RU2772781C2 - Compositions of anti-pd-1 antibodies - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions relates to treatment of cancer. The pharmaceutical composition for treating cancer includes 150 mg/ml anti-PD-1 antibody; 20 mM histidine buffer; 84 mg/ml trehalose; 0.2 mg/ml PS80 and 0.045 or 0.05 mg/ml EDTA, wherein said pharmaceutical composition has a pH of 5.0 +/- 0.5 . Also disclosed is another variant of implementation of the pharmaceutical composition.

EFFECT: composition with a high concentration of the antibody and reduced viscosity.

28 cl, 7 dwg, 26 tbl, 6 ex

Description

The technical field to which the invention belongs

The present invention relates to the field of pharmaceutical compositions of antibodies. In particular, the present invention relates to an anti-PD-1 antibody composition and its pharmaceutical preparation and use.

Prerequisites for the creation of the invention

Antibody therapeutics are typically administered on a regular basis and typically use a dose of several mg/kg by injection. Parenteral delivery is a common route of administration for a therapeutic antibody. For parenteral administration, relatively high concentrations of antibody compositions are desirable in order to minimize the volume of each dose.

The development of highly concentrated protein compositions can be challenging due to problems associated with the physical and chemical stability of the protein, manufacturing, storage and delivery of the protein composition. The increased viscosity of antibody formulations can cause problems ranging from drug formulation to drug delivery to the patient. Various attempts have been made to investigate the effect of viscosity-reducing agents on highly concentrated aqueous protein-containing compositions.

An anti-PD-1 antibody has been shown to be useful in the treatment of hyperproliferative disorders including type 1 cancer. There is a need for a stable, highly concentrated antibody formulation comprising an anti-PD-1 antibody having a suitable viscosity to meet the medical needs of patients suffering from PD-1 mediated conditions such as cancer.

The essence of the invention

Compositions are provided that include an anti-PD-1 antibody and excipients capable of reducing the viscosity of the composition comprising the antibody. Certain compositions have been shown to be effective in reducing viscosity. Preferably, the compositions provided herein exhibit viscosity properties suitable for achieving concentrations greater than 100 mg/mL for a drug product for use in therapeutic treatment.

This application provides pharmaceutical compositions that maintain high concentrations of bioactive antibody in solution and are suitable for parenteral administration, including subcutaneous, intravenous, intramuscular, intraperitoneal or intradermal injection. The compositions include an anti-PD-1 antibody, a disaccharide, a buffer, a chelating agent, and a polysorbate. In some embodiments, the pH of the composition may be between about 4.5 and 5.5. In some embodiments, the composition preferably has a viscosity ranging from about 1 centipoise (cP) to about 20 cP. In some embodiments, the preferred route of administration is subcutaneous injection.

In some embodiments, the composition may include or consist essentially of about 100 mg/ml to about 200 mg/ml of anti-PD-1 antibody, disaccharide, buffer, chelating agent, and polysorbate, and has a pH of about 4.5 to about 5, 5. In some embodiments, the composition may consist essentially of about 150 mg/ml of anti-PD-1 antibody, disaccharide, buffer, chelating agent, and polysorbate and has a pH of about 5.0.

In some embodiments, the composition does not include an antioxidant. In some embodiments, the implementation of the composition does not include methionine, such as, for example, without limitation, L-methionine or its pharmaceutically acceptable salt. In some embodiments, the composition does not include arginine.

In some embodiments, the composition may have a viscosity of less than about 50 centipoise, less than about 40 centipoise, less than about 30 centipoise, or less than about 20 centipoise at 20°C. In some embodiments, the implementation of the composition may have a viscosity of from about 5 to about 50 centipoise at 20°C. In some embodiments, the implementation of the composition may have a viscosity of from about 5 to about 40 centipoise at 20°C. In some embodiments, the implementation of the composition may have a viscosity of from about 5 to about 30 centipoise at 20°C. In some embodiments, the implementation of the composition may have a viscosity of from about 5 to about 20 centipoise at 20°C. In some embodiments, the implementation of the composition may have a viscosity of from about 10 to about 20 centipoise at 20°C. In some embodiments, the implementation of the composition may have a viscosity of from about 14 to about 16 centipoise at 20°C. In some embodiments, the implementation of the composition may have a viscosity of about 14 centipoise at 20°C.

