, such as radiation, surgery, etc. Dysregulated in surgery, chemotherapy, targeted therapy, and tumors When administered in combination with other signaling pathway inhibitors and other immune enhancing agents, It has been proposed that the combination of PD-1 antagonists may be potentiated. One such agent that has been identified is cytotoxic T-lymphocyte-associated antigen 4 (abbreviated as CTLA4).

[0010] Anti-CTLA for medical use, e.g., to treat various cancers and infectious diseases. 4. Stable formulation of antibodies and co-formulation with anti-human PD-1 antibodies There is a need for stable formulations of anti-CTLA4 antibodies that are The formulations exhibit an extended shelf life, are stable during storage and shipping, and preferably are self-administered. The drug was stored for several months under conditions typical for storage of the drug, i.e., in a syringe at refrigerator temperature. They exhibit stability for months to years, which translates into a long shelf life for the corresponding pharmaceutical products. Glass. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Bhambhani et al. (2012) J.Pharm.Sci. 101:1120 [Non-Patent Document 2] Grosso et al.,CTLA-4 blockade in tumor models: an overview of preclinical and translational research. Cancer Immun. 13:5(2013) [Non-Patent Document 3] Sharpe et al., The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nature Immunolog (2007) 8:239-245 Summary of the Invention [Means for solving the problem]

[0012] In one embodiment, the present invention provides a method for treating a cancer cell comprising: (i) an anti-CTLA4 antibody or an antigen-binding fragment thereof; (ii) a buffer; (iii) a non-reducing sugar; (iv) a non-ionic detergent; and an acid inhibitor. The present invention also includes formulations of anti-CTLA4 antibodies or antigen-binding fragments thereof that include a agonizing agent. In some embodiments, the formulation further comprises an anti-PD-1 antibody, such as pembrolizumab or nivolumab. In one embodiment, the formulation further comprises a chelator.

[0013] In one embodiment, the formulation comprises: (i) about 10 mg / ml to about 200 mg / ml of an antibody; (ii) about 5 mM to about 20 mM buffer. (iii) about 6% to about 8% weight / volume (w / v) non-reducing sugars; (iv) about 0.01 % to about 0.10% of a non-ionic surfactant; and (v) about 1 mM to about 20 mM of an anti- In another embodiment, the formulation comprises an anti-PD-1 antibody, such as pembrolizumab. In another 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. The pH is between 4.5 and 6.5. In certain embodiments, the pH of the formulation is about pH 5.0. In a further embodiment, the pH of the formulation is from about pH 5.3 to about pH 6.0. In another embodiment, the pH is 5.8. In another embodiment, the pH is 5.3. In one embodiment, the pH is 5.4. In one embodiment, the pH is 5.5. In a further embodiment, the pH is 5.6. In a further embodiment, the pH is 5.7. In an embodiment, the pH is 5.8.

[0014] In one embodiment of the formulation, the buffer is an L-histidine buffer or sodium acetate. The non-reducing sugar is sucrose, and the non-ionic surfactant is poly(ethylene glycol). sorbate 80, and the antioxidant is methionine or a pharma- ceutically acceptable salt thereof. In one embodiment, the antioxidant is L-methionine. In the present invention, the antioxidant is a pharma- ceutically acceptable salt of L-methionine, such as methionine HCl. It is.

[0015] In another embodiment, the formulation comprises: (i) about 10 mg / ml to about 200 mg / ml of an antibody; (ii) about 5 mM to about 20 mM L-His; (iii) about 6% to about 20 mM sodium acetate buffer; (iv) from about 0.01% to about 0.10% w / v of Polysol; and (v) about 1 mM to about 20 mM L-methionine. In some embodiments, 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 buffer is an L-histidine buffer. In one embodiment, the formulation contains 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 a further embodiment, the formulation contains polysorbate 80 in a weight ratio of about 0.02% w / v. In one embodiment, the anti-CTLA4 formulation contains sucrose at about 7% (w / v). Included by weight.

[0016] In an embodiment of the formulation, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is about 1 0 mg / ml to about 100 mg / ml. In another embodiment, the anti-CTLA4 antibody or its antigen-binding fragment, the concentration is about 10 mg / ml, 12.5 mg / ml, 15 mg / ml, 20mg / ml, 25mg / ml, 50mg / ml, 75mg / ml or 10 In one embodiment, the anti-CTLA4 antibody or its antigen-binding The concentration of the fragment is 25 mg / mL. In additional embodiments, the anti-CTLA4 antibody or or an antigen-binding fragment thereof, the concentration of which is about 50 mg / ml. The concentration of the CTLA antibody or antigen-binding fragment thereof is about 75 mg / mL. In one embodiment, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 100 mg / m It's L.

[0017] In one embodiment, about 25 mg / mL of an anti-CTLA4 antibody or antigen-binding fragment thereof. , 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / A formulation containing about 10 mM polysorbate 80 and about 10 mM L-methionine is provided.

[0018] In one embodiment, about 50 mg / mL of an anti-CTLA4 antibody or antigen-binding fragment thereof. , 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / A formulation containing about 10 mM polysorbate 80 and about 10 mM L-methionine is provided.

[0019] In one embodiment, about 75 mg / mL of an anti-CTLA4 antibody or antigen-binding fragment thereof. , 10 mM L-histidine buffer, about 7% w / v sucrose, about 0.02% w / A formulation containing about 10 mM polysorbate 80 and about 10 mM L-methionine is provided.

[0020] In one embodiment, about 100 mg / mL of an anti-CTLA4 antibody or antigen-binding fragment thereof. Pieces, 10 mM L-histidine buffer, approximately 7% w / v sucrose, approximately 0.02% w A formulation containing about 10 mM L-methionine and about 10 mM polysorbate 80 is provided.

[0021] In one embodiment of any of the above formulations, the formulation has a pH of about 5.3 to 5.8. In another embodiment, the formulation has a pH of about 5.5 to about 5.6. The formulation has a pH of about 5.5. In another embodiment, the formulation has a pH of about 5.6.

[0022] In one embodiment of any of the above formulations, the formulation comprises an anti-PD1 antibody or an antigen-binding fragment thereof. In one embodiment, the anti-PD1 antibody is pembrolizumab. In this case, the anti-PD1 antibody is nivolumab.

[0023] In another embodiment, the formulation may further comprise a chelator. In one embodiment, the chelator is DTPA. In one embodiment, the chelator is EDTA. In one embodiment, 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 the chelator. In one embodiment, the formulation contains about 15 μM of the chelator. In one embodiment, the formulation comprises about 20 μM of the chelator. In one embodiment, the formulation comprises about 25 μM of the chelator. In one embodiment, the formulation contains about 35 μM of the chelator. In one embodiment, the formulation comprises about 40 μM of the chelator. In one embodiment, the formulation comprises about 45 μM of the chelator. In one embodiment, the chelating agent is present in any of the amounts described above. In another embodiment, the chelator is DTPA present in any of the above amounts. The most common is EDTA, which is present in

[0024] In one embodiment, the formulation is contained in a glass vial. In another embodiment, the formulation is contained in an injection device. In one embodiment, the formulation is frozen to at least below -70°C. In embodiments, the formulation is a reconstituted solution from a lyophilized formulation.

[0025] In certain embodiments, the formulations are preferably stored at refrigerated temperatures (2-8° C.) for at least 3 months. Preferably, the patient is provided with a stable diet for a period of six months, more preferably one year, and even more preferably up to two years. In one embodiment of the formulation, after 12 months at 5° C., size exclusion chromatography The percent monomer of anti-CTLA4 antibody as determined by FTIR is ≥ 90%. In an embodiment, after 12 months at 5° C., the antibody titer determined by size exclusion chromatography is The percent monomer of the CTLA4 antibody is ≧95%. After 2 months, % heavy and light chain anti-CTLA4 antibodies as determined by reduced CE-SDS were ≥ In another embodiment of the formulation, after 12 months at 5° C., reduced CE-SDS The % heavy and light chains of the anti-CTLA4 antibody as determined by the antibody titer are ≧95%. After 12 months at 5°C, the anti-CTLA4 antibody titers determined by non-reduced CE-SDS were In another embodiment of the formulation, the % intact IgG is ≧90%. After immunization, the % intact IgG of anti-CTLA4 antibodies determined by non-reduced CE-SDS was ≥ 9. It is 5%.

[0026] In one embodiment of any of the above formulations, the formulation comprises three light chain CDRs and three heavy chain CDRs. and an anti-CTLA4 antibody or antigen-binding fragment thereof comprising the light chain CDRs, wherein the 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 heavy chain CDRs comprise CDRH1 of SEQ ID NO: 35, CDRH2 of SEQ ID NO: 36, and and CDHR3 of SEQ ID NO: 37. In another embodiment, the formulation comprises SEQ ID NO: 88 An anti-CTLA4 antibody comprising a heavy chain variable region and a light chain variable region comprising SEQ ID NO:48 or In another embodiment, the formulation comprises a heavy chain comprising SEQ ID NO: 99 and the sequence The present invention includes an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain comprising number 100.

[0027] In one embodiment, the present invention provides a method for treating a cancer cell 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; (ii) a buffer; (iii) (iv) a non-reducing sugar; (v) a non-ionic surfactant; and an antioxidant. or an antigen-binding fragment thereof and an anti-human PD-1 antibody or an antigen-binding fragment thereof In one embodiment, the co-formulation provides In one embodiment, the chelator is EDTA. In one embodiment, the chelator is DTPA. The ratio of anti-human PD-1 antibody to A4 antibody is 1:2. In another embodiment of the co-formulation, In this study, the ratio of anti-human PD-1 antibody to anti-CTLA4 antibody was 1:1. In yet another embodiment, the ratio of anti-human PD-1 antibody to anti-CTLA4 antibody is 2:1 In another embodiment of the co-formulation, an anti-human PD-1 antibody is In a further embodiment of the co-formulation, the ratio of In another embodiment, the ratio of anti-human PD-1 antibody to anti-CTLA4 antibody is 1:10. In a further embodiment, the ratio of anti-human PD-1 antibody to human PD-1 antibody is 8:1. The ratio of anti-human PD-1 antibody to TLA4 antibody is 8:3.

[0028] In one embodiment of the invention, the co-formulation comprises: (i) a concentration of from about 1 mg / ml to about 100 mg / ml (ii) about 1 mg / ml to about 10 (ii) about 5 mM to about 20 mM buffer; (iii) about 6% to about 8% weight / volume (w / v) non-reducing sugars; (iv) about 0.01% to about 0.10% non-ionic surfactant; and (v) about 1 mM to about 20 mM antioxidant. In one embodiment, the co-formulation further comprises a chelator. In one embodiment of the co-formulation, the chelator is DTPA. The ratio of anti-human PD-1 antibody to body is 1:2. In another embodiment of the co-formulation, The ratio of anti-human PD-1 antibody to anti-CTLA4 antibody is 1:1. In an embodiment, the ratio of anti-human PD-1 antibody to anti-CTLA4 antibody is 2:1. In another embodiment of the co-formulation, the ratio of anti-human PD-1 antibody to anti-CTLA4 antibody is In a further embodiment of the co-formulation, the anti-human CTLA4 antibody is In another embodiment, the ratio of anti-CTLA4 antibody to anti-PD-1 antibody is 1:10. In a further embodiment, the ratio of anti-CTLA to anti-human PD-1 antibody is 8:1. The ratio of anti-human PD-1 antibody to anti-human PD-4 antibody is 8:3. The formulation has a pH between 4.5 and 6.5. In another embodiment, the pH of the formulation is about p In a further embodiment, the pH of the formulation is about pH 5. 3 to about pH 5.8.

[0029] In one embodiment of the co-formulation, the buffer is a histidine buffer or a sodium acetate buffer. The sodium buffer is the non-reducing sugar is sucrose, and the non-ionic surfactant is polyisoprene. and the antioxidant is methionine or a pharma- ceutically acceptable salt thereof. In one embodiment, the antioxidant is L-methionine. The antioxidant may be, for example, a pharma- ceutically acceptable form of L-methionine, such as methionine HCl. It's salt.

[0030] In another embodiment, the co-formulation comprises: (i) about 1 mg / ml to about 100 mg / ml of anti-CT LA4 antibody or antigen-binding fragment thereof; (ii) about 1 mg / ml to about 100 mg / ml (iii) about 5 mM to about 20 mM of an anti-human PD-1 antibody or antigen-binding fragment thereof; L-histidine buffer or about 5 mM to about 20 mM sodium acetate buffer; (iv) about 6% to about 8% w / v sucrose; (v) about 0.01% to about 0.10% w / v / v polysorbate 80; and (vi) about 1 mM to about 20 mM L-methionine. In one embodiment, the co-formulation further comprises a chelator. In one embodiment, the chelator is DTPA. In one embodiment, the co-formulation comprises about 8 mM to about 12 mM L -histidine buffer. In another embodiment, the co-formulation comprises about 5 mM to about 10 In a further embodiment, the co-formulation comprises polysorbate 8. In one embodiment, the co-formulation comprises sucrose at a weight ratio of about 0.02% w / v. It contains approximately 7% (w / v) of

[0031] In an embodiment of the co-formulation, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is about In another embodiment, the anti-CTLA4 antibody is In another embodiment, the concentration of the antibody is from about 10 mg / ml to about 100 mg / ml. The concentration of the CTLA4 antibody or antigen-binding fragment thereof is about 10 mg / ml. In the embodiment, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 1.25 mg / m In another embodiment, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is In another embodiment, the anti-CTLA4 antibody or its antigen binding agent is The concentration of the synthetic fragment is 5 mg / ml. In a further embodiment, the concentration of the antigen-binding fragment is 12.5 mg / ml. The concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 25 mg / ml. In some embodiments, the concentration of the anti-CTLA4 antibody or antigen-binding fragment thereof is 50 mg / mL or In another embodiment, the anti-CTLA4 antibody or antigen-binding fragment thereof is 7 In another embodiment, the anti-CTLA4 antibody or antigen-binding fragment thereof is The concentration of the fragment is 100 mg / ml. In additional embodiments, the anti-CTLA4 antibody or or an antigen-binding fragment thereof, the concentration of which is about 50 mg / ml. The concentration of the CTLA4 antibody is 2.9 mg / mL. The antibody concentration is 7.9 mg / mL.

[0032] In embodiments of the co-formulation, the concentration of the anti-human PD-1 antibody is from about 1 mg / mL to about 100 In another embodiment, the concentration of the anti-human PD-1 antibody is about 10 mg / mL. In another embodiment, the concentration of the anti-human PD-1 antibody is from about 1 mg / ml to about 100 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 / g / ml. In another embodiment, the concentration of the anti-human PD-1 antibody is about 23.5 mg / ml. It is.

[0033] In one embodiment, the co-formulation comprises about 25 mg / mL of anti-PD1 antibody, about 12.5 mg / mL of 10 μg / mL anti-CTLA4 antibody, 10 mM L-histidine buffer, approximately 7% w / v cellulose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine Includes.

[0034] In one embodiment, the co-formulation comprises about 25 mg / mL of anti-PD1 antibody, about 25 mg / mL of anti-PD1 antibody, mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, approximately 7% w / v sucrose about 0.02% w / v polysorbate 80 and about 10 mM L-methionine. nothing.

[0035] In one embodiment, the co-formulation comprises about 25 mg / mL of anti-PD1 antibody, about 50 mg / mL of anti-PD1 antibody, mL of anti-CTLA4 antibody, 10 mM L-histidine buffer, approximately 7% w / v sucrose about 0.02% w / v polysorbate 80 and about 10 mM L-methionine. nothing.

[0036] In one embodiment, the co-formulation contains about 22.72 mg / mL of anti-PD1 antibody, about 2. 3mg / mL anti-CTLA4 antibody, 10mM L-histidine buffer, approximately 7% w / v of sucrose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine. Contains nin.

[0037] In one embodiment, the co-formulation contains about 2.27 mg / mL of anti-PD1 antibody, about 22. 7mg / mL anti-CTLA4 antibody, 10mM L-histidine buffer, approximately 7% w / v of sucrose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine. Contains nin.

[0038] In one embodiment, the co-formulation comprises about 23.5 mg / mL of anti-PD1 antibody, about 2.9 mg / mL of anti-PD1 antibody, mg / mL anti-CTLA4 antibody, 10 mM L-histidine buffer, approximately 7% w / v Sucrose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine Includes

[0039] In one embodiment, the co-formulation comprises about 21.1 mg / mL of anti-PD1 antibody, about 7.9 mg / mL anti-CTLA4 antibody, 10 mM L-histidine buffer, approximately 7% w / v Sucrose, about 0.02% w / v polysorbate 80 and about 10 mM L-methionine Includes

[0040] In one embodiment of any of the above formulations, the formulation comprises three light chain CDRs and three heavy chain CDRs. and an anti-CTLA4 antibody or antigen-binding fragment thereof comprising the light chain CDRs, wherein the 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 heavy chain CDRs comprise CDRH1 of SEQ ID NO: 35, CDRH2 of SEQ ID NO: 36, and and CDHR3 of SEQ ID NO: 37. In another embodiment, the formulation comprises SEQ ID NO: 88 An anti-CTLA4 antibody comprising a heavy chain variable region and a light chain variable region comprising SEQ ID NO:48 or In another embodiment, the formulation comprises a heavy chain comprising SEQ ID NO: 99 and the sequence The present invention relates to an anti-CTLA4 antibody or an antigen-binding fragment thereof, the anti-CTLA4 antibody comprising a light chain containing SEQ ID NO: 100. In one embodiment of any of the compositions, the anti-human PD-1 antibody or antigen-binding fragment thereof is and one light chain CDR and three heavy chain CDRs, wherein the light chain CDRs are RL1, CDRL2 of SEQ ID NO:2, CDRL3 of SEQ ID NO:3, and heavy chain CDR CDRH1 of SEQ ID NO:6, CDRH2 of SEQ ID NO:7, and CDHR3 of SEQ ID NO:8 In another embodiment, the formulation comprises a light chain variable region comprising SEQ ID NO:4 and a light chain variable region comprising SEQ ID NO:9. In another embodiment, the present invention includes an anti-human PD1 antibody or an antigen-binding fragment thereof, the anti-human PD1 antibody or the antigen-binding fragment thereof comprising a heavy chain variable region comprising wherein the formulation comprises an anti-human PD1 antibody comprising a light chain comprising SEQ ID NO:5 and a heavy chain comprising SEQ ID NO:10. In one embodiment of any of the above formulations, the formulation comprises an anti-human IgG antibody or an antigen-binding fragment thereof. In another embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof is pembrolizumab. The anti-human PD-1 antibody or antigen-binding fragment thereof is nivolumab.

[0041] In one embodiment of any of the above co-formulations, the formulation comprises: (i) three light chain CDRs and An anti-CTLA4 antibody or antigen-binding fragment thereof comprising three heavy chain CDRs and one light chain CDR. R is a CDRL1 of SEQ ID NO: 38, a CDRL2 of SEQ ID NO: 39, a CDRL3 of SEQ ID NO: 40 3, and the heavy chain CDRs comprise CDRH1 of SEQ ID NO: 35, CDRH2 of SEQ ID NO: 36 2 and CDHR3 of SEQ ID NO: 37, and (ii) an anti-human PD-1 antibody comprising three light chain CDRs and three heavy chain CDRs; or The antigen-binding fragment thereof, wherein the light chain CDRs are CDRL1 of SEQ ID NO: 1, CRL2 of SEQ ID NO: 2 and the heavy chain CDRs comprise the CDRs of SEQ ID NO:6, the DRL2 of SEQ ID NO:3, and the CDRL3 of SEQ ID NO:6. H1, CDRH2 of SEQ ID NO: 7 and CDHR3 of SEQ ID NO: 8. The present invention includes an antigen-binding fragment of the above.

[0042] In one embodiment of any of the above co-formulations, the formulation comprises: (i) a heavy chain comprising SEQ ID NO:88 An anti-CTLA4 antibody or an antigen thereof comprising a variable region and a light chain variable region comprising SEQ ID NO:48. and (ii) a light chain variable region comprising SEQ ID NO: 4 and SEQ ID NO: 9. The antibody includes an anti-human PD1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region.

[0043] In another embodiment of any of the above co-formulations, the formulation comprises: (i) a heavy chain comprising SEQ ID NO: 99 and and an anti-CTLA4 antibody or antigen-binding fragment thereof comprising a light chain comprising SEQ ID NO: 100; and (ii) an anti-human PD1 antibody comprising a light chain comprising SEQ ID NO:5 and a heavy chain comprising SEQ ID NO:10. Includes antibodies or antigen-binding fragments thereof.

[0044] In one embodiment, the formulation is contained in a glass vial. In another embodiment, the formulation is contained in an injection device. In one embodiment, the formulation is frozen to at least below -70°C. In embodiments, the formulation is a reconstituted solution from a lyophilized formulation.

[0045] In one embodiment, an effective amount of an anti-CTLA4 formulation or co-formulation described herein is administered. The treatment of chronic infections or other conditions in mammalian subjects (e.g., humans) in need thereof, including administering the Methods for treating cancer are provided. [Brief description of the drawings]

[0046] [Figure 1] Figure 1A shows the UV A350 absorbance of Formulation A1 over 8 weeks at 5°C, 25°C and 40°C. Figure 1B shows the UV A350 absorbance of Formulation A2 over 8 weeks at 5°C, 25°C and 40°C. [Diagram 2] FIG. 2 shows the UV A350 absorbance of formulations A1 and A2 for freeze-thaw, agitation and light stress tests. [Diagram 3] 3A and 3B show the % HMW data as determined by UP-SEC versus time at 5° C., 25° C. and 40° C. storage conditions for formulations A1 and A2, respectively. [Figure 4] 4A and 4B show the % monomer versus time data as determined by UP-SEC at 5° C., 25° C. and 40° C. storage conditions for formulations A1 and A2, respectively. [Diagram 5] FIG. 5 shows the % HMW determined by UP-SEC for formulations A1 and A2 for freeze-thaw, agitation and light stress tests. [Figure 6] FIG. 6 shows the % monomer determined by UP-SEC for formulations A1 and A2 for freeze-thaw, agitation and light stress tests. [Figure 7] 7A and 7B show the % acidic species versus time data as determined by HP-IEX at 5° C., 25° C. and 40° C. storage conditions for formulations A1 and A2, respectively. [Figure 8] 8A and 8B show the % basic species versus time data as determined by HP-IEX at 5° C., 25° C. and 40° C. storage conditions for formulations A1 and A2, respectively. [Figure 9]9A and 9B show the % main species versus time data as determined by HP-IEX at 5° C., 25° C. and 40° C. storage conditions for formulations A1 and A2, respectively. [Figure 10] FIG. 10 shows the % acidic species determined by HP-IEX for formulations A1 and A2 for freeze-thaw, agitation and light stress tests. [Figure 11] FIG. 11 shows the % basic species as determined by HP-IEX for formulations A1 and A2 for freeze-thaw, agitation and light stress tests. [Figure 12] FIG. 12 shows the % main species determined by HP-IEX for formulations A1 and A2 for freeze-thaw, agitation and light stress tests. [Figure 13] FIG. 13 shows the percent oxidation of LC-M4 (methionine oxidation) as determined by peptide mapping for formulations A1 and A2. [Figure 14] FIG. 14 shows the percent oxidation of HC-M34 (methionine oxidation) as determined by peptide mapping for formulations A1 and A2. [Figure 15] FIG. 15 shows the percent oxidation of HC-M250 (methionine oxidation) as determined by peptide mapping for formulations A1 and A2. [Figure 16] FIG. 16 shows the percent oxidation of HC-M426 (methionine oxidation) as determined by peptide mapping for formulations A1 and A2. [Figure 17] Figure 17A shows the UV A350 absorbance of Formulation B1 over 8 weeks at 5° C., 25° C. and 40° C. Figure 17B shows the UV A350 absorbance of Formulation B2 over 8 weeks at 5° C., 25° C. and 40° C. [Figure 18] FIG. 18 shows the UV A350 absorbance of formulations B1 and B2 for freeze-thaw, agitation and light stress tests. [Figure 19] 19A and 19B show the % HMW versus time data as determined by UP-SEC at 5° C., 25° C. and 40° C. storage conditions for formulations B1 and B2, respectively. [Figure 20] 20A and 20B show the % monomer versus time data as determined by UP-SEC at 5° C., 25° C. and 40° C. storage conditions for formulations B1 and B2, respectively. [Figure 21] FIG. 21 shows the % HMW as determined by UP-SEC for formulations B1 and B2 for freeze-thaw, agitation and light stress tests. [Figure 22] FIG. 22 shows the % monomer determined by UP-SEC for formulations B1 and B2 for freeze-thaw, agitation and light stress tests. [Figure 23] 23A and 23B show the % acidic species versus time data as determined by HP-IEX at 5° C., 25° C. and 40° C. storage conditions for Formulations B1 and B2, respectively. [Figure 24] 24A and 24B show the % basic species versus time data as determined by HP-IEX at 5° C., 25° C. and 40° C. storage conditions for formulations B1 and B2, respectively. [Diagram 25] 25A and 25B show the % main species versus time data as determined by HP-IEX at 5° C., 25° C. and 40° C. storage conditions for formulations B1 and B2, respectively. [Figure 26] FIG. 26 shows the % acidic species as determined by HP-IEX for formulations B1 and B2 for freeze-thaw, agitation and light stress tests. [Figure 27] FIG. 27 shows the % basic species as determined by HP-IEX for formulations B1 and B2 for freeze-thaw, agitation and light stress tests. [Figure 28] FIG. 28 shows the % main species determined by HP-IEX for formulations B1 and B2 for freeze-thaw, agitation and light stress tests. [Figure 29] FIG. 29 shows the percent oxidation of LC-M4 (methionine oxidation) as determined by peptide mapping for formulations B1 and B2. [Diagram 30]FIG. 30 shows the percent oxidation of HC-M34 (methionine oxidation) as determined by peptide mapping for formulations B1 and B2. [Diagram 31] FIG. 31 shows the percent oxidation of HC-M250 (methionine oxidation) as determined by peptide mapping for formulations B1 and B2. [Diagram 32] FIG. 32 shows the percent oxidation of HC-M426 (methionine oxidation) as determined by peptide mapping for formulations B1 and B2. [Diagram 33] FIG. 33 shows the KD data of the co-formulations indicating that the formulations are stable at three different pH values ​​(5.0, 5.5, and 6.0). [Diagram 34] FIG. 34 shows the amino acid sequences of the heavy and light chains of ipilimumab (SEQ ID NOs: 84 and 85, respectively). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] In one embodiment, the present invention provides an anti-CTLA4 antibody that contains methionine and its antigen binding. The present invention also provides a formulation comprising an anti-CTLA4 antibody or an anti-CTLA4 antibody fragment thereof that contains methionine. Co-formulations of an antigen-binding fragment and an anti-human PD-1 antibody or antigen-binding fragment thereof are also provided. In each case, the formulations and co-formulations may include a chelating agent.

[0048] I. Definitions and Abbreviations As used throughout this specification and the appended claims, the following abbreviations apply: To be: API Active Pharmaceutical Ingredient CDRs are defined using the Kabat numbering system unless otherwise indicated. Complementarity determining regions in immunoglobulin variable regions CHO Chinese hamster ovary CI Confidence Interval CTLA4 Cytotoxic T-lymphocyte-associated antigen 4 DTPA Diethylenetriaminepentaacetic acid EC50 Concentration that produces 50% efficacy or binding ELISA Enzyme-Linked Immunosorbent Assay FFPE Formalin-fixed Paraffin-embedded FR Framework Area HRP Horseradish Peroxidase HNSCC head and neck squamous cell carcinoma IC50 Concentration that produces 50% inhibition IgG Immunoglobulin G ICH International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use ce of Harmonization) IHC immunohistochemistry or immunohistochemistry mAb Monoclonal antibody MES 2-(N-morpholino)ethanesulfonic acid NCBI National Center for Biotechnology Information iotechnology Information) NSCLC Non-small cell lung cancer PCR Polymerase Chain Reaction PD-1 Programmed Death 1 (also known as Programmed Cell Death-1 and Programmed Death Receptor 1) ) PD-L1 Programmed Cell Death 1 Ligand 1 PD-L2 Programmed cell death 1 ligand 2 PS80 Polysorbate 80 TNBC Triple-negative breast cancer V H Immunoglobulin heavy chain variable region VK immunoglobulin kappa light chain variable region V L Immunoglobulin light chain variable region v / v volume per volume WFI water for injection w / v Weight per volume.

[0049] In order that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. As used herein, unless specifically defined elsewhere in this document, All other technical and scientific terms used herein are understood to be within the skill of those of ordinary skill in the art to which this invention pertains. It has a meaning that is universally understood.

[0050] As used throughout this specification and in the appended claims, the singular forms "a," "b," and "c" are used interchangeably. "a," ​​"an," and "the" are used to refer to plural words unless the context clearly dictates otherwise. Includes.

[0051] A reference to "or" indicates that the context clearly indicates one of the possibilities indicated. Unless otherwise indicated, either or both possibilities are indicated. In these cases, the word "and / or" is used to emphasize either or both possibilities. It was.

[0052] As used herein, "treating" or "treating" cancer refers to at least One positive therapeutic effect, e.g., reduction in the number of cancer cells, reduction in tumor size, and peripheral organ To achieve a reduction in the rate of cancer cell invasion or a reduction in the rate of tumor metastasis or tumor growth To achieve this, the formulations of the invention are administered to a subject having an immune condition or a cancer condition, or to a subject suffering from cancer or is intended to be administered to subjects diagnosed with a pathogenic infection (e.g., viral, bacterial, fungal). "Treatment" may include one or more of the following: inducing / enhancing an anti-tumor immune response; stimulating an immune response against pathogens, toxins and / or self-antigens; stimulating an immune response to bacterial infection; reducing the number of one or more tumor markers; inhibiting the proliferation or survival of tumor cells; eliminating or a reduction in the size of the tumor; a decrease in the level of one or more tumor markers; reducing the severity or duration of cancer; Extending the life of a patient compared with expected survival.

[0053] "Immune conditions" or "immune disorders" include, for example, pathological inflammation, inflammatory disorders, and autoimmune disorders. "Immune conditions" also include infectious diseases, persistent infections, and This refers to proliferative conditions such as cancer, tumors and angiogenesis that resist eradication by the immune system. "Cancer condition" includes, for example, cancer, cancer cells, tumors, Includes angiogenesis and precancerous conditions such as dysplasia.

[0054] Positive treatment outcomes in cancer can be measured in several ways (WA See Weber, J. Nucl. Med. 50:1S-10S (2009). For example, in terms of tumor growth inhibition, according to the NCI standard, T / C ≦ 42% is considered to be antitumor activity. T / C < 10% is considered a high level of antitumor activity, where T / C (%) = median treated tumor volume / median control tumor volume × 100. In some embodiments, the treatment achieved by administration of the formulation of the present invention improves progression-free survival ( PFS, disease-free survival (DFS) or overall survival (OS). is also known as "Time to Tumor Progression" This is called a complete response and refers to the period during and after treatment during which the cancer does not grow and patients achieve a complete response or This includes the time to partial response, as well as the time to stable disease. OS refers to the period during and after treatment during which a patient remains disease-free. refers to an increase in life expectancy compared to an untreated individual or patient. In certain embodiments of the use, the method may be ineffective in achieving a positive therapeutic effect in all patients. However, any statistical test known in the art, such as Student's t-test, chi 2 test, Mann and Whitney U test, Kruskal- Wallis test (H-test), Jonckheere-Terpstra test and In a statistically significant number of subjects, as determined by Wilcoxon tests, this is not the case. There will be.

[0055] The term "patient" (also referred to herein as "subject" or "individual") refers to a subject of the present invention. A mammalian animal that can be treated with the disclosed formulation (e.g., a rat, a mouse, a dog, a cat, a mouse ... In some embodiments, the patient is an adult. In other embodiments, the patient is a pediatric patient.

[0056] The term "antibody" refers to any form of antibody that exhibits the desired biological activity. This is used in the broadest sense and specifically includes, but is not limited to, monochrome Clonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized antibodies "Parent antibody" refers to any antibody that is suitable for the intended use, e.g. Prior to modification of an antibody, such as humanizing the antibody for use as a human therapeutic antibody, immunoassays may be performed. Antibodies are obtained upon exposure of the system to antigens.

[0057] Generally, the basic antibody structural unit comprises a tetramer. Each tetramer contains two identical polypeptides. It contains a peptide chain pair, each pair consisting of one "light" chain (approximately 25 kDa) and one "heavy" chain. The amino-terminal portion of each chain is primarily involved in antigen recognition. Each light chain / heavy chain contains a variable region consisting of about 100 to 110 amino acids that contribute to the The paired variable regions form the antibody binding site. Therefore, generally, an intact antibody The carboxy-terminal portion of the heavy chain is primarily responsible for effector functions. The constant regions involved can be defined. Typically, human light chains are kappa light chains and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha or and epsilon, which classify the antibody isotype as IgM, IgD, IgG, and I Within the light and heavy chains, there are approximately 12 variable and constant regions. The heavy chains are joined by a "J" region consisting of about 10 or more amino acids. In general, the term also includes the "D" region, which consists of: Fundamental Immunolo gy Ch.7(Paul, W., ed., 2nd ed. Raven Press, See NY (1989).

[0058] Typically, both the heavy and light chain variable domains contain complementary determining regions (CDRs). The nucleotide sequence of each of the three hypervariable regions is represented by relatively conserved framework regions (FRs). The CDRs are usually aligned with the framework regions and provide the link to a specific epitope. Generally, from N-terminus to C-terminus, the variable domains of both the light and heavy chains are Each sequence includes FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to domains is generally Sequences of Proteins ns of Immunological Interest ,Kabat,et al .;National Institutes of Health,Bethesda ,Md.;5 th ed.;NIH Publ.No.91-3242(1991);K abat(1978) Adv.Prot.Chem.32:1-75;Kabat,e t al.,(1977) J.Biol.Chem.252:6609-6616;C hothia,et al.,(1987) J.Mol.Biol.196:901- 917 or Chothia, et al., (1989) Nature 342:8 As defined in 78-883.

[0059] An antibody that "specifically binds" to a particular target protein is one that specifically binds to that target protein compared to other proteins. Although antibodies that exhibit preferential binding to a target, this specificity does not require absolute binding specificity. Antibodies are not designed to detect unwanted, undesired, binding-related events that may signal the presence of the target protein in a sample, e.g., a false positive. A compound is said to be "specific" for its intended target if it can be determined without producing unexpected results. It is believed that the antibodies or binding fragments thereof useful in the present invention can bind to non-target proteins. At least 2 times stronger than the affinity, preferably at least 10 times stronger, more preferably or at least 20 times stronger, and most preferably at least 100 times stronger. As used herein, a given amino acid has a specific function to bind to a target protein. A polypeptide comprising the amino acid sequence of a mature human CTLA4 or human PD-1 molecule. If an antibody binds to a protein that contains that sequence but does not bind to a protein that lacks that sequence, then the antibody is It is said to specifically bind to a polypeptide containing the .

[0060] A "chimeric antibody" is an antibody in which some portions of the heavy and / or light chains are identical to those of a particular species (e.g., human). Derived antibodies or corresponding sequences in antibodies belonging to a particular antibody class or subclass The remainder of the chain(s) is identical or homologous to an antibody from another species (e.g., mouse) or a sequence identical or homologous to a corresponding sequence in an antibody belonging to another antibody class or subclass. and such antibodies so long as they exhibit the desired biological activity. It also refers to a fragment of.

[0061] As used herein, "co-formulated" or " "Co-formulation" or "coformulati "Coformulated" means to combine ingredients together in a single formulation. Either the two drugs are mixed together, stored, and then mixed before administration, or administered in a single vial rather than separately. They are formulated together in a container or vessel (e.g., an injection device) and are combined At least two different antibodies or their antigen-binding products are stored as In one embodiment, the co-formulation refers to a co-immunoglobulin fragment of two different antibodies or their antigen-binding fragments. It contains a synthetic fragment.

[0062] The term "pharmacologically effective amount" or "effective amount" refers to an amount sufficient to treat a disease or condition. By this term is meant an amount of a therapeutic composition or formulation that is introduced into a patient sufficient to achieve this level of efficacy. It will be appreciated that dosages may vary depending on patient characteristics such as age and weight.