In some embodiments, the polysorbate concentration may be from about 0.01 to about 0.3 mg/mL. In some embodiments, the polysorbate concentration is about 0.2 mg/mL. In some embodiments, the polysorbate is polysorbate 80.

In some embodiments, the disaccharide may be trehalose. In some embodiments, the trehalose is trehalose dihydrate. In some embodiments, the concentration of trehalose may be about 1 mg/mL to about 100 mg/mL. In some embodiments, the concentration of trehalose is about 84 mg/mL. In other embodiments, the concentration of trehalose is about 50 mg/mL.

In other embodiments, the disaccharide agent may be sucrose. In some embodiments, the concentration of sucrose may be from about 1 mg/mL to about 100 mg/mL. In some embodiments, the sucrose concentration is about 50 mg/mL.

In some embodiments, the buffer may be a histidine buffer. In some embodiments, the concentration of histidine buffer may be from about 1.0 to about 30 mM. In some embodiments, the concentration of the histidine buffer is approximately 20 mM histidine.

In other embodiments, the buffer may be an acetate buffer. In some embodiments, the acetate buffer concentration may be from about 1.0 to about 30 mM. In some embodiments, the acetate buffer concentration is approximately 20 mM acetate.

In some embodiments, the chelating agent may be EDTA, including, for example, without limitation, disodium EDTA and disodium EDTA dihydrate. In some embodiments, the concentration of EDTA may be from about 0.01 to about 0.3 mg/mL. In some embodiments, the concentration of EDTA may be about 0.01 mg/ml, about 0.05 mg/ml, about 0.1 mg/ml, about 0.15 mg/ml, about 0.2 mg/ml, about 0 .25 mg/ml or approximately 0.3 mg/ml. In some embodiments, the EDTA concentration is about 0.04, about 0.045, or about 0.05 mg/mL.

In some embodiments, the antibody concentration may be from about 100 mg/mL to about 150 mg/mL. In some embodiments, the antibody concentration may be about 130 mg/ml, about 135 mg/ml, and about 140 mg/ml. In some embodiments, the antibody concentration is about 150 mg/mL. In some embodiments, the antibody concentration is about 120 mg/mL.

In some embodiments, the implementation of the composition may further include arginine. In some embodiments, the concentration of arginine is from about 25 mM to about 300 mM, preferably about 50 mM, about 100 mM, about 150 mM, about 200 mM, or about 250 mM.

In some embodiments, the implementation of the composition may further include proline. In some embodiments, the concentration of proline is from about 25 mM to about 300 mM, preferably about 100 mM or about 200 mM.

In some embodiments, the implementation of the composition essentially consists of about 150 mg/ml anti-PD-1 antibody; about 20 mM histidine buffer; approximately 84 mg/ml trehalose dihydrate; about 0.2 mg/ml PS80; and approximately 0.05 mg/ml EDTA. In some embodiments, the composition has a pH of 5.0 +/- 0.5.

In some embodiments, the implementation of the composition essentially consists of approximately 150 mg/ml anti-PD-1 antibody; about 20 mM histidine buffer; about 100 mm arginine HCl, about 50 mg/ml trehalose dihydrate; about 0.2 mg/ml PS80; and approximately 0.05 mg/ml EDTA. In some embodiments, the composition has a pH of 5.0 +/- 0.5 and a viscosity of about 10 centipoise to about 16 centipoise at 20°C. In some embodiments, the implementation of the composition has a viscosity of approximately 15 centipoise at 20°C.

In some embodiments, the composition essentially consists of about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 145 mg/ml, about 148 mg/ml , about 149 mg/ml, about 150 mg/ml, about 151 mg/ml or about 152 mg/ml antibody, about 20 mM histidine buffer, about 84 mg/ml trehalose dihydrate, about 0.2 mg/ml PS80, about 0.05 mg/ml EDTA, and the composition has a pH of 5.0 +/- 0.5. In some embodiments, the implementation of the composition has a viscosity of from about 10 centipoise to about 16 centipoise at 20°C.