[0063] The term "about" refers to the amount of a substance or composition (e.g., mM or M), the percentage of a formulation component, -Percentage (v / v or w / v), pH of the solution / formulation, or steps in the method When modifying the values ​​of parameters to be characterized, for example, the preparation, properties, etc. of a substance or composition Through typical measurement, manipulation and sampling techniques involved in the evaluation and / or use through instrumental errors in these procedures; to make or use the compositions; or may arise through differences in manufacture, source, or purity of the components employed to carry out the method. In certain embodiments, "about" refers to ±0.1%, 0.5%, It can mean a variation of 1%, 2%, 3%, 4%, 5% or 10%.

[0064] As used herein, "x % (w / v)" is equal to x g / 100 ml ( For example, 5% w / v is equal to 50 mg / ml).

[0065] The formulations of the present invention contain biologically active antibodies and fragments thereof when reconstituted or in liquid form. Includes.

[0066] The terms "cancer," "cancerous," or "malignant" refer to a condition that is typically characterized by unregulated cell growth. Cancer refers to or describes a physiological condition in mammals in which the cancer is present. Examples of cancer include, but are not limited to, Cancers such as, but not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastrointestinal (digestive tract) cancer, kidney cancer, ovarian cancer, liver cancer Liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer , prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, polymorphonuclear nephropathy Glioblastoma, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon carcinoma and head and neck cancer Includes neck cancer.

[0067] “Chothia” is Al-Lazikani et al., JMB 273:9 By "numbering" is meant the antibody numbering system described in J. Immunol. 27-948 (1997).

[0068] As used herein, "Kabat" refers to the methodology used by Elvin A. Kabat (1991 ) Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health,Bethesda,M d.) The immunoglobulin alignment and numbering system developed by It means.

[0069] "Growth inhibitory agent" as used herein refers to an agent that inhibits growth, either in vitro or in vivo. In one embodiment, the present invention relates to a cancer cell line, comprising a medicament for treating cancer, the cancer cell line ... A proliferation inhibitor is a compound or composition that inhibits cell proliferation. Therefore, a proliferation inhibitor is a compound or composition that inhibits cell proliferation in the S phase. The percentage of cells that overexpress such genes is significantly reduced. Examples include agents that block cell cycle progression (outside of S phase), such as G1 arrest and These include agents that induce M-phase arrest and block M-phase inhibition. Classical M-phase blockers include the vincas (vincristine, vinblastine and vinblastine), taxanes, and doxorubicin, epirubicin, Agents that arrest G1 include topo II inhibitors such as daunorubicin and etoposide. It also extends to S-phase arrest, and is mediated by, for example, dacarbazine, mechlorethamine, and cisplatin. For more information, see The Molecular In Basis of Cancer, edited by Mendelsohn and Israel, , Murakami et al., “Cell cycle regulation, onc The article was titled “Genes, and Antineoplastic Drugs.” (WB Saunders: Philadelphia, 1995).

[0070] The terms "CTLA4-binding fragment," "antigen-binding fragment thereof," "binding fragment thereof" or The "fragment" binds to an antigen (human CTLA4) and inhibits its activity (e.g., human C The biological activity of blocking the binding of TLA4 to its natural ligands is still substantially Thus, the term "antibody fragment" or "antibody derivative" includes fragments or derivatives of antibodies that retain their original structure. A TLA4-binding fragment is a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of CTLA4 antibody fragments include Fab, Fab', and F(ab') 2 and F Typically, a binding fragment or derivative will retain at least some of its CTLA4 inhibitory activity. The 10% of the target molecule is retained in the 10% ELISA kit. Although any binding fragment that binds to the nucleotide sequence is useful, in some embodiments, a binding fragment or The derivatives have at least 25%, 50%, 60%, 70%, 80% or more of their CTLA4 inhibitory activity. %, 90%, 95%, 99% or 100% (or more). In some embodiments, the antigen-binding fragment has affinity for the antigen and for an unrelated antigen. At least 2 times stronger than the with at least 20 times greater affinity, and most preferably at least 100 times greater affinity. In one embodiment, the antibody has an affinity for, for example, an antibody that is capable of binding to, or is capable of binding to, an antibody. Finite is about 10 9 Stronger than liter / mol. Munsen et al. 980) Analyt.Biochem. 107:220-239. Also, CTLA 4. The binding fragment may be a variant having conservative amino acid substitutions that do not substantially alter its biological activity. It is also intended that the term "common" can include the term "common" or "non-common".

[0071] The terms "PD-1 binding fragment," "antigen-binding fragment thereof," "binding fragment thereof" or " The fragment thereof binds to an antigen (human PD-1) and inhibits its activity (e.g., human PD- 1 to PDL1 and PDL2) is still substantially active. Therefore, the term "antibody fragment" or "PD A -1 binding fragment refers to a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab', and F(ab') 2 and Fv fragments. Typically, a binding fragment or derivative retains at least 10% of its PD-1 inhibitory activity. Any binding affinity that has sufficient affinity to exert a desired biological effect. Although fragments are useful, in some embodiments, binding fragments or derivatives are D-1 inhibitory activity of at least 25%, 50%, 60%, 70%, 80%, 90%, 95% , 99% or 100% (or more). Thus, an antigen-binding fragment has an affinity for the antigen that is less than or equal to the affinity for an unrelated antigen. at least twice as strong, preferably at least 10 times as strong, more preferably at least 20 times as strong, Most preferably, the binding occurs with at least 100-fold greater affinity. In some embodiments, the antibody has an affinity of about 1, as determined, for example, by Scatchard analysis. 0 9 Stronger than liter / mol. Munsen et al.(1980) Anal yt.Biochem. 107:220-239. In addition, the PD-1 binding fragment Variants having conservative amino acid substitutions that do not substantially alter biological activity can be included. It is also intended that

[0072] A "human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. The antibody may be produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" or a "rat antibody" may contain mouse glycosylation. These refer to antibodies that contain only mouse or rat immunoglobulin sequences, respectively.

[0073] A "humanized antibody" refers to an antibody that has been engineered to have a sequence derived from a non-human (e.g., murine) antibody as well as from a human antibody. This term refers to a form of antibody that contains a minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody contains at least one, and typically two, variable domains. and wherein the hypervariable domains correspond to those of non-human immunoglobulins. All or substantially all of the loops and all or substantially all of the FR regions are human. The humanized antibody also contains the immunoglobulin constant region (Fc) of the humanized antibody. The human antibody may comprise at least a portion of a human immunoglobulin, typically that of a human immunoglobulin. The humanized form generally contains the same CDR sequences as the parent rodent antibody, but has the affinity Certain amirites may be used to improve performance, improve stability, or for other reasons. Acid substitutions may be included.

[0074] The antibodies of the present invention may also have modified (e.g., or blocked) antibodies. O2003 / 086310;WO2005 / 120571;WO2006 / 005770 2;Presta (2006) Adv.Drug Delivery Rev. 5 8:640-656. Such modifications can enhance or suppress various responses of the immune system. It can be used to control the immune system and has potential beneficial effects in diagnosis and therapy. Changes in the c region include amino acid changes (substitutions, deletions and insertions), glycosylation or Modifications to the Fc can also be made to therapeutic antibodies, including deglycosylation, and adding multiple Fcs. The half-life of the antibody in the antibody can also be altered, with a longer half-life allowing for less frequent dosing and Presta (2005) J. All See Cell Clin. Immunol. 116:731 at 734-35. I want to.

[0075] A "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. Fully human antibodies can be produced in mice, mouse cells, or hybridomas derived from mouse cells. Similarly, a "mouse antibody" may contain mouse glycosylation when produced by immunization with a mouse. A fully human antibody is an antibody that contains only human immunoglobulin sequences. Phage display in transgenic animals carrying phospho-germline sequences or by other molecular biology methods.

[0076] "Hypervariable region" refers to the amino acid residues of an antibody that are involved in antigen-binding. The regions are selected from amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., Kabat Numbering system (Kabat et al. (1991) Sequences of Proteins of Immunological Interest,5t h Ed. Public Health Service, National Ins. Mild symptoms measured by the Department of Health, Bethesda, Md. Residues 24-34 (CDRL1), 50-56 (CDRL2), and 8 in the chain variable domain 9-97 (CDRL3) and residues 31-35 in the heavy chain variable domain (CDRH1); 50-65 (CDRH2) and 95-102 (CDRH3)), and / or Amino acid residues from the “hypervariable loop” (i.e., residues 26–32 in the light chain variable domain) (L1), 50-52 (L2) and 91-96 (L3) in the heavy chain variable domain 26-32 (H1), 53-55 (H2) and 96-101 (H3) (Chothia and Lesk (1987) J.Mol.Biol. 196:901-917 As used herein, the term "framework" or "FR" residues include refers to those variable residues other than the hypervariable region residues defined herein as CDR residues. Domain residues are referred to. CDR and FR residues are referred to according to the standard sequence definition of Kabat. Kabat et al. (1987) Sequences of P roteins of Immunological Interest,Nation al Institutes of Health, Bethesda Md.

[0077] "Conservatively modified variants" or "conservative substitutions" refer to amino acid substitutions known to those of skill in the art. This refers to the substitution of a sequence, even in an essential region of a polypeptide, that generally alters the bioactivity of the resulting molecule. Such exemplary substitutions may be made without altering the biological activity of the compound. This is carried out in accordance with that described in.

[0078] Table 1. Exemplary conservative amino acid substitutions [Table 1]

[0079] In addition, one of skill in the art will generally recognize single amino acid substitutions in non-essential regions of a polypeptide. does not substantially alter biological activity. See, e.g., Watson et al. (1987) Molecular Biology of the Gene,Th e Benjamin / Cummings Pub.Co.,p.224(4th Ed .) for reference.

[0080] As used throughout this specification and claims, the phrase "consisting essentially of" "consists essentially of" or "consists essentially of "consist essentially of" or "consist essentially of" Variations such as "sisting essentially of" are The inclusion of a recited element or group of elements and the prescribed dosing regimen, method or The recited elements and properties do not materially change the basic or novel characteristics of the composition. It is noted that other elements, similar or different, may be included. A binding compound consisting essentially of a recited amino acid sequence will have substantially the same properties as the binding compound. It may also include one or more amino acids that do not affect the function of the polypeptide, which includes the substitution of one or more amino acid residues. Includes.

[0081] "Comprising" or "comprise" "comprises" or "comprised of" Such variations are not intended to be construed as construing the same principle as the general rule, but rather as a general rule where the context requires otherwise, either by explicit language or necessary implication. Unless otherwise required, the present specification and claims are used in an inclusive sense throughout, i.e. That is, the existence of a stated feature does not necessarily imply the function or utility of any of the embodiments of the invention. The present invention is not intended to exclude the presence or addition of further features which may substantially enhance the functionality of the present invention.

[0082] "Isolated antibody" and "isolated antibody fragment" refer to a purified state and in this context In this study, the named molecule is synthesized from other biological molecules, such as nucleic acids, proteins, lipids, and carbohydrates. Or it means substantially free of other materials, such as cell debris and growth medium. Generally, the term "isolated" refers to an experimental or therapeutically isolated binding compound described herein. The complete absence of such substances, unless present in an amount that would substantially interfere with the therapeutic use of the substance; or It is not intended to refer to the absence of water, buffers or salts.

[0083] As used herein, "monoclonal antibody" or "mAb" or "Mab" refers to refers to a population of substantially homogeneous antibodies, i.e., the amino acid sequence of the antibody molecules that make up the population. The sequences are identical except for potential naturally occurring mutations that may exist in small amounts. Reclonal) antibody preparations are typically prepared by isolating their variable domains, especially their CD R contains many different antibodies with different amino acid sequences, which are often The modifier "monoclonal" refers to a substantially homogeneous antibody. It refers to the characteristics of antibodies as obtained from a body population and indicates that they are anti- It is not to be construed that whole body production is required. For example, the monoclonal antibodies used in accordance with the present invention may be Clonal antibodies were prepared as described by Kohler et al. (1975) Nature 256: 495, or by recombinant DNA techniques. (See, for example, U.S. Patent No. 4,816,567.) "Nonoclonal antibodies" can also be derived from phage antibody libraries, e.g., Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597 The antibodies can be isolated using techniques described in Presta (2005) J. Allergy C See also Lin. Immunol. 116:731.

[0084] "Tumor" as applied to a subject diagnosed with or suspected of having cancer means any A malignant or potentially malignant neoplasm or tissue mass of any size, including primary tumors and secondary Solid tumors include neoplasms. A solid tumor is an abnormal increase in tissue that does not usually contain cysts or areas of liquid. Different types of solid tumors are classified according to the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (blood tumors) Cancers generally do not form solid tumors (National Cancer Institute, tute, Dictionary of Cancer Terms).

[0085] 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 measured by various methods known in the art, for example, By measuring the size of the tumor(s) when removed from the subject, for example with calipers. or while in the body, imaging techniques such as bone scan, ultrasound, CT or M This can be determined by using an RI scan.

[0086] As used herein, a "variable region" or "V region" refers to a region that has sequence variation among different antibodies. This refers to the segment of an IgG chain that is at Kabat residues 109 and It spans 113 in the heavy chain.

[0087] The term "buffer" refers to an agent that maintains the solution pH of the formulation of the present invention within an acceptable range, or or, in the case of the lyophilized formulation of the present invention, an agent that provides an acceptable solution pH prior to lyophilization. do.

[0088] The terms "lyophilized," "lyophilized," and "freeze-dried" "Freeze-dried" means that the material to be dried is first frozen and then cooled. It also refers to the process of removing frozen solvent by sublimation in a vacuum environment. To improve the stability of the dried product, excipients are pre-lyophilized. It may be included in the ED formulation.

[0089] The term "pharmaceutical formulation" refers to a form in which the active ingredient is capable of being administered. A preparation that does not contain additional components that are toxic to the subject to which it is administered. The terms "formulation" and "pharmaceutical formulation" are used interchangeably throughout.

[0090] "Pharmaceutically acceptable" means a compound that is suitable for use in a subject to provide an effective dose of the active ingredient employed. It can be administered reasonably well and is "generally recognized as safe," i.e., physiologically tolerated. and when administered to humans, they have been known to cause allergic reactions such as acute gastric congestion or similar untoward effects. It refers to excipients (vehicles, additives) and compositions that typically do not cause a reaction. In this regard, the term refers to any product or service that has been approved by a federal or state regulatory agency or is a U.S. Pharmacopoeia or other generally recognized for use in animals, and more particularly in humans. Refers to molecular entities and compositions listed in the Pharmacopoeias.

[0091] The "reconstituted" formulation is prepared by subjecting the lyophilized protein preparation to a concentration sufficient to disperse the protein in the reconstituted formulation. The reconstituted formulation is prepared by dissolving the agent in a diluent. It is suitable for parenteral administration and may also be suitable for subcutaneous administration.

[0092] "Reconstitution time" refers to the time it takes for the lyophilized formulation to rehydrate with the solution to become a clear, particulate-free solution. is the time required for

[0093] A "stable" formulation is one in which the protein therein exhibits its physical stability and / or These proteins essentially retain their chemical stability and / or biological activity. Various analytical techniques for measuring stability are available in the art, including Pep Tide and Protein Drug Delivery,247-301,V incent Lee Ed.,Marcel Dekker,Inc.,New Yo rk, NY, Pubs. (1991) and Jones, A. Adv. Drug Reviewed in Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. In an embodiment, the stable formulation has significant shelf life at refrigerated temperatures (2-8° C.) for at least 12 months. In another embodiment, the stable formulation is one in which no change is observed. 8°C) for at least 18 months. In this study, stable formulations showed no significant changes at room temperature (23-27°C) for at least three months. In another embodiment, the stable formulation is a formulation that is stable at room temperature (23 to 27° C.) for a short time. In another embodiment, the stable formulation is one in which no significant changes are observed for at least six months. A suitable formulation is one that shows no significant changes for at least 12 months at room temperature (23-27°C). In another embodiment, the stable formulation is a formulation that has a shelf life of at least 1 The stability criteria for antibody formulations are: Typically, 10% of antibody monomers as measured by SEC-HPLC Typically, the formulation is degraded by visual analysis to less than 5%. They are colorless or clear to slightly opalescent. Typically, the concentration, pH and osmolality of the formulation are The titers typically vary by 60-14% of the control or reference. 0%, preferably in the range of 80-120%. Typically, antibody clipping, i.e. That is, the % low molecular weight species, as determined, for example, by HP-SEC, should be less than 10%, preferably less than 5%. Typically, only antibody agglutination, as determined by, for example, HP-SEC, is observed. The percent high molecular weight species observed is no more than 10%, preferably no more than 5%.

[0094] The antibodies may be assayed for color and / or clarity by visual examination, or by UV light scattering, size exclusion spectroscopy, or by immunofluorescence. Aggregation, precipitation and / or precipitation as measured by chromatography (SEC) and dynamic light scattering. If the physical stability or denaturation of the pharmaceutical preparation is not significantly increased, the The change in protein structure was observed by fluorescence spectroscopy, which determines the tertiary structure of proteins. and can be assessed by FTIR spectroscopy, which determines protein secondary structure.

[0095] If an antibody does not exhibit significant chemical changes, it is considered to have "indicated its chemical stability" in a pharmaceutical formulation. Chemical stability is the ability to detect and quantify chemical changes in protein conformation. This can be assessed by degradation processes that often change the chemical structure of proteins. This can be achieved by hydrolysis or clipping (e.g. size exclusion chromatography and SD S-PAGE), oxidation (e.g., by mass spectrometry or MALDI / Assessed by methods such as peptide mapping with TOF / MS), deamidation (e.g. ion exchange chromatography, capillary isoelectric focusing, peptide mapping isomerization (evaluated by methods such as isoaspartic acid measurement) and isomerization (isoaspartic acid measurement). (evaluated by measuring the paraginyl content, peptide mapping, etc.).

[0096] The antibody may be used in a pharmaceutical formulation prepared according to the biological activity of the antibody at a given time. Within a predefined range of biological activity, a pharmaceutical preparation is deemed to have "its biological stability." The biological activity of the antibody is determined, for example, by antigen binding assays. It is possible.

[0097] The term "isotonic" means that the subject formulation has essentially the same osmotic pressure as human blood. Isotonic preparations generally have an osmotic pressure of about 270 to 328 mOsm. Slightly hypotonic pressures are The osmolarity is 250-269, and the slightly hypertonic pressure is 328-350 mOsm. For example, it can be measured using a vapor pressure or ice-freezing type osmometer.

[0098] II. Formulations and Co-formulations of the Invention In one embodiment, the present invention provides an anti-CTLA4 antibody that specifically binds to human CTLA4. or an antigen-binding fragment thereof as an active pharmaceutical ingredient. The inclusion of methionine in such a formulation is consistent with the presence of methionine in the Fc region of the anti-CTLA4 antibody. This reduces the oxidation of methionine residues that cause

[0099] In one embodiment, the present invention also provides a co-formulation of an anti-CTLA4 antibody with an anti-PD-1 antibody. The main degradation pathway of pembrolizumab is at methionine 105( Met105) (e.g., M105 of SEQ ID NO: 10) upon peroxide stress, These included oxidation of Met105 and Fc methionine residues upon exposure to light and Pembrolizumab was tested under the maximum stress conditions for degradation levels. However, surface plasmon resonance (SPR) showed that the activity of the A reduction in affinity for PD-1 was observed for samples that had been treated with Exposed methionine residues or methionine residues in the CDRs can alter the biology of antibodies through oxidation. The addition of methionine to the pembrolizumab heavy chain CDRs may affect the targeting activity. It is possible to reduce the oxidation of Met105 in

[0100] Anti-PD-1 antibodies and antigen-binding fragments thereof In one embodiment, the present invention provides a method for the preparation of a PD-1 antibody comprising administering to a subject a subject in need thereof a human PD-1 antibody as an active pharmaceutical ingredient (PD-1 API). An anti-human PD-1 antibody or an antigen-binding fragment thereof that specifically binds to PD-1 (e.g., a human or or a humanized anti-PD-1 antibody) Also provided are stable biological formulations comprising the fragments, as well as methods for using the formulations of the invention. The anti-PD-1 antibodies or antigen-binding fragments thereof are used in the co-formulations and methods of the invention. In certain embodiments, the PD-1 API is pembrolizumab and and nivolumab. In a specific embodiment, the anti-PD-1 antibody is In an alternative embodiment, the anti-PD-1 antibody is nivolumab. Table 2 shows exemplary anti-human PD-1 antibodies pembrolizumab and nivolumab. The amino acid sequences of the mabs are provided. Alternative PD- 1 antibodies and antigen-binding fragments are shown in Table 3.

[0101] As used herein, "pembro" is alternatively referred to herein as "pembro" rolizumab (formerly known as MK-3475, SCH900475 and lambrolizumab) (known as 2, pp. 161-162 (2013) and having the structure described in Table 2 It is a humanized IgG4 mAb containing the amino acid sequences and CDRs of the heavy and light chains of Embrolizumab is approved for use in patients with KEYTRUDA(TM) who have a prescribing indication. nformation) (Merck & Co., Inc., Whitehouse Station, NJ USA; first US approval in 2014, updated in September 2017 As of 2018, it has been approved by the U.S. FDA for use in the treatment of unresectable or metastatic melanoma. for the treatment of patients with recurrent or metastatic head and neck squamous cell carcinoma (HN SCC), classical Hodgkin lymphoma (cHL), urothelial carcinoma, gastric cancer, microsatellite Treatment of certain patients with multiple sclerosis instability-positive (MSI-H) and non-small cell lung cancer has been approved for.

[0102] In some embodiments, anti-human PD-1 antibodies for use in the co-formulations of the invention The antibody or antigen-binding fragment thereof has three light chains, CDRL1, CDRL2 and CDRL3. The CDRs and / or the three heavy chain CDRH1, CDRH2 and CDRH3 Contains R.

[0103] In one embodiment of the invention, CDRL1 is SEQ ID NO:1 or the barrier sequence of SEQ ID NO:1. CDRL2 is SEQ ID NO:2 or a variant of SEQ ID NO:2, and CDRL3 is is SEQ ID NO:3 or a variant of SEQ ID NO:3.

[0104] In one embodiment, CDRH1 is SEQ ID NO:6 or a variant of SEQ ID NO:6. wherein CDRH2 is SEQ ID NO: 7 or a variant of SEQ ID NO: 7, and CDRH3 is SEQ ID NO: SEQ ID NO: 8 or a variant of SEQ ID NO: 8.

[0105] In one embodiment, the three light chain CDRs are set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. No. 3, and the three heavy chain CDRs are SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. be.

[0106] In an alternative embodiment of the invention, CDRL1 is SEQ ID NO:11 or a variant of SEQ ID NO:11. and CDRL2 is SEQ ID NO:12 or a variant of SEQ ID NO:12; DRL3 is SEQ ID NO:13 or a variant of SEQ ID NO:13.

[0107] In one embodiment, CDRH1 is SEQ ID NO:16 or a variant of SEQ ID NO:16. and CDRH2 is SEQ ID NO: 17 or a variant of SEQ ID NO: 17, and CDRH3 is is SEQ ID NO:18 or a variant of SEQ ID NO:18.

[0108] In one embodiment, the three light chain CDRs are set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. No. 3, and the three heavy chain CDRs are SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. be.

[0109] In an alternative embodiment, the three light chain CDRs are SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13. SEQ ID NO: 13, and the three heavy chain CDRs are SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: Column number 18.

[0110] In a further embodiment of the invention, CDRL1 is SEQ ID NO:21 or a sequence similar to SEQ ID NO:21. and CDRL2 is SEQ ID NO:22 or a variant of SEQ ID NO:22; CDRL3 is SEQ ID NO:23 or a variant of SEQ ID NO:23.

[0111] 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 CDRH 3 is SEQ ID NO:26 or a variant of SEQ ID NO:26.

[0112] In another embodiment, the three light chain CDRs are selected from SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. No. 23, and the three heavy chain CDRs are SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: The number is 26.

[0113] Some of the anti-human PD-1 antibodies and antigen-binding fragments of the present invention comprise a light chain variable region and In some embodiments, the light chain variable region comprises a heavy chain variable region of SEQ ID NO: 4 or and the heavy chain variable region is SEQ ID NO: 9 or a variant of SEQ ID NO: 9. In a further embodiment, the light chain variable region comprises a variant of SEQ ID NO: 14 or and the heavy chain variable region comprises a variant of SEQ ID NO: 14, and the heavy chain variable region comprises a variant of SEQ ID NO: 19 or SEQ ID NO: In a further embodiment, the heavy chain variable region comprises a variant of SEQ ID NO:27. or a variant of SEQ ID NO: 27, and the light chain variable region comprises SEQ ID NO: 28 or is a variant of SEQ ID NO: 28, SEQ ID NO: 29 or a variant of SEQ ID NO: 29, or In such an embodiment, the variant includes SEQ ID NO: 30 or a variant of SEQ ID NO: 30. The light or heavy chain variable region sequence of the ant may have one, two, three, four or five amino acid substitutions. In some embodiments, the substitutions are identical to the reference sequence in the framework region. In some embodiments, one, two, three, four or more amino acids are present within the CDRs (i.e., outside the CDRs). or five amino acid substitutions are conservative substitutions.

[0114] In one embodiment of the co-formulation of the invention, the anti-human PD-1 antibody or antigen-binding fragment The antibody comprises a light chain variable region comprising or consisting of SEQ ID NO: 4 and a light chain variable region comprising or consisting of SEQ ID NO: 9. In a further embodiment, the anti-human PD-1 antibody or The antigen-binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO: 14, and a light chain variable region comprising the sequence The heavy chain variable region comprises or consists of number 19. One embodiment of the formulation of the present invention wherein the anti-human PD-1 antibody or antigen-binding fragment comprises or comprises SEQ ID NO:28. and a heavy chain variable region comprising or consisting of SEQ ID NO: 27. In a further embodiment, the anti-human PD-1 antibody or antigen-binding fragment comprises SEQ ID NO: a light chain variable region comprising or consisting of SEQ ID NO:29, and a light chain variable region comprising or consisting of SEQ ID NO:27 In another embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region consisting of the sequence A light chain variable region comprising or consisting of SEQ ID NO: 30, and a light chain variable region comprising or consisting of SEQ ID NO: 27 It comprises a heavy chain variable region consisting of this.

[0115] In another embodiment, the co-formulations of the invention comprise the V L Domain or V H Domain At least 95%, 90%, 85%, 80%, 75% or 50% sequence identity with one of the Same-sex VL Domain and / or V H Anti-human PD-1 antibody with domain or comprises an antigen binding protein and exhibits specific binding to PD-1. Therefore, the anti-human PD-1 antibody or antigen-binding protein of the co-formulation of the invention is V with up to 4 or 5 or more amino acid substitutions L and V H Domain It contains an agonist and exhibits specific binding to PD-1.

[0116] In any of the above embodiments, the PD-1 API specifically binds human PD-1. The antibody may be a full-length anti-PD-1 antibody or an antigen-binding fragment thereof. In the present invention, the PD-1 API includes IgM, IgG, IgD, IgA, and IgE. It is a full-length anti-PD-1 antibody selected from any class of immunoglobulins. , the antibody is an IgG antibody. 1 , IgG 2 , IgG 3 and IgG 4 Includes Any isotype of IgG can be used. Different constant domains are described herein. V provided L and V H For example, the antibody (or fragment) of the present invention may be If a particular application is to seek altered effector function, heavy chain constants other than IgG1 may be used. Common domains can be used. IgG1 antibodies have long half-lives and are known to inhibit, for example, complement activation and Although it provides effector functions such as antibody-dependent cellular cytotoxicity, such activities are not limited to any particular antibody. In such cases, it may be undesirable to use, for example, an IgG4 constant domain. Main can be used.

[0117] In an embodiment of the invention, the PD-1 API has the amino acid sequence set forth in SEQ ID NO:5. A light chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 10. In an alternative embodiment, the anti-PD-1 antibody comprises a heavy chain comprising or consisting of: The PD-1 API comprises or consists of the sequence of amino acid residues set forth in SEQ ID NO:15. and a heavy chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 20. In a further embodiment, the PD-1 API is an anti-PD-1 antibody comprising the sequence a light chain comprising or consisting of the sequence of amino acid residues set forth in SEQ ID NO: 32; 31. An anti-PD-1 antibody comprising a heavy chain comprising or consisting of an amino acid sequence as set forth in In additional embodiments, the PD-1 API comprises the amino acid sequence set forth in SEQ ID NO:33. a light chain comprising or consisting of the sequence of residues set forth in SEQ ID NO: 31; and an anti-PD-1 antibody comprising a heavy chain comprising or consisting of the sequence of: In one embodiment, the PD-1 API comprises the sequence of amino acid residues set forth in SEQ ID NO:34 or and a light chain comprising or comprising the sequence of amino acid residues set forth in SEQ ID NO: 31. In some co-formulations of the invention, the anti-PD-1 antibody comprises a heavy chain consisting of -1 API is pembrolizumab or a biosimilar of pembrolizumab. In some of the coformulations of the invention, the PD-1 API is nivolumab or a vaccinia It is an iosimilar.

[0118] Typically, the anti-PD-1 antibodies and antigen-binding fragments of the invention or the anti-CTLA4 antibodies of the invention and antigen-binding fragment amino acid sequence variants are similar to those of a reference antibody or antigen-binding fragment (e.g. For example, heavy chain, light chain, V H , V L or a humanized sequence) More preferably at least 80%, more preferably at least 85%, more preferably at least at least 90%, most preferably at least 95, 98 or 99% amino acid sequence identity. As used herein, identity or homology with respect to sequences is defined as aligning sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity; Residues of anti-PD-1 after not considering any conservative substitutions as part of the sequence identity It is defined as the percentage of amino acid residues in the candidate sequence that are identical. Any terminal, C-terminal or internal extensions, deletions or insertions may be used without regard to sequence identity or homology. It is not to be construed as having any influence.

[0119] Sequence identity is the degree to which the amino acids of two polypeptides match when the two sequences are optimally aligned. The degree of identity at corresponding positions. Sequence identity can be determined using the BLAST algorithm. The parameters of the algorithm can be determined for each reference sequence. The sequences are chosen to give the maximum match between each other over the entire length of the The literature concerns the BLAST algorithm, which is often used for sequence analysis: ST ALGORITHMS:Altschul,SF,et al.,(1990 ) J.Mol.Biol. 215:403-410;Gish,W.,et al. ,(1993) Nature Genet. 3:266-272;Madden,T. .L.,et al.,(1996) Meth.Enzymol. 266:131- 141;Altschul,S.F.,et al.,(1997) Nucleic Acids Res. 25:3389-3402;Zhang,J.,et al., (1997) Genome Res. 7:649-656;Wootton,J.C .,et al.,(1993) Comput.Chem. 17:149-163; Hancock,J.M. et al.,(1994) Comput.Appl.B iosci. 10:67-70;ALIGNMENT SCORING SYSTEM S:Dayhoff,M.O.,et al.,“A model of evolut ionary change in proteins.” in Atlas of Protein Sequence and Structure,(1978) vo l.5,suppl.3. M.O.Dayhoff(ed.),pp.345-352 ,Natl.Biomed.Res.Found.,Washington,DC;Sc hwartz,R.M.,et al.,“Matrices for detecti ng distant relationships.” in Atlas of P rotein Sequence and Structure,(1978) vol .5,suppl.3.”M.O.Dayhoff(ed.),pp.353-358, Natl.Biomed.Res.Found.,Washington,DC;Alt schul,S.F.,(1991) J.Mol.Biol. 219:555-56 5;States,D.J.,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;A LIGNMENT STATISTICS:Karlin,S.,et al.,(19 90) Proc.Natl.Acad.Sci.USA 87:2264-2268; Karlin,S.,et al.,(1993) Proc.Natl.Acad.S ci.USA 90:5873-5877;Dembo,A.,et al.,(199 4) Ann.Prob. 22:2022-2039; and Altschul, S. F. “Evaluating the statistical significan ce of multiple distinct local alignments .” in Theoretical and Computational Meth ods in Genome Research(S.Suhai,ed.),(199 7) pp.1-14, Plenum, New York.

[0120] Similarly, any classification of light chains may be used in the compositions and methods herein. In particular, kappa, lambda or variants thereof can be used in the compositions and methods of the present invention. The present invention is useful in

[0121] Table 2. Exemplary PD-1 Antibody Sequences [Table 2] TIFF0007653465000003.tif114163

[0122] Table 3. Additional PD-1 antibodies and methods useful in the co-formulations, methods and uses of the invention. antigen-binding fragment [Table 3]

[0123] In some embodiments of the co-formulations of the invention, the PD-1 API (i.e., anti-PD- 1) antibody or antigen-binding fragment thereof) at a concentration of about 25 mg / mL to about 100 mg / mL In alternative embodiments, the API is present at about 10 mg / mL, about 25 mg / mL , about 50 mg / mL, about 75 mg / mL, or about 100 mg / mL.

[0124] Anti-CTLA4 antibodies and antigen-binding fragments thereof The present invention relates to a method for the preparation of a CTLA4-based pharmaceutical composition comprising administering to a subject a therapeutically effective amount of CTLA4. Binding anti-CTLA4 antibodies or antigen-binding fragments thereof (e.g., human or humanized anti-CTL The present invention provides stable biological preparations comprising the A4 antibody, as well as methods for using the preparations of the present invention. do.

[0125] The present invention also relates to (i) an anti-CTLA4 antibody or an antigen-binding fragment (e.g., a human or humanized anti-CTLA4 antibody); and (ii) a human P A stable biosynthetic antibody comprising an anti-human PD-1 antibody or an antigen-binding fragment thereof that specifically binds to PD-1. The present invention provides a pharmaceutical co-formulation comprising any anti-CTLA4 antibody or antigen-binding fragment thereof. The compositions and methods of the present invention can be used in the formulations and methods of the present invention, including those shown in Tables 4-8 and FIG. Exemplary anti-CTLA4 antibodies useful in the formulations and methods of the invention, including co-formulations: and the amino acid sequences of the antigen-binding fragments are provided.

[0126] In one embodiment of the formulation, including the co-formulation, the anti-CTLA-4 antibody is a human monoclonal antibody. The monoclonal antibody 10D1, now known as ipilimumab, was developed by Yervoy (trademark ), which is protected by U.S. Patent No. 6,984,720 and WHO Drug Information 19(4):61 (2005). In one embodiment, the anti-CTLA-4 antibody is tremelimumab, which is a CP-675,20 6, also known as U.S. Patent Application Publication No. 2012 / 263677 or PCT International As described in application publication no. WO2012 / 122444 or 2007 / 113648A2 It is an IgG2 monoclonal antibody that has been described.

[0127] In one of the formulations, including the co-formulation, the anti-CTLA-4 antibody is and a light chain comprising the amino acid sequence set forth in SEQ ID NO:85. In some embodiments, the CTLA4 antibody is a monoclonal antibody having the sequence 84 and / or an antigen-binding fragment of SEQ ID NO: 85, wherein the antigen-binding fragment The fragment specifically binds to CTLA4.

[0128] In one embodiment of the formulations of the invention, including co-formulations, the anti-CTLA-4 antibody is The anti-CTLA-4 antibody disclosed in International Patent Publication No. WO2016 / 015675A1 In one embodiment, the antibody is an anti-CTLA4 antibody or an antigen-binding fragment thereof. The antibody is a monoclonal antibody comprising the following CDRs: HCDR1 containing the amino acid sequence GFTFSDNW (SEQ ID NO:35) HCDR2 containing the amino acid sequence IRNKPYNYET (SEQ ID NO:36) HCDR3 containing the amino acid sequence TAQFAY (SEQ ID NO:37) and / or LCDR1 containing the amino acid sequence ENIYGG (SEQ ID NO:38) LCDR2 containing the amino acid sequence GAT (SEQ ID NO:39) QNVLRSPFT (SEQ ID NO: 40); QNVLSRHPG (SEQ ID NO: 41); if or QNVLSSRPG (SEQ ID NO: 42). .

[0129] In one embodiment of the formulations of the invention, including co-formulations, an anti-CTLA4 antibody or The antigen-binding fragment of comprises a variable heavy chain and a variable light chain. The variable and light chains are selected from the VH and VL sequences of 8D2 / 8D2(RE) or variants thereof. In another embodiment, the variable heavy and light chains comprise the VH and VL of 8D2H1L1. and VL sequences or variants thereof. The variable light chain comprises the VH and VL sequences of 8D2H2L2 or variants thereof. In an embodiment, the variable heavy and variable light chains are the VH and VL sequences of 8D3H3L3 or In a further embodiment, the variable heavy chain and the variable light chain are 8D 2H2l17 VH and VL sequences or variants thereof. 8D2 / 8D2(RE), 8D2H1L1, 8D2H2L2, 8D2H2L15 or The methionine at position 18 in either variant of 8D2H2L17 independently binds leucine The amino acids are substituted with amino acids selected from valin, isoleucine, valine, isoleucine and alanine. In another embodiment of the variant, 8D2 / 8D2(RE), 8D2H1L1, 8D2H The 18th amino acid sequence of either the 2L2, 8D2H2L15 or 8D2H2L17 variants Thionine is replaced by leucine.