In some embodiments, the composition essentially consists of about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 145 mg/ml, about 148 mg/ml , approximately 149 mg/ml, approximately 150 mg/ml, approximately 151 mg/ml or approximately 152 mg/ml anti-PD-1 antibody, approximately 20 mM histidine buffer, approximately 84 mg/ml trehalose, approximately 0.2 mg/ml ml PS80, approximately 0.05 mg/ml EDTA, and the composition has a pH of 5.0 +/- 0.5.

In some embodiments, the composition essentially consists of about 150 mg/ml anti-PD-1 antibody, about 20 mM histidine buffer, about 84 mg/ml trehalose dihydrate, about 0.2 mg/ml PS80, about 0.05 mg/ml ml of EDTA, and the composition has a pH of 5.0 +/- 0.5.

In some embodiments, the antibody may be a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is of the subclass IgG ₁ , IgG ₂ , IgG _2Δa , IgG ₃ , IgG ₄ , IgG _4Δb , IgG _4Δc , IgG ₄ S228P, IgG _4Δb S228P, and IgG _4Δc S228P human. In some embodiments, the antibody is of the IgG4 isotype and includes a stabilized hinge region, such as S228P.

In some embodiments, the antibody may be PF-06801591, nivolumab, pembrolizumab, cemiplimab, or spartalizumab. In other embodiments, the antibody may be an antigen-binding portion of PF-06801591, nivolumab, pembrolizumab, cemiplimab, or spartalizumab.

In some embodiments, the antibody may comprise a heavy chain variable region (VH) comprising a first VH complementarity determining region (CDR1), VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2; and/or a light chain variable region (VL) comprising the VL CDR1, VL CDR2, and VL CDR3 of the VL sequence shown in SEQ ID NO: 3. In some embodiments, the VH CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4, VH CDR2 includes the amino acid sequence shown in SEQ ID NO: 5 and VH CDR3 includes the amino acid sequence shown in SEQ ID NO: 6, VL CDR1 includes the amino acid sequence shown in SEQ ID NO: 7, VL CDR2 includes the amino acid sequence shown in SEQ ID NO: 7 SEQ ID NO: 8, and VL CDR3 includes the amino acid sequence shown in SEQ ID NO: 9.

In some embodiments, the VH region includes the amino acid sequence shown in SEQ ID NO: 2, or a variant with one or more conservative amino acid substitutions in residues that are not in the CDR, and/or the VL region includes the amino acid sequence shown in SEQ ID NO. :3, or a variant thereof with one or more amino acid substitutions for amino acids that are not in the CDR. In some embodiments, the antibody may include an amino acid sequence that is at least 90% identical to the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 2 and an amino acid sequence that is at least 90% identical to the amino acid sequence of the light chain variable region. chain shown in SEQ ID NO: 3. In some embodiments, the antibody may comprise a heavy chain variable region sequence comprising the amino acid sequence shown in SEQ ID NO: 10, a light chain variable region sequence comprising the amino acid sequence shown in SEQ ID NO: : 11. In some embodiments, the antibody comprises a VH region produced by an expression vector with ATCC Accession No. PTA-121183. In some embodiments, the antibody comprises a VL region produced by an expression vector with ATCC Accession No. PTA-121182. In some embodiments, the antibody is an antibody that specifically binds to PD-1 and competes with and/or binds to the same PD-1 epitope as the antibodies described herein.

In some embodiments, the composition essentially consists of about 150 mg/ml PF-06801591, about 20 mM histidine buffer, about 84 mg/ml trehalose dihydrate, about 0.2 mg/ml PS80, and about 0.05 mg/ml EDTA; wherein the composition has a pH of 5.0 +/- 0.1 and does not contain any methionine or arginine. In some embodiments, the composition essentially consists of about 150 mg/ml nivolumab, about 20 mM histidine buffer, about 84 mg/ml trehalose dihydrate, about 0.2 mg/ml PS80, and about 0.05 mg/ml EDTA; wherein the composition has a pH of 5.0 +/- 0.1 and does not contain any methionine or arginine. In some embodiments, the composition essentially consists of about 150 mg/ml pembrolizumab, about 20 mM histidine buffer, about 84 mg/ml trehalose dihydrate, about 0.2 mg/ml PS80, and about 0.05 mg/ml EDTA; wherein the composition has a pH of 5.0 +/- 0.1 and does not contain any methionine or arginine.

In some embodiments, the composition may not be lyophilized. In other embodiments, the composition may be lyophilized.

Also provided herein are methods for treating a condition in a subject, which include: administering to a subject in need thereof an effective amount of a pharmaceutical composition described herein. In some embodiments, the condition is cancer. In some embodiments, the cancer is selected from the group consisting of gastric cancer, sarcoma, lymphoma, leukemia, head and neck cancer, nasopharyngeal cancer, thymus cancer, epithelial cancer, epithelial ovarian cancer, salivary gland cancer, liver cancer, gastric cancer, thyroid cancer. gland, lung cancer (including, for example, without limitation, non-small cell lung cancer), ovarian cancer, fallopian tube cancer, breast cancer (including, for example, without limitation, triple negative breast cancer), prostate cancer, esophageal cancer, cancer pancreas, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, oral cavity cancer, skin cancer, peritoneal cancer and melanoma. In some embodiments, the subject is a previously treated adult patient with locally advanced or metastatic melanoma, head and neck squamous cell carcinoma (SCHNC), ovarian carcinoma, sarcoma, or relapsed or refractory classical Hodgkin's lymphoma (cHL). In some embodiments, the cancer may be platinum-resistant and/or platinum-refractory cancer, such as, for example, platinum-resistant and/or refractory ovarian cancer, platinum-resistant and/or refractory breast cancer, or platinum-resistant and/or or refractory lung cancer.

In some embodiments, the anti-PD-1 antibody pharmaceutical composition provided herein is administered at a dose of about 25 mg to about 1000 mg, preferably about 50 mg, about 100 mg, about 125 mg, about 150 mg, about 200 mg , about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 525 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, or about 800 mg. In some embodiments, the composition is administered at a dose of about 0.5 mg/kg to about 15 mg/kg, preferably about 0.5 mg/kg, about 1.0 mg/kg, about 3.0 mg/kg, about 5 .0 mg/kg or approximately 10 mg/kg. In some embodiments, the composition is administered once every 7, 14, 21, or 28 days. In some embodiments, the composition is administered subcutaneously. In other embodiments, the composition is administered intravenously. In some embodiments, the composition is administered as a single 2 ml subcutaneous injection. In some embodiments, the composition is administered once every three weeks. In some embodiments, the composition is administered once every four weeks. In some embodiments, the composition is administered at a dose of 300 mg subcutaneously. In some embodiments, the composition is administered as a 300 mg subcutaneous dose once every 28 days.

Also provided herein are methods for inhibiting the growth or progression of a tumor in a subject who has a tumor, comprising administering to the subject an effective amount of a pharmaceutical composition described herein.

Also provided herein are methods for inhibiting or preventing cancer cell metastasis in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition described herein.

Also provided herein are methods for inducing tumor regression in a subject that has a PD-1 expressing tumor, comprising administering to the subject an effective amount of a pharmaceutical composition described herein.

In some embodiments, the implementation of the antibody described in this application, you can enter the subject parenterally. In some embodiments, the subject is a human.

In some embodiments, the method may further comprise administering an effective amount of at least one other therapeutic agent. In some embodiments, the therapeutic agent is, for example, crizotinib, palbociclib, talazoparib, an anti-CTLA4 antibody, an anti-4-1BB antibody, an anti-OX40 antibody, a second anti-PD-1 antibody, a CAR-T cell, or a chemotherapeutic agent.

Also provided herein are uses of any of the anti-PD-1 antibody pharmaceutical compositions provided herein in the manufacture of a medicament for the treatment of cancer or for inhibiting tumor growth or progression in a subject in need thereof.