[0130] In one embodiment of the formulation, including the co-formulation, an anti-CTLA4 antibody or its antigen-binding domain is The synthetic fragment is 8D2H2L2 or a variant thereof, and the possible 8D2H2L2 variant is The methionine at position 18 in the variable heavy chain (VH) amino acid sequence can be independently replaced by leucine, valine, The amino acid is substituted with an amino acid selected from isoleucine and alanine.

[0131] Table 4: Exemplary anti-CTLA4 antibody sequences [Table 4] TIFF0007653465000006.tif233169

[0132] In another embodiment of the formulations of the invention, including co-formulations, an anti-CTLA4 antibody or The antigen-binding fragment comprises the VH and VL chain sequences of 8D2 / 8D2(RE) variant 1. In a further embodiment, the anti-CTLA4 antibody or antigen-binding fragment thereof is 8D2H. 1L1 comprises the VH and VL chain sequences of variant 1. In another embodiment, the anti-CTLA 4 The antibody or antigen-binding fragment thereof has the VH and VL chain sequences of 8D2H2L2 variant 1. In a further embodiment, the anti-CTLA4 antibody or antigen-binding fragment thereof comprises a sequence comprising: In another embodiment, the VH and VL chain sequences of a variant of 8D2H2L15 are included. The anti-CTLA4 antibody or antigen-binding fragment thereof may comprise the VH and VL chain sequences or 8D2H2 Includes l17 variants.

[0133] In one embodiment of the formulations of the invention, including co-formulations, the anti-CTLA4 antibody is PCT International Application No. PCT / CN2016 / 09635 filed on August 23, 2016 7 or any of the anti-CTLA4 antibodies or antigen-binding fragments thereof described in In one embodiment, the anti-CTLA4 antibody comprises the following VH and VL chain amino sequences: The murine antibody 4G10, which contains 4G1A, as well as a humanized version of this antibody.

[0134] Table 5: Mouse anti-CTLA4 antibodies [Table 5]

[0135] In one embodiment of the formulations of the invention, including co-formulations, the anti-CTLA4 antibody is A monoclonal antibody containing the CDRs: GYSFTGYT (SEQ ID NO: 57) or GYTX 1 N (SEQ ID NO:58) X 1 is M, V, L, I, G, A, S, T) DR1; 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 QKF X 4 G (SEQ ID NO:60) (wherein X 1 is N, D;X 2 is T, D, E, G or A Yes;X 3 is A or N;X 4 is Q or M) HCDR2 containing; Select from LDYRSY (SEQ ID NO: 61) or ARLDYRSY (SEQ ID NO: 62) HCDR3 comprising an amino acid sequence to be and / or TGAVTTSNF (SEQ ID NO: 63) or GSSTGAVTTSNFX 1 N(distribution Column number 64) (where X 1 is P or A) DR1; 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; X 2 is S or P) an LCDR2 comprising an amino acid sequence of choice; ALX 1 YSNHX 2 (SEQ ID NO:66) (wherein X 1 is W or M if X is any amino acid other than C; 2 is W or any other than M or C any amino acid); or ALX 1 YSNHX 2 V (SEQ ID NO:67) (wherein X 1 is W or any amino acid other than M or C, and X 2 is W or or any amino acid other than M or C) LCDR3.

[0136] In another embodiment, the humanized VH sequence of the 4G10 antibody comprises any of the following VH sequences: include: Table 6: Exemplary anti-CTLA4 antibody sequences [Table 6]

[0137] In another embodiment of the formulation of the invention, including a co-formulation, the humanized VL sequence of the 4G10 antibody is The string contains one of the following VL sequences: Table 7: Exemplary anti-CTLA4 antibody sequences [Table 7]

[0138] In some embodiments, the anti-CTLA4 antibody comprises the VH and VH of 4G10H1L1. In another embodiment, the anti-CTL The A4 antibody contains variable heavy and light chains corresponding to the VH and VL sequences of 4G10H3L3. In one embodiment, the anti-CTLA4 antibody comprises the VH and VL of 4G10H3L3. In another embodiment, the antibody comprises a variable heavy and variable light chain sequence corresponding to the VL and VL sequences. The CTLA4 antibody has variable heavy and variable nucleotide sequences corresponding to the VH and VL sequences of 4G10H5L3. Contains a variant light chain sequence.

[0139] Table 8: Exemplary anti-CTLA4 antibody sequences [Table 8]

[0140] [Table 9]

[0141] In another embodiment of the formulations of the invention, including co-formulations, the anti-CTLA-4 antibody is Limumab, tremelimumab, or 8D2 / 8D2(RE) or 8D2 / 8D2(R E) Variant 1, 8D2H1L1 or 8D2H1L1 Variant 1, 8D2H2L 2 or 8D2H2L2 variant 1, 8D3H3L3, 8D2H2L15 or 8D2H2L15 variant 1, 8D2H2L17 or 8D2H2L17 variant 1, 4G10H1L1 or its variants, 4G10H3L3 or its variants 4G10H3L3 or its variants and 4G10H5L3 or its variants The antibody cross-competes with any of the above antibodies, including the agonists, for binding to human CTLA-4. or an antibody or an antibody that binds to the same epitope region of human CTLA-4 as the above. It is a prototypical binding fragment.

[0142] formulation In some embodiments of the invention, the formulations described herein are capable of preventing antibody aggregates (high molecular weight antibodies). Minimize the formation of molecular weight species and particulates, high and low molecular weight species, and reduce methionine residues. Oxidation of the group is minimized to ensure that the antibody retains biological activity over time.

[0143] In one embodiment, the present invention provides various anti-CTLA4 antibodies or antigen-binding fragments thereof. For example, the present invention includes formulations comprising: (i) an anti-CTLA4 antibody or antigen-binding fragment thereof; (ii) a buffer (e.g., L-histidine or acetate); (iii) a non-reducing sugar (e.g., (iv) non-ionic surfactants (e.g., polysorbate 80); and and (v) an antioxidant (e.g., L-methionine). In one embodiment, the formulation further comprises an anti-PD-1 antibody. In one embodiment, the formulation further comprises a chelator. In one embodiment, the chelator is diethylenetriaminepentaacetic acid (DTP A).

[0144] In one embodiment, the present invention also provides an anti-CTLA4 antibody or antigen-binding fragment thereof and and various co-formulations of an anti-human PD-1 antibody or antigen-binding fragment thereof. In one embodiment, the present invention provides: (i) an anti-CTLA4 antibody or an antigen-binding fragment thereof; (iii) a buffer (e.g., L-His; thiamine or acetate), (iv) non-reducing sugars (e.g., sucrose), (v) non-ionic interfaces (vi) an antioxidant (e.g., L-methionine In one embodiment, the formulation includes a chelator (e.g., DTPA). ).

[0145] The pharmaceutical formulations described herein may include a buffer. The term "buffer" means , an agent that maintains the solution pH of the liquid formulations described herein within an acceptable range, or In the case of the lyophilized formulations described herein, the permissible Included are agents that provide an acceptable solution pH.

[0146] Buffers that are useful in the pharmaceutical formulations and methods of the present invention include succinate ( Sodium succinate or potassium succinate), L-histidine, phosphate (sodium phosphate (Tris(hydroxymethyl)aminomethane), Diethanolamine, citrate (sodium citrate), acetate (sodium acetate) In one embodiment of the invention, the buffer is present in the formulation at about 1 to 20 mM (1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 In a specific embodiment of the invention, the bat In another embodiment, the buffer is L-hisitidine. It is a styrene buffer.

[0147] In one embodiment, the buffer has a pH in the range of about 4.5 to about 6.5. In another embodiment, the pH is in the range of about 5.0 to 6.0. In one embodiment, the pH range is about 5.3 to 5.8. In another embodiment, the pH range is about 5. In arriving at an exemplary formulation, histidine and Acetate buffers were investigated for suitability. The pH range was between 5.5 and 6.0. When a range is recited, such as "pH," the range is intended to include the recited value. For example, the range of about 5.0 to about 6.0 is 5.0, 5.1, 5.2, 5.3, 5.4, Includes 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0. For lyophilized formulations Unless otherwise indicated, pH refers to the pH after reconstitution. pH is typically measured at 25° C. and standard As used herein, pH is measured using a standard glass bulb pH meter. A solution containing "histidine buffer of pH X" is a solution containing a histidine buffer at pH X. The term "anti-CTL" refers to a solution containing the anti-CTL antigen, i.e., pH is intended to refer to the pH of the solution. Several implementations of co-formulations containing higher concentrations of anti-human PD-1 antibodies compared to the A4 antibody In this form, the pH of the co-formulation is about 5.0.

[0148] In one embodiment of the invention, anti-CTLA4 formulations and anti-CTLA4 and anti-human The co-formulation of PD-1 includes a non-reducing sugar. As used herein, "non-reducing sugar" refers to Containing or being converted to contain a free aldehyde group or a free ketone group These sugars cannot act as reducing agents because they cannot form a reducing agent. Examples of non-reducing sugars are Examples of sugars include, but are not limited to, disaccharides such as sucrose and trehalose. In certain embodiments, the non-reducing sugar is about 1-10% (w / v) (1, 2, 3, 4, 5, 6, 7 In another embodiment, non-reducing sugars are present in an amount of about 6% to about 8%, 9%, or 10%. In a further embodiment, the non- In a further embodiment, the reducing sugar is present in an amount of about 6% (w / v). % (w / v). In a further embodiment, the non-reducing sugars are present in an amount of about 8% (w / v). In one embodiment, the non-reducing sugar is sucrose, trehalose or is raffinose. In another embodiment, the non-reducing sugar is sucrose. In an embodiment, the sucrose is present at 6-8% w / v. The sucrose is present at 6% (w / v). In one embodiment, the sucrose is present at 7% ( In one embodiment, sucrose is present at 8% (w / v). .

[0149] The formulations described herein also include a surfactant. Surfactants are surface active agents that are amphiphilic in nature. To provide a soluble form of the compound, to reduce and / or prevent aggregation, or to improve the solubility of the compound, e.g. by purification, filtration, Prevents damage to proteins during processing conditions such as lyophilization, transportation, storage and delivery In one embodiment of the present invention, In the present invention, surfactants may be useful to provide additional stability to the active ingredient(s). .

[0150] Non-ionic surfactants that may be useful in the formulations and co-formulations described herein Agents include, but are not limited to, polyoxyethylene sorbitan fatty acid esters. (Polysorbate, sold under the trade name Tween® (Unique (Ma Americas LLC, Wilmington, DE), which is a polysorbate Polysorbate-20 (Polyoxyethylene sorbitan monolaurate), Polysorbate-40 ( Polyoxyethylene sorbitan monopalmitate), Polysorbate-60 (Polyoxy Ethylene sorbitan monostearate) and polysorbate-80 (polyoxyethylene sorbitan monooleate); polyoxyethylene alkyl ethers, e.g. Brij 58 (Uniquema Americas LLC, Wilm ington, DE) and Brij® 35; poloxamers (e.g., poloxamers mer 188; Triton® X-100 (Union Carbide C orp., Houston, TX) and Triton® X-114; NP4 0;Span 20, Span 40, Span 60, Span 65, Span 80 and Span 85; Copolymers of ethylene and propylene glycol (e.g., nonionic surfactants such as pl uronic® series, such as pluronic® F68, pl uronic(registered trademark) 10R5, pluronic(registered trademark) F108, plur onic F127, pluronic F38, pluroni cL44, pluronic L62, etc. (BASF Corp ., Ludwigshafen, Germany); and sodium dodecyl sulfate ( In one embodiment, the non-ionic surfactant comprises polysorbate 8. 0 or polysorbate 20. In one embodiment, the nonionic surfactant is In another embodiment, the non-ionic surfactant is polysorbate 20. The rating is 80.

[0151] The amount of nonionic surfactant included in the formulation of the present invention may be varied to provide the desired function. an amount sufficient to administer the active pharmaceutical ingredient (i.e., an anti-CTLA4 antibody or its antigen binding agent) to the subject or an anti-CTLA4 antibody or an antigen-binding fragment thereof and an anti-human PD-1 antibody The minimum amount required to stabilize the antibody or its antigen-binding fragment in the formulation. All percentages listed for Polysorbate 80 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, the surfactant is present in the formulation at 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 0.0 35%, or from about 0.02% to about 0.03%. In an alternative embodiment, the surfactant is present in an amount of about 0.02%. , about 0.01%, about 0.015%, about 0.025%, about 0.03%, about 0.035% or is present in an amount of about 0.04%.

[0152] In an exemplary embodiment of the invention, the surfactant is polysorbate 20 or polysorbate 21. and Lubet 80. In the present invention, the surfactant is polysorbate 80.

[0153] In a specific embodiment, the formulations of the invention, including co-formulations, contain from about 0.01% to about 0 In a further embodiment, the composition described herein comprises 0.04% w / v polysorbate 80. The formulations described contain polysorbate 80 at 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 yet another embodiment, the amount of polysorbate 80 is about 0.025% w / v. In an embodiment, the amount of polysorbate 80 is about 0.03% w / v. In one embodiment, the amount of polysorbate 80 is about 0.035% w / v. In a further embodiment, the amount of polysorbate 80 is about 0.04% w / v. In certain embodiments, the amount of polysorbate 80 is about 0.045% w / v. The formulation of the present invention contains about 0.02% w / v polysorbate 80.

[0154] The formulations of the present invention, including co-formulations, also contain methionine or a pharma- ceutically acceptable salt thereof. In one embodiment, the methionine is L-methionine. In this case, methionine may be a pharma- ceutically acceptable salt of L-methionine, e.g., methionine HCl. In one embodiment, methionine is present in the formulation at about 1 to 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, methionine is present at a concentration of about 5 mM to In another embodiment, the methionine is present in an amount of about 10 mM (5, 6, 7, 8, 9, and 10 mM). Thionine is present at approximately 10 mM.

[0155] The formulations and co-formulations described herein may further comprise a chelating agent. In one embodiment, the chelating agent is present in the formulation at about 1-50 μM (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 μM). In an embodiment, the chelating agent is DTPA. In another embodiment, the chelating agent is In some additional embodiments, DTPA is present in the following amounts: 1, 5, 1 Either 0, 15, 20, 25, 30, 35, 40, 45 or 50 μM is an antioxidant that can be present in any of the formulations described in

[0156] Freeze-dried composition Lyophilized formulations of therapeutic proteins offer several advantages. Generally, these formulations offer better chemical stability and therefore increased half-life than solution formulations. Lyophilized formulations may also be formulated in different concentrations depending on clinical factors, such as route of administration or dosage. For example, lyophilized formulations can be reconstituted at higher concentrations (i.e., 100% ethanol) if necessary for subcutaneous administration. It can be reconstituted in small doses (e.g., small amounts) or in low concentrations if administered intravenously. Higher concentrations are also possible, particularly The injection volume must be minimized, especially when high dosages are required for a given subject. One such lyophilized antibody formulation is available in the United States. 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. Pat. No. 7,247,707. No. 6,399,623, which is incorporated herein by reference in its entirety.

[0157] Typically, the lyophilized formulation is a drug product (DP, in an exemplary embodiment a humanized anti-PD- 1) antibody pembrolizumab or its antigen-binding fragment) at high concentrations. The DP is prepared in anticipation of reconstitution with a small amount of water. For dilution, subsequent dilution with water or isotonic buffer can be easily used thereafter. Typically, excipients are included in the lyophilized formulations of the invention, e.g., high D for subcutaneous administration. P concentration that results in a formulation that is approximately isotonic upon reconstitution. Reconstitution with a larger amount of water to give a given concentration necessarily reduces the tonicity of the reconstituted solution. However, such reduction is of little importance in non-subcutaneous administration, e.g., intravenous administration. If isotonicity at lower DP concentrations is desired, the lyophilized powder can be mixed with a standard small amount of water. It can be reconstituted and then further diluted with an isotonic diluent such as 0.9% sodium chloride. .

[0158] The lyophilized formulation of the present invention is a pre-lyophilization solution. Lyophilization of the solution (freeze-drying It is formed by freeze-drying. This is achieved by freezing the formulation followed by sublimation of water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic or collapse temperature of the formulation. The shelf temperature for drying is set at a suitable pressure, typically ranging from about 50 to 250 mTorr. The drying temperature ranges from about -30 to 25°C (the product remains frozen during the primary drying process). The size and type of the preparation and container (e.g., glass vial) that will hold the sample The type, as well as the volume of liquid, will determine the time required for drying, which can range from a few hours to This can last for several days (e.g., 40-60 hours). The secondary drying stage depends mainly on the type of container and Depending on the size and size of the protein employed, the reaction may be carried out at temperatures between about 0 and 40°C. The secondary drying time is dictated by the desired residual moisture level in the product and is typically less than 10 minutes. Typically, the moisture content of the lyophilized formulation is less than about 5%, preferably less than about 10%. The pressure can be the same as that employed during the primary drying step. Freeze-drying conditions vary depending on formulation and vial size. There are times when I do.

[0159] In some instances, the reconstitution of proteins is performed to avoid a translocation step. It may be desirable to lyophilize the protein formulation in a container. The containers can be, for example, 3, 5, 10, 20, 50 or 100 cc vials.

[0160] The lyophilized formulations of the present invention are reconstituted prior to administration. , at concentrations of 25, 30, 40, 50, 60, 75, 80, 90 or 100 mg / mL , or higher concentrations, e.g., 150 mg / mL, 200 mg / mL, 250 mg / mL Alternatively, it may be reconstituted at 300 mg / mL, up to about 500 mg / mL. In one embodiment, the protein concentration after reconstitution is about 10 to 300 mg / ml. In an embodiment, the protein concentration after reconstitution is about 20 to 250 mg / ml. In one embodiment, the protein concentration after reconstitution is about 150-250 mg / ml. In one embodiment, the protein concentration after reconstitution is about 180-220 mg / ml. In one embodiment, the protein concentration after reconstitution is about 50 to 150 mg / mL. In one embodiment, the protein concentration after reconstitution is about 100 mg / ml. In one embodiment, the protein concentration after reconstitution is about 75 mg / ml. In one embodiment, the protein concentration after reconstitution is about 50 mg / ml. In one embodiment, the protein concentration after reconstitution is about 25 mg / ml. A low protein concentration is particularly useful when the reconstituted formulation is intended for subcutaneous delivery. However, for other routes of administration, such as intravenous administration, lower protein concentrations may be desirable. In some cases, the concentration may be increased (for example, about 5 to 50 mg / mL).

[0161] Reconstitution is generally carried out at a temperature of about 25° C. to ensure complete hydration, but may be carried out at any desired temperature. Other temperatures may be used if desired. The time required for reconstitution will vary depending on, for example, the type of diluent, Dependent on the amount of excipient(s) and protein. Exemplary diluents include sterile water, staphylococcus aureus for injection, and nasturtium diluent. Bacterial water with fluid (BWFI), pH buffer solution (e.g. phosphate buffered saline), sterile saline solution , Ringer's solution or glucose solution.

[0162] liquid composition Liquid antibody formulations are produced by purifying drug substance in liquid form (e.g. Take an anti-humanized PD-1 (e.g., pembrolizumab in an aqueous formulation) and mix it with the desired In this embodiment, the buffer can be exchanged into a There is no lyophilization step. The drug substance in the final buffer is concentrated to the desired concentration. Excipients such as sucrose and polysorbate 80 are added to the drug substance, which Dilute to final protein concentration with appropriate buffer. Final formulated drug The material is filtered using a 0.22 μm filter and placed in the final container (e.g. glass vial). It is filled inside.

[0163] III.How to use The present invention also provides a method of treating cancer in a subject, comprising administering to a subject an effective amount of a formulation of the present invention. i.e., comprising administering to a subject any of the formulations described herein. In some specific embodiments of the method, the formulation is administered intravenously. In other embodiments, the formulation is administered to the subject via subcutaneous administration. In one embodiment, the present invention relates to a method for administering any of the formulations of the present invention to a patient. The present invention also includes a method of treating cancer in a human patient, comprising:

[0164] In any of the methods of the present invention, the cancer is 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 gland cancer Salivary cancer, prostate cancer (e.g. hormone-refractory prostate cancer) ), pancreatic cancer, colon cancer, esophageal cancer, liver cancer, thyroid cancer, glioblastoma, glioma and and other neoplastic malignancies.

[0165] In some embodiments, the lung cancer is non-small cell lung cancer.

[0166] In an alternative embodiment, the lung cancer is small cell lung cancer.

[0167] In some embodiments, the lymphoma is Hodgkin's lymphoma.

[0168] In other embodiments, the lymphoma is non-Hodgkin's lymphoma. In this case, the lymphoma is mediastinal large B-cell lymphoma.

[0169] In some embodiments, the breast cancer is triple-negative breast cancer.

[0170] In a further embodiment, the breast cancer is ER+ / HER2- breast cancer.

[0171] In some embodiments, the bladder cancer is urothelial cancer.

[0172] In some embodiments, the head and neck cancer is nasopharyngeal carcinoma. In one embodiment, the cancer is thyroid cancer. In another embodiment, the cancer is salivary gland cancer. In other embodiments, the cancer is squamous cell carcinoma of the head and neck.

[0173] In one embodiment, the present invention provides a method for the treatment of a human cancer comprising administering to a patient a formulation of the present invention. The present invention includes a method for treating metastatic non-small cell lung cancer (NSCLC) in a patient. In terms of morphology, patients had high PD-L1 expression (tumor proportion score (TPS) ≥ 50%) In another embodiment, the patient has a tumor and has not been previously treated with platinum-containing chemotherapy. had tumors that expressed PD-L1 (TPS ≥ 1%) and had been previously treated with platinum-containing chemotherapy In yet other embodiments, the patient expresses PD-L1 (TPS > 1%). ) tumors that have not been previously treated with platinum-containing chemotherapy. In most cases, patients had disease progression during or after platinum-containing chemotherapy. In an embodiment, PD-L1 TPS is determined by an FDA-approved test. In certain embodiments, the patient's tumor does not have EGFR or ALK genomic abnormalities. In certain embodiments, the patient's tumor has an EGFR or ALK genomic abnormality, EGFR or ALK abnormality(s) prior to receiving anti-PD-1 antibody or its antigen-binding fragment The disease had progressed at the time of or after treatment for

[0174] In some embodiments, the cancer is characterized by high levels of microsatellite instability (MS The patient has metastatic colorectal cancer with IH.

[0175] In some embodiments, the cancer is characterized by high levels of microsatellite instability (MS The patient has metastatic colorectal cancer with IH.

[0176] In some embodiments, the cancer is characterized by high levels of microsatellite instability (MS It is a solid tumor with IH.

[0177] In some embodiments, the cancer is characterized by a high mutational burden. It is a solid tumor that has a den.

[0178] In some embodiments, the cancer is melanoma, non-small cell lung cancer, recurrent or refractory cancer. Classical Hodgkin's lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal cancer, gastric cancer, and and hepatocellular carcinoma.

[0179] In other embodiments of the above methods of treatment, the cancer is a hematological malignancy. In this study, hematological malignancies included acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AM), and L), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large cell type DLBCL, EBV-positive DLBCL, primary mediastinal large B-cell lymphoma lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma Myeloid leukemia (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia -1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL) HL) or small lymphocytic lymphoma (SLL).

[0180] Disease-free survival and overall survival in relation to the presence of tumor-infiltrating lymphocytes in biopsy or surgical material Malignancies that demonstrate improvement in duration include melanoma, colorectal cancer, liver cancer, and kidney cancer. Cancers such as gastric / esophageal cancer, breast cancer, pancreatic cancer and ovarian cancer can be treated with the methods and methods described herein. These cancer subtypes are subject to immune regulation by T lymphocytes. In addition, the antibodies described herein can be used to inhibit proliferation. Also included are refractory or recurrent malignancies which may be treated with chemotherapy.

[0181] Additional cancers that may benefit from treatment with the formulations described herein include For example, Kaposi's sarcoma, liver cancer, nasopharyngeal cancer, lymphoma, cervical cancer, vulvar cancer, anal cancer, Viruses known to be implicated in the causation of rectal, penile and oral cancers Persistent infections, such as human immunodeficiency virus, hepatitis A, B and C viruses, Eps including those associated with persistent infections such as Tyne and Barr virus and human papillomavirus. nothing.

[0182] The formulations can also be used to prevent or treat infections and infectious diseases. Therefore, the present invention provides a method for the treatment of a mammal comprising administering to the subject an effective amount of a formulation of the present invention. Methods for treating a chronic infection in a subject are provided. In certain embodiments, the formulation is administered to the subject via intravenous administration. In this case, the formulation is administered to the subject by subcutaneous administration.

[0183] These agents act alone to stimulate immune responses to pathogens, toxins, and self-antigens. or in combination with a vaccine. including but not limited to human immunodeficiency virus, hepatitis A, B and C viruses , Epstein-Barr virus, human cytomegalovirus, human papillomavirus, and Stimulating an immune response to viruses that are infectious to humans, including rabies and herpes viruses. The antagonist anti-PD-1 antibodies or antibody fragments can be used to target bacteria or It can be used to stimulate immune responses to infections with fungal parasites and other pathogens. Hepatitis B and C viruses, as well as HIV infections, are chronic viral infections. It is among those considered infectious diseases.

[0184] The formulations of the invention may be administered to a patient in combination with one or more "additional therapeutic agents." Targeted therapeutic agents include biotherapeutics (including but not limited to VEGF, EGFR, Her2 / n eu, VEGF receptor, other growth factor receptors, CD20, CD40, CD-40L, OX -40, 4-1BB and ICOS), immunogenic agents (e.g., attenuated Antigen-presenting cells, e.g., dendritic cells, that have been pulsed with a tumor-derived antigen, tumor-derived antigen, or nucleic acid. inflammatory cells, immune stimulatory cytokines (e.g., IL-2, IFNα2, GM-CSF), and and immunostimulatory cytokines such as, but not limited to, GM-CSF. The target cell may be a cell transfected with a gene that modulates the expression of the target gene.

[0185] As noted above, in some embodiments of the methods of the present invention, the methods further comprise administering to the patient an additional therapeutic agent. In certain embodiments, 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, or an anti-CD27 antibody or an antigen-binding fragment thereof. In an embodiment, the additional therapeutic agent is a Newcastle Disease Virus vector expressing IL-12. In a further embodiment, the additional therapeutic agent is dinaciclib. In further embodiments, the additional therapeutic agent is a STING agonist.

[0186] Suitable routes of administration include, for example, intramuscular, subcutaneous, as well as intrathecal, direct intracerebroventricular, intravenous, and intravenous. The drug may be administered by a variety of conventional methods, including intravenous, intraperitoneal, parenteral, and intravenous delivery. It can be administered parenterally, intraarterially or intravenously. The volume of solution must be limited. Modes of administration that are not available (e.g., subcutaneous administration) are lyophilized formulations to allow reconstitution at high concentrations. is required.

[0187] The choice of dosage of the additional therapeutic agent will depend on the serum or tissue turnover rate of the agent, the level of symptoms, , the immunogenicity of the product, and the accessibility of the target cells, tissues or organs in the individual being treated. The dosage of the additional therapeutic agent will depend on several factors, including the acceptable level of side effects. Therefore, the amount of each additional therapeutic agent (e.g., a biotherapeutic agent or The dosage and frequency of administration of the therapeutic agent (or chemotherapy agent) will depend on the particular therapeutic agent, the severity of the cancer being treated, and The choice of appropriate doses of antibodies, cytokines and small molecules is dependent in part on the clinical trial and patient characteristics. Guidance is available in the literature. For example, Wawrzynczak (1996) A ntibody Therapy,Bios Scientific Pub.Ltd, Oxfordshire,UK;Kresina(ed.)(1991) Monocl onal Antibodies,Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach(ed.)(1993 ) Monoclonal Antibodies and Peptide Ther apy 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;B eniaminovitz et al.(2000) New Engl.J.Med 342:613-619;Ghosh et al.(2003) New Eng. lJMed. 348:24-32;Lipsky et al. (2000) N ew Engl.J.Med. 343:1594-1602;Physicians' Desk Reference 2003(Physicians' Desk Re reference,57th Ed);Medical Economics Compa ny;ISBN:1563634457;57th edition(November Determination of an appropriate dosing regimen is up to the clinician, e.g., known or believed in the art to affect treatment or using parameters or factors predicted to affect treatment, which For example, the patient's medical history (e.g., prior treatments), the type and stage of the cancer being treated, and In particular, the present invention relies on biomarkers of response to one or more of the therapeutic agents in the combination therapy.

[0188] To facilitate the selection of a pharma- ceutically acceptable carrier or excipient for an additional therapeutic agent Various references are available, e.g., Remington's Pharmac eutical Sciences and US Pharmacopeia:N ational Formulary,Mack Publishing Compan y, Easton, PA (1984); Hardman et al. (2001) G odman and Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill,New Yo rk,NY;Gennaro(2000) Remington:The Scienc. e and Practice of Pharmacy, Lippincott, Wi. lliams,and Wilkins,New York,NY;Avis et a l.(eds.)(1993) Pharmaceutical Dosage For ms:Parental Medications,Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmace utical Dosage Forms:Tablets,Marcel Dekke r,NY;Lieberman et al.(eds.)(1990) Pharma ceutical Dosage Forms:Disperse Systems,M arcel Dekker,NY;Weiner and Kotkoskie(200 0) Excipient Toxicity and Safety,Marcel See Dekker, Inc., New York, NY.

[0189] The pharmaceutical antibody formulations may be administered by continuous infusion or, for example, daily, 1 to 7 times per week for a week. Doses can be administered at intervals of 2 weeks, 3 weeks, monthly, bimonthly, etc. The dosing protocol should determine the maximum dosage or frequency of dosing that avoids significant undesirable side effects. The total weekly dosage is generally at least 0.05 μg / kg, 0.2 μg / kg, 0.5μg / kg, 1μg / kg, 10μg / kg, 100μg / kg, 0 .2mg / kg, 1.0mg / kg, 2.0mg / kg, 10mg / kg, 25mg / k g, 50 mg / kg body weight or more. For example, Yang et al. 3) 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.Psy ch. 67:451-456;Portielji et al.(20003) C See Ancer Immunol. Immunother. 52:133-144. The desired dose of a small molecule therapeutic, e.g., a peptidomimetic, natural product, or organic compound, is , approximately the same for an antibody or polypeptide on a moles / kg basis.

[0190] Embodiments of the invention also include (a) therapeutic (e.g., treatment of the human body); (b) pharmaceutical; ) inducing or enhancing an antitumor immune response; (d) determining the number of one or more tumor markers in a patient; (e) stopping or slowing the growth of tumors or blood cancers; f) halting or slowing the progression of CTLA4 or PD-1 associated disease; (g) stopping or slowing progression of cancer; (h) CTLA4 or PD-1-related (i) inhibiting the proliferation or survival of tumor cells; (j) inhibiting one or more cancers (k) removing or reducing the size of a cytoplasmic lesion or tumor; (l) reducing the progression, onset or severity of A4 or PD-1 associated disease; The severity or duration of clinical symptoms of CTLA4- or PD-1-related disease such as (m) reducing the expected survival of a patient compared to the expected survival of a similar untreated patient (n) to delay the onset of a cancerous condition or other CTLA4- or PD-1-related disease (o) inducing a complete or partial remission; (o) treating cancer; or (p) treating a chronic infection. (i) for use in, or (ii) for use as a medicament or composition for, the treatment of or (iii) one or more of the compounds described herein for use in the preparation of a medicament. This includes biological preparations of the above.

[0191] General method Standard methods in molecular biology are described by Sambrook, Fritsch and Ma niatis (1982&1989 2 nd Edition, 2001 3 rd Ed ition) Molecular Cloning,A Laboratory Ma nual,Cold Spring Harbor Laboratory Press ,Cold Spring Harbor,NY;Sambrook and Russ ell(2001) Molecular Cloning, 3 rd ed., Cold Spring Harbor Laboratory Press,Cold Spr. ing Harbor,NY;Wu(1993) Recombinant DNA,V ol.217, Academic Press, San Diego, CA) Standard methods are also described by Ausbel, et al. (2001) Curren t Protocols in Molecular Biology,Vols.1- 4. John Wiley and Sons, Inc. New York, NY These include bacterial cells and DNA mutagenesis (Vol. 1), mammalian cells and and cloning in yeast (Vol. 2), glycoconjugate and protein expression (Vol. .3) and Bioinformatics (Vol.4).

[0192] Protein analysis including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization The purification method was described (Coligan, et al. (2000) Curr ent Protocols in Protein Science,Vol.1,J ohn Wiley and Sons, Inc., New York). chemical analysis, chemical Chemical modifications, post-translational modifications and production of fusion proteins, protein glycosylation have been described. (e.g., Coligan, et al. (2000) Current Pro tocols in Protein Science,Vol.2,John Wil ey and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Bi ology, 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) BioDirectory, Piscataway,N .J., pp.384-391). Polyclonal and monoclonal antibodies Production, purification and fragmentation of clonal antibodies have been described (Coligan, et al. (2001) Current Protocols in Immunology,V ol.1, John Wiley and Sons, Inc., New York; arlow and Lane(1999) Using Antibodies,Co ld Spring Harbor Laboratory Press,Cold S Spring Harbor, NY; Harlow and Lane, NY. Standard techniques for characterization of receptor interactions are available (e.g., Coliga, et al. (2001) Current Protocols in Immu Nology, Vol. 4, John Wiley, Inc., New York. (I want to be).

[0193] Monoclonal, polyclonal and humanized antibodies can be prepared. (See, for example, Sheperd and Dean (eds.) (2000) Monocl onal Antibodies,Oxford Univ.Press,New Yo rk,NY;Kontermann and Dubel(eds.) (2001) Antibody Engineering,Springer-Verlag,New York;Harlow and Lane(1988) Antibodies A Laboratory Manual,Cold Spring Harbor La Boratory Press, Cold Spring Harbor, NY,pp. 139-243;Carpenter,et al.(2000) J.Immunol 165:6205;He,et al.(1998) J.Immunol. 16 0:1029;Tang et al. (1999) J.Biol.Chem. 27 4:27371-27378;Baca et al.(1997) J.Biol.C hem. 272:10678-10684;Chothia et al.(1989 ) Nature 342:877-883;Foote and Winter(19 92) J.Mol.Biol. 224:487-499;USPat.No.6 ,329,511).

[0194] An alternative method for humanization is the use of human antibody libraries displayed on phage or transfected human antibodies. The first step is to use a human antibody library in a transgenic mouse (Vaugha n 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) I mmunol.Today 21:371-377;Barbas et al.(20 01) Phage Display:A Laboratory Manual,Co ld Spring Harbor Laboratory Press,Cold S pring Harbor, New York; Kay et al. (1996) P Hage Display of Peptides and Proteins:A Laboratory Manual,Academic Press,San Die go,CA;de Bruin et al.(1999) Nature Biote chnol. 17:397-399).

[0195] Purification of the antigen is not necessary for antibody production. Immunizing an animal with cells bearing the desired antigen is the same as Spleen cells are then isolated from the immunized animal and fused with a myeloma cell line. Hybridomas can be produced by the above method (e.g., Meyaard et al. (1997) Immunity 7:283-290;Wright et al. 2000) Immunity 13:233-242; Preston et al. l.;Kaithamana et al.(1999) J.Immunol. 16 3:5157-5164).

[0196] Antibodies can be combined with, for example, small molecule drugs, enzymes, liposomes, and polyethylene glycol (PEG). The antibodies can be used as therapeutic agents, diagnostic agents, kits, or for other purposes. These are useful for binding, for example, to dyes, radioisotopes, enzymes, or metals, such as colloidal gold. (See, e.g., Le Doussal et al. (1991) J. Immunol. 146:169-175;Gibellini et al.(19 98) J.Immunol. 160:3891-3898;Hsing and B ishop(1999) J.Immunol. 162:2804-2811;Eve rts et al. (2002) J. Immunol. 168:883-889 Please refer to.

[0197] Flow cytometry methods, including fluorescence activated cell sorting (FACS), are available. (See, for example, Owens, et al. (1994) Flow Cytometry P principles for Clinical Laboratory Practice ce, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2 nd ed.;Wiley-Liss ,Hoboken,NJ;Shapiro(2003) Practical Flow Cytometry,John Wiley and Sons,Hoboken,N (See, for example, J. Nucleic acid primers and probes for use as diagnostic agents. Fluorescent reagents suitable for modification of nucleic acids, polypeptides, and antibodies, including antibodies, are available. (Molecular Probesy(2003) Catalog,Mole cular Probes, Inc., Eugene, OR; Sigma-Aldric h(2003) Catalog, St. Louis, MO).