Also provided are pharmaceutical compositions of anti-PD-1 antibodies for use in the treatment of cancer or for inhibiting tumor growth or progression in a subject in need thereof. In some embodiments, the cancer is, for example, without limitation, gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, thymus cancer, epithelial cancer, salivary gland cancer, liver cancer, gastric cancer, thyroid cancer, lung cancer (including, for example, non-small cell lung carcinoma), ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, cancer colon, oral cancer, skin cancer and melanoma.

Also provided herein are methods for enhancing the immunogenicity or therapeutic effect of a vaccine for the treatment of cancer in a mammal, in particular a human, which method comprises administering to the mammal receiving the vaccine an effective amount of an anti-PD-1 antibody composition provided in the present disclosure.

Also provided herein are methods for treating cancer in a mammal, particularly a human, which method comprises administering to the mammal (1) an effective amount of a vaccine capable of inducing an immune response against cancer cells and (2) an effective amount of an anti-PD pharmaceutical composition. -1 antibody provided in this disclosure. In some embodiments, the composition is administered as a subcutaneous bilateral dose of about 125 to about 300 mg. In some embodiments, the composition is administered as a subcutaneous bilateral dose of 300 mg.

Brief description of the drawings

Fig. 1 is a graph comparing the viscosity of anti-PD-1 antibody formulations at various pH values.

Fig. 2 is a graph comparing the viscosity of anti-PD-1 antibody formulations with varying concentrations of arginine HCl.

Fig. 3 is a graph comparing the viscosity of an anti-PD-1 antibody formulation at various pH values.

Fig. 4 is a graph comparing the viscosity of anti-PD-1 antibody formulations when 100 mM arginine HCl is added, with or without proline.

Fig. 5 is a graph comparing the viscosity of anti-PD-1 antibody formulations 7 and 8.

Fig. 6 is a graph showing the heterogeneity of glycans in the anti-PD-1 antibody mAb7.

Fig. 7 is a graph showing the thermal properties of various anti-PD-1 antibodies in formulation 7.

Detailed description of the invention

The present application discloses compositions having reduced viscosity. Preferably, the compositions stably maintain high concentrations of the bioactive antibody in solution and are suitable for parenteral administration, including either subcutaneous, intravenous, intramuscular, intraperitoneal, or intradermal injection.

General Methods

In carrying out the present invention in practice will be used, unless otherwise indicated, the usual methods of molecular biology (including recombinant methods), microbiology, cell biology, biochemistry and immunology, which are within the competence of specialists in this field. Such methods are fully described in the literature, for example, in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995.

Definitions

The following terms, unless otherwise indicated, are to be understood as having the following meanings: The term "isolated molecule" (where a molecule is, for example, a polypeptide, polynucleotide, or antibody) is a molecule that, by virtue of its origin or the source from which it is derived , (1) is not associated with naturally associated components that accompany it in its natural state, (2) is essentially free of other molecules from the same species, (3) is expressed by a cell from another species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system other than the cell from which it naturally originates will be "isolated" from its naturally associated components. The molecule can also be made substantially free of naturally occurring components by isolation using purification methods well known in the art. The purity or homogeneity of molecules can be determined by a number of methods well known in the art. For example, analysis of the purity of a polypeptide sample can be performed using polyacrylamide gel electrophoresis and staining of the polypeptide imaging gel using techniques well known in the art. For some purposes, higher resolution can be achieved using HPLC or other purification means well known in the art.

As used herein, the terms "preparation" or "composition" when referring to an antibody are intended to describe the antibody in combination with a pharmaceutically acceptable excipient comprising at least one tonicity regulator, at least one buffer, at least one chelating agent. , at least one surfactant, where the pH is as defined above.

The terms "pharmaceutical composition" or "pharmaceutical preparation" refer to preparations which are in a form which renders effective the biological activity of the active ingredients.

"Pharmaceutical acceptable excipients" (carriers, additives) are those substances that can be safely administered to a subject to provide an effective dose of the active ingredient used. The term "excipient" or "carrier" in the context of this application refers to an inert substance that is commonly used as a diluent, filler, preservative, binder or stabilizer for drugs. In the context of the present application, the term "diluent" refers to a pharmaceutically acceptable (safe and non-toxic for human administration) solvent and useful in the present invention to obtain liquid compositions. Examples of diluents include, but are not limited to, sterile water and bacteriostatic water for injection (BWFI).