[0198] Standard methods for immune system histology have been described (e.g., Muller-Harmel et al. ink(ed.) (1986) Human Thymus: Histopathol ogy and Pathology,Springer Verlag,New Yo rk, NY; Hiatt, et al. (2000) Color Atlas of Histology,Lippincott,Williams,and Wilkin s,Phila,PA;Louis,et al.(2002) Basic Hist ology:Text and Atlas,McGraw-Hill,New York (See, for example, antigenic fragments, leader sequences, protein fold changes, etc.) Software for determining binding, functional domains, glycosylation sites and sequence alignments. Software packages and databases are available (e.g., GenBank, vector NTI (registered trademark) Suite (Informax, Inc., Beth esda,MD);GCG Wisconsin Package(Accelrys, Inc., San Diego, CA); DeCypher (registered trademark) (TimeLo gic Corp.,Crystal Bay,Nevada);Menne,et a l.(2000) Bioinformatics 16:741-742;Menne ,et al.(2000) Bioinformatics Application s Note 16:741-742;Wren, et al. (2002) Comp ut.Methods Programs Biomed. 68:177-181;v on Heijne(1983) Eur.J.Biochem. 133:17-21 ;von Heijne(1986) Nucleic Acids Res. 14: (See 4683-4690.)

[0199] Analysis method A suitable analytical method for assessing product stability is size exclusion chromatography. (SEC), Dynamic Light Scattering (DLS), Differential Scanning Calorimetry (DSC), iso-asp Quantitation, titer, UV at 340 nm, UV spectroscopy and FTIR. SEC (J. Pharm.Scien.,83:1645-1650,(1994);Pharm.R es.,11:485(1994);J.Pharm.Bio.Anal.,15:19 28(1997);J.Pharm.Bio.Anal.,14:1133-1140( 1986) measured the percent monomer in the product and provided information on the amount of soluble aggregates. DSC (Pharm.Res.,15:200(1998);Pharm.Res. ,9:109(1982)) provides information on protein denaturation temperatures and glass transition temperatures. DLS (American Lab., November (1991)) is a method to measure the average spread of The diffusion coefficient is measured and gives information on the amount of soluble and insoluble aggregates. UV measures the scattered light intensity at 340 nm and determines the amount of soluble and insoluble aggregates. UV spectroscopy measures the absorbance at 278 nm and gives information about protein concentration. Provides information. FTIR (Eur. J. Pharm. Biopharm., 45:231 (1998);Pharm.Res.,12:1250(1995);J.Pharm. Scien.,85:1290(1996);J.Pharm.Scien.,87:1 069 (1998)) measured the IR spectrum in the amide 1 region and determined the protein 2 Gives information on the secondary structure.

[0200] The iso-asp content in the sample was determined using Isoquant Isoaspartate De The assay is performed using the Detection System (Promega). To specifically detect the presence of isoaspartic acid residues in target proteins, Protein isoaspartyl methyltransferase (PIMT) is used. From S-adenosyl-L-methionine at the carboxyl position to isoaspartic acid It catalyzes the transfer of methyl groups, producing S-adenosyl-L-homocysteine ​​(S This is a relatively small molecule that is usually isolated and provided in the kit. Quantitation can be achieved by reverse phase HPLC using SAH HPLC standards.

[0201] The titer or biological identity of an antibody can be measured by its ability to bind to an antigen. Specific binding of an antibody to its antigen can be determined by any method known to those of skill in the art, for example, EL It can be quantified by immunoassays such as ISA (enzyme-linked immunosorbent assay). do.

[0202] All publications mentioned herein are incorporated by reference in their entirety for purposes of illustration only and are not intended to be limiting unless otherwise specified. For purposes of describing and disclosing the materials, they are incorporated by reference.

[0203] As different embodiments of the invention are described herein with reference to the accompanying drawings, The invention is not limited to those precise embodiments, and various modifications thereto are contemplated. Modifications and variations will be apparent to those skilled in the art within the scope of the invention as defined in the appended claims. It is understood that various modifications may be made without departing from the spirit and scope of the present invention. EXAMPLES

[0204] Example 1 Stability of anti-CTLA4 antibody formulations with and without methionine This study examined the effect of 10 mM L-methionine on the stability of anti-CTLA4 antibody formulations. The present study was carried out to investigate the effect of L-methionine on the anti-CTLA4 formulation. The effects of the following stresses were evaluated:

[0205] (1) 5±3℃ (ambient humidity), 25℃ (60% relative humidity), 40℃ (75% relative humidity) ) for up to 3 months of heat stress. (2) Stirring stress in horizontal position (300 rpm for 3 days). (3) Freeze-thaw stress (freezing at -80°C to 18-22°C (thawing at room temperature for 4 hours) 5 thaw cycles). (4) ICH conditions under light stress (0.2×ICH, 0.5×ICH, 1×ICH) ).

[0206] Based on the data, formulations containing L-methionine are more effective than their non-L-methionine counterparts. It is more stable than formulations that

[0207] Materials and Methods The following liquid formulation was prepared using the following CDRs on an IgG1 backbone: CDRH1 of SEQ ID NO: 35, CDRH2 of SEQ ID NO: CDRH2 of SEQ ID NO:36, CDRH3 of SEQ ID NO:37, CDRL1 of SEQ ID NO:38, Prepared with an anti-CTLA4 antibody having a CDRL2 of SEQ ID NO: 9 and a CDRL3 of SEQ ID NO: 40 The sequences of the variable heavy and light chains of the anti-CTLA4 antibody are SEQ ID NO: 88 and SEQ ID NO: 9, respectively. and 48. Each formulation was filled at 1 mL into 2 mL Type 1 glass vials. A total of 36 vials were filled for formulation A1 and 19 vials for formulation A2. The target pH of the agent was 5.5.

[0208] Table 10: Anti-CTLA4 Antibody Formulations [Table 10]

[0209] The vials were then stored 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: : The photostability test was performed using 1 mL liquid formulations of A1 and A2 in glass vials at room temperature. , performed under 0.2×ICH; 0.5×ICH; and 1×ICH.

[0210] Protein concentration was determined by using UV absorbance at 280 nm.

[0211] The samples were equilibrated to room temperature and a turbidity test was performed on the samples by measuring the absorbance at 350 nm in a spectrophotometer ( A350).

[0212] The samples were assessed for purity by size exclusion chromatography (SEC), where The percentage of monomer, as well as the percentage of high molecular weight species (HMW) and late eluting peaks ( The percentage of LMW species was determined by ultra-performance size exclusion chromatography (UP -SEC) was performed by dissolving the sample in a mobile phase (50 mM sodium phosphate, 450 mM arginine monohydrochloride). The diluted sample was diluted to 5.0 mg / mL in 100 mM NaCl, pH 7.0. was injected into a UPLC equipped with a Waters BEH200 column and UV detector ( Proteins in the sample were separated by size and analyzed by UV absorption at 280 nm. Detected.

[0213] Ion exchange chromatography was used to assess chemical stability and to characterize the electrical conductivity over time. An ion-exchange HPLC method was performed to monitor changes in the cargo variant profile. A Dionex ProPac WCX-10 column and a 280 nm UV detector were used. The experiment was carried out using 100 μg of 100% ethanol. Samples were diluted in purified water and 80 μg was injected for analysis. The mobile phases used for the IEX analysis of the samples were as follows: Mobile phase A: 20 mM MO PS, pH 7.2; Mobile phase B: 50 mM sodium phosphate, 60 mM sodium chloride p The assay consisted of a gradient of 20 mM MOPS, pH 7.2 to 8.0. A mobile phase gradient of 50 mM sodium phosphate, 60 mM NaCl, pH 8.0 was used. UV detection is performed at 280 nm. These methods are considered equivalent. The results are expressed as relative percentages based on the total area of ​​the chromatogram.

[0214] Peptide mapping was performed by Lys-C digestion. Samples were loaded onto the Q Exactive. Data analysis was performed using PinPoint software. It was done.

[0215] The results of the test are set out in the table below: Table 11 [Table 11]

[0216] Table 12 [Table 12]

[0217] Table 13: [Table 13]

[0218] Table 14 [Table 14]

[0219] Table 15 [Table 15]

[0220] Table 16 [Table 16]

[0221] Table 17 [Table 17]

[0222] Table 18 [Table 18]

[0223] result Protein concentration measured by UV absorbance at 280 nm for each test condition and duration. There was no measurable change in substance concentration between formulations.

[0224] There was no measurable change in pH between formulations for the conditions and duration tested.

[0225] Turbidity (A350) data is shown in Figures 1A and 1B. At 40°C, both formulations The turbidity tended to increase over the 8-week period. There was no substantial change in turbidity up to the 8 week time point. As shown in FIG. was slightly greater (but consistent) for all light stress conditions compared to formulation A1. The turbidity increased. The samples of formulation A1 were subjected to freeze-thaw (up to 5 times) or agitation stress (300 rpm for up to 3 days) did not change the turbidity of the samples compared to the control (T0) samples. Formulation A2 was not subjected to freeze-thaw or agitation stress. Therefore, the turbidity data Based on this, formulation A1, which contains L-methionine, is slightly preferred.

[0226] As shown in Figures 3A, 3B, 4A and 4B, the percentage of HMW and molar fractions were UP-SEC analysis of the samples to determine the percentage of mers showed that at 40°C, both formulations The increase in %HMW peak and %LMW peak (and the resulting At 25°C, both formulations showed the same tendency of decreasing the monomer peak. The results showed a similar tendency, but the change was smaller than that at 40°C. At 5°C, no substantial change was observed. As shown in Figure 5, formulation A2 had a significantly higher solubility than formulation A1 in all tested A slightly larger but consistent increase in %HMW for all light stress conditions (and The freeze-dried samples of formulation A1 show a slightly larger but consistent decrease in % monomer in response to the freeze-dried samples. Thawing (up to 5 times) or agitation stress (300 rpm, up to 3 days) treatments were performed in the same manner as the control (T0 ) sample did not change % HMW or % monomer (see Figures 5 and 6 Formulation A2 was not subjected to freeze-thaw or agitation stress. Based on the P-SEC data, formulation A1, which contains L-methionine, is slightly preferred.

[0227] As shown in Figures 7A, 7B, 8A, 8B, 9A and 9B, the HP-IEX data At 40°C, both formulations showed an increasing trend in the % acidic peak and % basic peak up to 12 weeks. At 25°C, both showed a corresponding decrease in the main peak. The formulations showed a similar trend, but the changes were smaller than those at 40°C. At 5°C, the % basic pyridine A slight increase in the peak and a corresponding decrease in the main peak were observed in the 8% of formulation A2. No substantial changes were observed for Formulation 1 or Formulation 2 except at the week time point. Formulation A This trend was not observed at 12 weeks / 5°C because two samples were not tested. As shown in Figures 10-12, formulation A2 showed slightly greater growth rates under all light stress conditions. A large but consistent increase in the % acidic and % basic peaks (and correspondingly, a slight A rather large but consistent % main peak reduction) and a corresponding % main peak reduction As shown in Figures 10 to 12, the formulation A1 sample Treatment with freeze-thaw (up to 5 times) or agitation stress (300 rpm, up to 3 days) was similar to that of the control. Compared to the (T0) sample, the % acidic peak, % basic peak, or % main peak were changed. (Formulation 2 was not subjected to freeze-thaw or agitation stress). Therefore, HP - Based on the IEX data, formulation A1, which contains L-methionine, is slightly preferred.

[0228] Monoclonal antibodies often contain CD4+, which may be susceptible to oxidation under light stress. It has methionine residues in the R and Fc regions. In the case of anti-CTLA4 antibodies, LC-M4 , HC-M34, HC-M250 and HC-M426 are easily oxidized under light stress. Peptide mapping studies may be performed at 40°C or 2x / 0.5x / 1x. Changes in the oxidation levels of these residues were determined following 8-week exposure to ICH light stress treatment.

[0229] Percentage oxidation of residues LC-M4, HC-M34, HC-M250 and HC-M426 The results of peptide mapping experiments showing oxidation are shown in Figures 13, 14, 15, and 16, respectively. As depicted in FIG.

[0230] As shown in Figures 13 to 16, under light stress conditions, certain methionine residues were oxidized. Notably, residues HC-M250 and HC-M426 were In the light stress treatment of 0.5× and 1× ICH, the oxidation level was significantly increased. However, the presence of 10 mM L-methionine in formulation A1 reduced the L-methionine The lower oxidation levels of M250 and M426 compared to formulation A2, which did not contain Therefore, based on the peptide mapping data, formulation 1 (LM et) appears to be slightly more favorable.

[0231] Based on a comparison of the analytical data from the above studies, Formulation A1 exhibited the following characteristics in comparison to Formulation A1: (i) (ii) increased turbidity for all light stress conditions; and (iii) flocculation for all light stress conditions. (iii) a smaller increase in glycerin levels (%HMW) for all light stress conditions; (iv) a slightly smaller but consistent decrease in the main peak after light stress Groups HC M250 and HC M426 exhibited a smaller increase in oxidation level.

[0232] Example 2 Screening of anti-CTLA4 antibody formulation buffers The study was carried out in the presence of sucrose, polysorbate 80 and L-methionine. SEQ ID NO:88 in different viable formulation buffers (L-histidine and acetate) and a light chain variable region comprising SEQ ID NO:48. The purpose of this study is to compare the protein-protein interactions of the two formulations (colloid and The thermal stability of the 100% 15 ... The diffusion interaction parameter (K D ) is positive (K D >0) The repulsive protein-protein interactions indicated by The K of both formulations at three different pH values ​​(pH 5, 5.5, and 6) D At least Each was measured three times (N=3), and the standard deviation was obtained. Based on data not shown or Figure Y, anti-CTLA4 antibodies are stable in the pH range of 5.0 to 6.0. It is stable in both L-histidine and acetate buffers over a period of time. The two formulations were then examined for further thermal stability at pH 5.5 at 5°C, 25°C and 40°C. Evaluated.

[0233] To evaluate the stability of the formulations, two anti-CTLA4 formulations (L-histidine buffer and The effects of the following stresses on the endothelial cells (sodium citrate and acetate buffer) were evaluated:

[0234] (1) 5±3℃ (ambient humidity), 25℃ (60% relative humidity), 40℃ (75% relative humidity) ) for up to 3 months of heat stress. (2) Stirring stress in horizontal position (300 rpm for 3 days). (3) Freeze-thaw stress (freezing at -80°C to 18-22°C (thawing at room temperature for 4 hours) 5 thaw cycles). (4) ICH conditions under light stress (0.2×ICH, 0.5×ICH, 1×ICH) ).

[0235] Based on the data, anti-CTLA4 antibodies are well tolerated in L-histidine and acetate buffers. It is stable in both the 1 and 2 directions.

[0236] Materials and Methods The following liquid formulation was prepared using the following CDRs on an IgG1 backbone: HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: HCDR2 of SEQ ID NO:36, HCDR3 of SEQ ID NO:37, LCDR1 of SEQ ID NO:38, SEQ ID NO:3 Prepared with an anti-CTLA4 antibody having LCDR2 of SEQ ID NO: 9 and LCDR3 of SEQ ID NO: 40 Each formulation was filled in 1 mL of a 2 mL type 1 glass vial. 72 batches of each formulation were manufactured. The table below lists the composition of the two formulations. 7% (w / v) of glucose was added as a stabilizer; PS-80 was effective in preventing agitation-induced stress. and L-methionine, which provides stability against light stress conditions. and is an antioxidant since it reduces the oxidation of methionine under reduced pressure (see Example 1 above). sea ​​bream).

[0237] Table 19: Formulations [Table 19]

[0238] The vials were then stored under three different storage conditions: 5°C (ambient humidity), 25°C (60% relative humidity), The plates were incubated at 40°C (75% relative humidity) and 40°C (75% relative humidity). Data were collected as follows: Ta: [Table 20] [Table 21]

[0239] The photostability test was performed using 1 mL liquid formulations of B1 and B2 in glass vials at room temperature. , performed under 0.2×ICH; 0.5×ICH; and 1×ICH.

[0240] Protein concentration was determined by using UV absorbance at 280 nm.

[0241] The samples were equilibrated to room temperature and a turbidity test was performed on the samples by measuring the absorbance at 350 nm in a spectrophotometer ( A350).

[0242] The samples were assessed for purity by size exclusion chromatography (SEC), where The percentage of monomer, as well as the percentage of high molecular weight species (HMW) and late eluting peaks (L The percentage of MW species was determined by ultra-performance size exclusion chromatography (UP- SEC was carried out by dissolving the sample in a mobile phase (50 mM sodium phosphate, 450 mM arginine monohydrochloride The diluted sample was diluted to 5.0 mg / mL in 1000 mM NaCl (pH 7.0). , and injected into a UPLC equipped with a Waters BEH200 column and UV detector (6 Proteins in the sample were separated by size and detected by UV absorption at 280 nm. I put it out.

[0243] Ion exchange chromatography was used to assess chemical stability and to characterize the electrical conductivity over time. An ion-exchange HPLC method was performed to monitor changes in the cargo variant profile. A Dionex ProPac WCX-10 column and a 280 nm UV detector were used. Samples were diluted in purified water and 80 μg was injected for analysis. The mobile phases used for the IEX analysis of the heat-stable samples were as follows: Mobile phase A: 20 ml M MOPS, pH 7.2; Mobile phase B: 50 mM sodium phosphate, 60 mM sodium chloride The assay consisted of a gradient of 20 mM MOPS, pH 7.0. Mobile phase gradient from 0.2 to 50 mM sodium phosphate, 60 mM NaCl, pH 8.0 The assay is performed using a ELISA kit. UV detection is performed at 280 nm. These methods are equivalent. and the results are expressed as relative percentages based on the total area of ​​the chromatogram. It is expressed as:

[0244] Peptide mapping was performed by Lys-C digestion. Samples were analyzed using a Q Exactive Data analysis was performed using PinPoint software. It was done.

[0245] The results of the test are set out in the table below: Table 22 [Table 22]

[0246] Table 23 [Table 23]

[0247] Table 24 [Table 24]

[0248] Table 25 [Table 25]

[0249] Table 26 [Table 26]

[0250] Protein concentration measured by UV absorbance at 280 nm for each test condition and duration. There was no measurable change in substance concentration between formulations.