An "antibody" is an immunoglobulin molecule capable of specifically binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the context of this application, this term includes not only intact polyclonal or monoclonal antibodies, but also, unless otherwise indicated, any of their antigen-binding part that competes with an intact antibody for specific binding, fusion proteins, including an antigen-binding part, and any another modified configuration of the immunoglobulin molecule that includes an antigen recognition site. Antigen binding portions include, for example, Fab, Fab', F(ab') ₂ , Fd, Fv, domain antibodies (dAb, eg shark and camel antibodies), fragments including complementarity determining regions (CDRs), single chain variable fragments antibodies (scFv), maxibodies, minibodies, intraantibodies, diabodies, tribodies, tetrabodies, v-NARs and bis-scFvs, and polypeptides that contain at least an immunoglobulin portion that is sufficient to confer specific antigen-binding properties to the polypeptide. An antibody includes an antibody of any class such as IgG, IgA or IgM (or a subclass thereof) and the antibody need not be of any particular class. Depending on the amino acid sequence of the heavy chain constant region, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions that correspond to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and spatial configurations of the various classes of immunoglobulins are well known.

The “variable region” of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. As is known in the art, each heavy and light chain variable region consists of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions, and contributes to the formation of the antigen-binding site of antibodies. If variants of the variable region in question are desired, in particular with a substitution of amino acid residues outside the CDR (i.e., in the framework region), the appropriate amino acid substitution, preferably a conservative amino acid substitution, can be identified by comparing the variable region in question with the variable regions of other antibodies that contain the sequences of CDR1 and CDR2 of the same canonical class as the variable region in question (Chothia and Lesk, J Mol Biol 196 (4): 901-917, 1987).

In some embodiments, final clarification of the CDR and identification of the residues constituting the binding site of the antibody is performed by establishing the structure of the antibody and/or establishing the structure of the antibody-ligand complex. In some embodiments, this can be done by any of a variety of methods known to those skilled in the art, such as X-ray crystallography. In some embodiments, various analysis methods may be used to identify or approximate CDR regions. Examples of such methods include, but are not limited to, Kabat determination, Chothia determination, AbM determination, contact determination, and conformational determination.

The Kabat definition is the standard for numbering residues in an antibody and is commonly used to identify CDR regions. See, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the positions of certain structural loop regions. See, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. For determination by the AbM method, an integrated set of computer programs created by the Oxford Molecular Group is used, which model the structure of antibodies. See, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; “AbM™, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular Ltd. AbM definition models the tertiary structure of an antibody from a primary sequence using a combination of knowledge base information and ab initio methods, such as those described by Samudrala et al., 1999, “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. The contact definition is based on the analysis of existing complex crystal structures. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to above as "conformational definition" of CDRs, positions of CDRs can be identified as residues that contribute enthalpy to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Some other definitions of CDRs boundaries may not be in strict accordance with one of the above approaches, but will nonetheless overlap with at least a portion of Kabat's CDRs, although they may be shortened or lengthened to account for predicted or obtained experimental data, which specific residues or groups of residues do not significantly affect antigen binding. In the context of this application, the term CDR may refer to CDRs determined using any approach known in the art, including combinations of approaches. The methods used in the present invention may use CDRs determined using any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, contact, and/or conformational definitions.

As known in the art, "constant region" of an antibody refers to an antibody light chain constant region or an antibody heavy chain constant region, alone or in combination.

In the context of the present application, the term "monoclonal antibody" refers to an antibody derived from a population of essentially homogeneous antibodies, ie. the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The term "monoclonal" indicates the nature of an antibody as derived from a substantially homogeneous population of antibodies and should not be construed as requiring the production of an antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be prepared by recombinant DNA methods such as those described in US Pat. No. 4,816,567. Monoclonal antibodies are also can be isolated from phage libraries obtained using the methods described, for example, in McCafferty et al., 1990, Nature 348:552-554. In the context of the present application, a "humanized" antibody refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen- binding antibody subsequences) that contain a minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the CDR of the recipient are replaced by residues from the CDR of a non-human antibody (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and activity. The humanized antibody may include residues that are not found in the recipient antibody or in the imported CDR or framework sequences, but which are included to further improve and optimize the performance of the antibody.