[0251] There was no measurable change in pH between formulations for the conditions and duration tested.

[0252] The turbidity (A350) data is shown in Figures 17A, 17B and 18. In comparison, at 40°C, both formulations showed a trend towards increasing turbidity up to the 8-week point. At 25°C and 5°C, both formulations showed substantial changes in turbidity up to the 8-week time point. As shown in FIG. 14, formulation B1 had a higher solubility in the sera than formulation B2 under light stress conditions. There was a slightly greater but consistent increase in turbidity after the conditions (0.5×ICH and 1×ICH). It was also observed that the samples showed a tendency to freeze-thaw (up to 5 times) or agitation stress (300r pm, for up to 3 days) treatment did not change the turbidity of the samples compared to the control (T0) samples.

[0253] As shown in Figures 19A, 19B, 20A and 20B, the percentage of HMW and UP-SEC analysis of the samples to determine the percentage of monomer at 40°C. Both formulations showed an increase in %HMW peak and %LMW peak (and At 25°C, both showed a tendency toward a decrease in the monomer peak (resulting in a decrease in the monomer peak). The formulations showed similar trends, but the changes were smaller than those at 40°C. At 5°C, there was no substantial change. As shown in Figures 21 and 22, formulation B2 had a similar effect to formulation B1. In comparison, a slightly larger (but consistent) % for all light stress conditions tested. The increase in HMW (and correspondingly, slightly larger but consistent decrease in % monomer) The samples were subjected to freeze-thawing (up to 5 times) or agitation stress (300 rpm, up to 3 days). The treatment did not change the % HMW or % monomer compared to the control (T0) sample (Figure 21). and 22).

[0254] As shown in Figures 23A, 23B, 24A, 24B, 25A and 25B, HP- IEX data showed that at 40°C, both formulations showed % peak acidity and % peak basicity up to 8 weeks. This indicates that the increase in the main peak was accompanied by a corresponding decrease in the main peak. However, formulation B2 had a slightly larger but consistent % main peak compared to formulation B1. At 25°C, both formulations showed similar trends, but the changes were smaller than those at 40°C. As shown in Figures 26, 27, and 28, under light stress conditions (0.5 × IC After 1×ICH and 1×H, formulation B1 showed a slightly larger but consistent % acidic peak. While formulation B1 showed a slightly greater (but consistent) increase in the % basic peak, formulation B2 showed a However, the corresponding % main peak reductions for the two formulations were comparable. Samples were subjected to freezing and thawing (up to 5 times) or agitation stress (300 rpm, up to 3 days). Changes in % acidic peak, % basic peak, or % main peak compared to the control (T0) sample (Figures 26-28).

[0255] Peptide mapping studies were performed at 40°C or .2× / 0.5× / 1×ICH light scattering. The changes in the oxidation levels of these residues upon exposure to stress treatment for 8 weeks were determined.

[0256] Percentage oxidation of residues LC-M4, HC-M34, HC-M250 and HC-M426 The results of peptide mapping experiments showing oxidation are shown in Figures 29, 30, 31, and 32, respectively. As shown in FIG.

[0257] As shown in Figures 29-32, under light stress conditions, certain methionine residues were oxidized. Notably, residues HC-M250 and HC-M426 were significantly increased in both formulations. In mice, treatment with 0.5× and 1× ICH light stress significantly increased the oxidation level. However, formulation B1 showed significantly lower oxidation levels of M250 and M426 compared to formulation B2. This resulted in a smaller increase in

[0258] Based on a comparison of the analytical data from the above studies, formulation B1 (L-histidine buffer) compared to formulation B2 (acetate buffer), (i) reduced aggregation across all light stress conditions; (ii) slightly less increase in glycerol levels (%HMW) at 40°C, but consistent (iii) the decrease in the main peak after light stress, and (iv) the increase in the residues HCM250 and The oxidation levels of HCM426 and HCM426 showed a smaller increase. Based on protein interaction and stability data, the anti-CTLA4 antibody is L-histidine. It has been shown to be stable in both ginseng and acetate buffers.

[0259] Example 3 Co-formulation of anti-CTLA4 and anti-PD-1 antibodies Co-formulation of two antibodies in a single formulation may be more convenient for patients and may improve patient compliance. Based on protein-protein interactions (shown below), All coformulations (shown below) were found to be stable over a pH range of 5.0 to 6.0. Therefore, the three coformulations at pH 5.5 (P1C1, P1C2, and P2C1) were selected and incubated at 5°C, 25°C and 40°C along with two controls (anti-PD1 and anti-CTLA4). Further thermal stability at 100° C. was evaluated.

[0260] The study included the following IgG1 scaffold co-formulated with pembrolizumab at various concentrations: The following CDRs: HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, H of SEQ ID NO: 37 CDR3 of SEQ ID NO: 38, LCDR1 of SEQ ID NO: 39 and LCDR2 of SEQ ID NO: 40 To evaluate the stability of anti-CTLA4 antibodies with LCDR3.

[0261] Table 27 [Table 27]

[0262] The following liquid formulations were prepared:

[0263] Table 28 [Table 28]

[0264] Each formulation was filled in 1 mL in a 2R vial. Stability was evaluated by visual inspection, PD350 Turbidity, protein concentration, microflow imaging ing) (MFI) (fine particle assessment), mixed mode size exclusion chromatography (S EC (assessment of aggregation), cIEF (assessment of charge variants), IEX (assessment of charge variants) Anti-CTLA4 and anti-PD1 Since they co-elute during UP-SEC, anti-PD1 and anti-CTLA4 are mixed in the case of co-formulation. The stability of the co-formulations was evaluated by partitioning in 2-mode SEC. The thermal stability protocol used in the qualitative test is as follows:

[0265] Table 29 [Table 29]

[0266] Colloidal and thermal stability for the different coformulations (three coformulations and two controls) were analyzed. The indicated protein-protein interactions were measured. The diffusion interaction parameters (K D ) is positive, K D Repulsive protein-protein interactions indicated by >0 The interaction indicates a stable formulation with low aggregation tendency. The Kd of all formulations in (5 and 6) was measured at least three times each and standard deviations were obtained. As shown in Figure 33, combos P1C1, P2C2 and P1C2 all have Plus K D Pembrolizumab ( PD1) and pembrolizumab-enriched combo (P2C1) are The CTLA4-enriched combination is more stable at pH 5.0 than the combination (P1C2). The combination (P1C2) is equally stable at pH 5.0 and pH 5.5. Coformulations with multiple pembrolizumab fractions were equally stable at pH 5.0 and pH 5.5. It is more stable at pH 5.0 compared to co-formulations with higher CTLA4 fractions at do.

[0267] 12-month stability results At 12 months, total protein concentration (as determined by UV280) was significantly higher in both conditions. No significant changes in the oxidative stress response (data not shown) were observed.

[0268] Turbidity changes: Turbidity changes were observed up to 12 months in the following order: 5 for each formulation ℃<25℃<40℃. Data on the rate of turbidity change up to 6 months at 40℃ are It appeared to be directly proportional to protein concentration (data not shown).

[0269] The number of fine particles (measured by MFI) in all formulations under all conditions was fairly low. (Data not shown). The quality of the co-formulated samples was evaluated. One milliliter of sample was drawn into a pipette tip. The solution is then gently pumped through the flow cell of the microscope system (150 micron depth of field). The particles were counted and photographed with a digital camera at a flow rate of 0.17 mL / min. As the sample passes through the flow cell, continuous brightfield images are captured in real time. The output at the end of the analysis is particle count and particle concentration data. FI images can also be analyzed using the system software to measure different morphological parameters, e.g. For example, they can be processed for size, strength, transparency and shape.

[0270] The % acidity indicating deamidation for each of the anti-PD-1 and anti-CTLA4 antibodies was An increase in charge variants was observed at higher temperatures (25°C and 40°C) for each antibody. The ion-exchange HPLC method was performed using a Dionex ProPac WCX-10 column. and a UV detector at 280 nm. The mobile phase used for the IEX analysis of the heat-stable samples was Mobile phase (Mobile phase A: 24 mM MES pH 6, 4% acetonitrile; Mobile phase B: 20 ​​mM NaPO4, 95 mM NaCl pH 8, 4% acetonitrile) A summary of the normalized cIEF data (baseline, 3 months, and 6 months) is provided below. can be.

[0271] [Table 30] TIFF0007653465000033.tif132152

[0272] Aggregation was measured using UPSEC and mixed mode SEC. PD1 antibodies co-elute during UP-SEC, but they are resolved by mixed-mode SEC The HMW and LMW aggregates increased with temperature. SEC results show that a very low percentage of aggregate species (%HMW) was observed at 5°C and 25°C. The results showed that the levels were detected up to 12 months after the start of the study, with no significant changes compared to the baseline.

[0273] Table 31 [Table 31] TIFF0007653465000035.tif22168

[0274] Mixed-mode SEC was used to resolve aggregates and oxidized species for two antibodies in a co-formulation. The results are shown in the table below. Pembrolizumab was analyzed by oxidizing species 1 and 2 at high temperatures. increased, reflecting oxidation of M105 on one and two arms, respectively. M105 is located in the CDR3 of pembrolizumab. Methionine residues in the bases or CDRs affect the biological activity of antibodies through oxidation Methionine may reduce the oxidation of Met105 in the heavy chain CDR of pembrolizumab. The small change in oxidative species compared to the starting point indicates that the co-formulation is stable up to 12 months at 5°C. This indicates that the setting is correct.

[0275] [Table 32]

[0276] Based on 12-month data, antibodies when co-formulated performed similarly to single antibody formulations. The co-formulations performed well in solution. The protein spreading was indicative of colloidal and thermal stability. Repulsive protein-protein interactions measured by the diffusion interaction parameter kD It has been shown to be stable at pH 5.0 to 6.0.

[0277] Example 4 Further co-formulations The study included the following IgG1 scaffold co-formulated with pembrolizumab at various concentrations: The following CDRs: HCDR1 of SEQ ID NO: 35, HCDR2 of SEQ ID NO: 36, H of SEQ ID NO: 37 CDR3 of SEQ ID NO: 38, LCDR1 of SEQ ID NO: 39 and LCDR2 of SEQ ID NO: 40 To evaluate the stability of anti-CTLA4 antibodies with LCDR3.

[0278] Table 32 [Table 33]

[0279] The formulations were prepared as liquid formulations as follows:

[0280] Table 33 [Table 34]

[0281] The formulations were stored under three different conditions: 5°C (ambient humidity), 25°C (60% relative humidity) and The 75 / 200 formulation was also exposed to light stress (0 ICH , 0.5×ICH or 1×ICH).

[0282] result The results showed that increasing the methionine concentration resulted in a visually significant increase in the MFI under all conditions. This indicates that the amount of microparticles that cannot be absorbed by the methionine receptor decreases (data not shown). When the concentration of methionine was increased, the % HMW species observed by UP-SEC decreased at 40°C. Increasing the temperature results in a slight decrease in turbidity at 40°C.

[0283] [Table 35]

[0284] Example 5: Long-term stability of CTLA4 monotherapy The following liquid formulations were used: anti-CTLA4 antibodies with the following CDRs: 100 mg / mL anti-CTLA 4 antibodies, 10 mM L-histidine buffer, 7% w / v sucrose, 0.02% w / The product was prepared at pH 5.5 using 100 ml of polysorbate 80. and dispensed into 2R type I glass with aluminum seal. Fill volume was 2. The sample was evaluated for stability under the following conditions: .

[0285] (i)5℃ / environmental humidity (ii) 25°C / 60% relative humidity (iii) 40°C / 75% relative humidity.

[0286] Samples were tested at baseline and at 1, 3, 6, 9 and 12 months. The results are set forth in the following table.

[0287] Table 34 [Table 36]

[0288] Table 35 [Table 37]

[0289] Table 36 [Table 38]

[0290] result: As shown in the data above, no significant changes were observed when the formulation was stored at 5°C. Such formulations are expected to be stable for up to 24 months at 5°C. The biological potency of the formulation as determined by A showed that no significant changes in potency were observed under all conditions. There was no change in pH with storage time or conditions.

[0291] Protein concentrations of samples were 1.0-1.5% under all storage conditions and across all three conditions tested. No significant changes in stability were observed up to 2 months.

[0292] Charge variants (% acidic variants, % main and % basic variants) were analyzed using HP-I No significant changes were observed over a 12-month period at 5°C. At 25°C, the % acidic variants increased over 12 months, while the % total main peak These results were consistent with those obtained by considering storage conditions. This is not unexpected. At 40°C, the % total main peak was 6 months after the start of the experiment. The % acidic variants showed a significant decrease by 0.05, whereas the % basic variants showed a significant increase. These results are not unexpected considering the storage conditions.

[0293] Purity was determined by UP-SEC (% HMW species, % monomer and % LMW species). The stability of the monomer or %HMW species at 5°C remained unchanged for up to 12 months. No peaks were detected over 12 months at 5°C. In this case, there was no change in the HMW species over 12 months, % monomer decreased slightly, and the LMW species At 40°C, the %HMW species increased slightly up to 6 months, and the %mono The mers were reduced to less than 85% at 6 months. These results were unexpected given the storage conditions. isn't it.

[0294] There was no change in pH with storage time or conditions.

[0295] Example 6 Addition of a chelator as an optional excipient The stability of the formulations was evaluated in the presence or absence of a chelator (DTPA). To evaluate the stability, the two formulations were filled into vials and stored at 5°C (ambient humidity), 25°C, and 10°C. (60% relative humidity) and 40°C (75% relative humidity) for 12 weeks, protected from light The two formulations were as follows: [Table 39]

[0296] Colloidal stability of the samples was evaluated by size exclusion chromatography (SEC) for purity. where the percentage of monomer, as well as the percentage of high molecular weight species (HMW) and The percentage of late eluting peaks (LMW species) was determined. is aggregates), % monomer and LMW (low molecular weight species) levels C data is in the table below.

[0297] Table 37 [Table 40]

[0298] Percentage of HMW and Percentage of Monomer as shown in the table above UP-SEC analysis of samples to determine the solubility of both formulations at 5°C, 25°C and 40°C for 12 h. Increase in %HMW peak and %LMW peak (and resulting % At 25°C, both formulations showed similar trends. At 5°C, no substantial change was observed. Based on the data, formulation 1 (without DTPA) was significantly more effective than formulation 2 (with DTPA). In addition, formulation 1 showed a slight increase in %HMW and %LMW compared with formulation 2. showed a greater decrease in % monomer.

[0299] HP-IEX analysis of the samples to determine their chemical stability was performed at 5°C, 25°C and At 40°C, both formulations showed a significant increase in the % acidic peak and the resulting % mono At 25°C, both formulations showed similar trends. However, the change was smaller than that at 40°C. At 5°C, no substantial change was observed. (data not shown).

[0300] The results in the table below show a trend towards decreased PS80 concentrations over time up to the 8 week point. At 25°C, both formulations showed similar trends, but the changes were smaller than at 40°C. At 40°C, no substantial changes were observed. 4) showed less PS8 than formulation 1 (anti-CTLA4 without DTPA) Decomposition of 0 was observed. [Table 41]

[0301] Therefore, DTPA also provides even greater stability with the formulations described herein. It can be provided.

Claims

1. (i) 1 mg / ml to 50 mg / ml of an anti-CTLA4 antibody, wherein the anti-CTLA4 antibody comprises a heavy chain comprising SEQ ID NO:99 and a light chain comprising SEQ ID NO:100; (ii) 1 mg / ml to 25 mg / ml of an anti-human PD1 antibody, wherein the anti-human PD-1 antibody comprises a light chain comprising SEQ ID NO:5 and a heavy chain comprising SEQ ID NO:10; (iii) 8 mM to 12 mM L-histidine buffer; (iv) 6% to 8% weight / volume (w / v) sucrose; (v) 0.02% w / v Polysorbate 80; and (vi) 5 mM to 10 mM L-methionine, wherein the formulation has a pH between 5.0 and 6.

0.

2. The formulation of claim 1 comprising 10 mg / ml to 50 mg / ml of anti-CTLA4 antibody.

3. The formulation of claim 1, comprising 10 mg / ml to 25 mg / ml of anti-human PD-1 antibody.

4. The formulation of any of claims 1 or 3, comprising an anti-human PD-1 antibody that is pembrolizumab.

5. 5. The formulation of any of claims 1-4, wherein the ratio of anti-PD1 antibody to anti-CTLA4 antibody is 1:2, 1:1, 2:1, 10:1, 1:10, 8:3 or 8:

1.

6. 6. The formulation of claim 5, wherein the ratio of anti-PD1 antibody to anti-CTLA4 antibody is 8:

1.

7. The formulation of claim 1, comprising 10 mM L-methionine.

8. The formulation of claim 1, wherein the formulation has a pH between 5.5 and 5.

6.

9. A formulation according to any one of claims 1 to 8, wherein the ratio of anti-PD1 antibody to anti-CTLA4 antibody is 1:

2.

10. 10. The formulation of any of claims 1 to 9, wherein the concentration of the anti-CTLA4 antibody 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 or 50 mg / ml.

11. The formulation of any one of claims 1 to 9, wherein the concentration of the anti-CTLA4 antibody is 50 mg / ml.

12. 12. The formulation of any of claims 1-11, wherein the concentration of the anti-PD1 antibody is 25 mg / ml, 22.7 mg / ml, 2.27 mg / ml, 21.1 mg / ml or 23.5 mg / ml.

13. The formulation of any one of claims 1 to 11, wherein the concentration of the anti-PD1 antibody is 25 mg / ml.

14. A formulation of any of claims 1 to 12 comprising 23.5 mg / ml anti-PD1 antibody and 2.9 mg / ml anti-CTLA4 antibody, 10 mM L-histidine buffer, 7% w / v sucrose, 0.02% w / v polysorbate 80 and 10 mM L-methionine.

15. 15. The formulation of any of claims 1-14, further comprising a chelator, wherein the chelator is diethylenetriaminepentaacetic acid (DTPA).

16. The formulation of any of claims 1 to 15, wherein the formulation is contained in a glass vial or an injection device.

17. 17. The formulation of any of claims 1 to 16, which is a liquid formulation, or which is frozen at least below -70°C, or which is a reconstituted solution from a lyophilized formulation.

18. Use of a formulation according to any of claims 1 to 17 for the preparation of a medicament for treating cancer or for treating a chronic infection.

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