A "human antibody" is an antibody that has an amino acid sequence that matches that of an antibody produced by a human and/or that is generated using any of the methods for producing a human antibody disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising antigen-binding residues of a non-human species.

In the context of the present application, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro non-specific or site-directed mutagenesis or by in vivo somatic mutation), for example, in CDRs and especially in CDR3. However, the term "human antibody" in the context of the present application is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "chimeric antibody" refers to antibodies in which the variable region sequences are from one species and the constant region sequences are from another species, such as an antibody in which the e variable region sequences are from a mouse antibody and the constant region sequences are from a human antibodies.

In the context of the present application, a "humanized" antibody refers to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other antigen -binding antibody subsequences) that contain a minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit, with the desired specificity, affinity and activity. In some cases, the framework region (FR) residues of the Fv human immunoglobulin are replaced by corresponding residues from a non-human species. In addition, the humanized antibody may include residues that do not occur in either the recipient antibody or the imported CDR or framework sequences, but are included to further improve and optimize the performance of the antibody. Typically, a humanized antibody will include substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the CDR regions correspond to non-human immunoglobulin regions and all or substantially all of the FR regions are derived from the consensus sequence human immunoglobulin. A humanized antibody will also optimally include at least a portion of the constant region or domain (Fc) of an immunoglobulin, typically a human immunoglobulin. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are altered from the parent antibody, also referred to as one or more CDRs "derived from" one or several CDRs from the original antibody.

In the context of the present application, the term "mAb7" is used as referring to an anti-PD-1 antibody comprising the amino acid sequence of the heavy chain and light chain variable region shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

Amino acid sequence of mAb7 heavy chain variable region:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSLTNYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTLVTVSS (SEQ ID NO: 2)

mAb7 light chain variable region amino acid sequence:

DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKPGQPPKLLIYWTSYRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPHTFGGGTKVEIK (SEQ ID NO: 3)

Generation and characterization of mAb7 are described in the Examples of WO2016/092419, the entire content of which is incorporated herein by reference. In some embodiments, the term "mAb7" refers to an immunoglobulin encoded by (a) a mAb7 light chain variable region-encoding polynucleotide that has ATCC No. PTA-121182, and (b) a polynucleotide encoding the mAb7 heavy chain variable region, which has the deposit number ATCC No. PTA-121183.

The term "epitope" refers to that portion of a molecule that can be recognized and bound by an antibody at one or more antigen-binding sites on the antibody. Epitopes often consist of surface groups of molecules, such as amino acids or sugar side chains, and have certain spatial structural characteristics as well as certain charge characteristics. In some embodiments, the epitope may be a protein epitope. Protein epitopes may be linear or conformational. In a linear epitope, all points of interaction between a protein and an interacting molecule (eg, an antibody) are located linearly along the protein's primary amino acid sequence. A "non-linear epitope" or "conformational epitope" includes non-contiguous polypeptides (or amino acids) in an antigenic protein to which an antibody specific for that epitope binds. The term "antigenic epitope" in the context of the present application is defined as the portion of an antigen to which an antibody can specifically bind, which can be determined by any method well known in the art, such as conventional immunoassays. Once a desired epitope has been determined on an antigen, antibodies to that epitope can be generated, for example, using the methods described herein. Alternatively, generation and characterization of antibodies can provide information about the desired epitopes during the research process. Based on this information, antibodies can then be competitively screened for binding to the same epitope. An approach to achieve this includes performing competition and cross-competition studies to look for antibodies that compete or cross-compete with each other for binding to PD-1, eg, antibodies compete for binding to an antigen.

In the context of this application, the terms "isolated antibody" or "purified antibody" refers to an antibody that, by virtue of its origin or the source from which it is derived, has one to four of the following characteristics: (1) not associated with naturally associated components, concomitant to it in its natural state, (2) is free from other proteins from the same species, (3) is expressed by a cell from another species, or (4) is not naturally present.

An antibody is "substantially pure", "substantially homogeneous", or "substantially purified" when at least about 60-75% of the sample exhibits a single antibody species. Substantially pure antibody can typically be about 50%, 60%, 70%, 80%, or 90% w/w of the antibody sample, more typically about 95%, and will preferably be greater than 99% pure. The purity or homogeneity of antibodies can be determined by various methods well known in the art, such as polyacrylamide gel electrophoresis or HPLC.

The term "antibody" refers to an antibody that binds to a target and prevents or reduces the biological effect of that target. In some embodiments, the term may mean an antibody that prevents the target, such as PD-1, to which it binds, from performing a biological function.

An antibody that "preferentially binds" or "specifically binds" (used interchangeably herein) to an epitope is a term well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is referred to as exhibiting "specific binding" or "preferential binding" if it interacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and/or with greater affinity than with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds to it with greater affinity, avidity, more easily and/or for longer duration than with other substances. For example, an antibody that specifically or preferentially binds to a PD-1 epitope is an antibody that binds to a sequence of that epitope with greater affinity, avidity, more readily, and/or longer duration than it binds to other sequences. It should also be understood from this definition that, for example, an antibody (or fragment or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, “specific binding” or “preferential binding” does not necessarily require (though may include) exclusive binding. Typically, but not necessarily, "binding" means predominantly binding.

In the context of this application, "immunospecific" antibody binding refers to an antigen-specific binding interaction that occurs between the antigen-binding site of an antibody and a specific antigen recognized by that antibody (i.e., the antibody interacts with a protein in an ELISA or other immunoassay and does not interact with a detectable way with unrelated proteins).

The term "compete" as used herein in relation to an antibody means that the first antibody or antigen-binding fragment thereof binds to an epitope in a manner sufficiently similar to the binding of the second antibody or antigen-binding fragment thereof, thus the result of binding of the first an antibody with its cognate epitope is markedly reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternatively, it may be, although not necessarily, that the binding of the second antibody to its epitope is also markedly reduced in the presence of the first antibody. That is, the first antibody can inhibit the binding of the second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its corresponding epitope. However, when each antibody markedly inhibits the binding of another antibody to its cognate epitope or ligand to the same, greater or lesser extent, the antibodies are said to "cross-compete" with each other for binding to their respective epitope(s). Both competitive and cross-competing antibodies are covered by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), those skilled in the art will recognize from the disclosure provided herein that such competing and/ or cross-competing antibodies are covered or may be useful for the methods disclosed in the present invention.

In the context of this application, the term "PD-1" refers to any form of PD-1 and its variants, which retain at least part of the activity of PD-1. Unless otherwise noted, for example when specifically referring to human PD-1, PD-1 includes the native PD-1 sequence of all mammalian species, eg, human, canine, feline, equine, and bovine. One exemplary human PD-1 can be found as Uniprot Accession Number Q15116 (SEQ ID NO: 1).

MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA

TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE

VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI

GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVVP CVPEQTEYAT

IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL (SEQ ID NO: 1).

In the context of the present application, "anti-PD-1 antibody" refers to an antibody that is capable of inhibiting the biological activity of PD-1 and/or downstream events mediated by PD-1. Anti-PD-1 antibodies encompass antibodies that block, antagonize, suppress or reduce (to any extent, including substantially) the biological activity of PD-1, including downstream events mediated by PD-1, such as binding to PD-L1 and downstream signals, PD-L2 binding and downstream signals, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10, and inhibition of antitumor immune responses. For the purposes of the present invention, it is to be clearly understood that the term “anti-PD-1 antibody” (used interchangeably with the term “anti-PD-1 antibody”) encompasses all previously defined terms, names and functional states and characteristics, whereby PD- 1 as such, the biological activity of PD-1 or the consequences of biological activity is essentially nullified, reduced or neutralized to any significant extent. In some embodiments, an anti-PD-1 antibody binds to PD-1 and activates an antitumor immune response. Examples of anti-PD-1 antibodies are presented in this application.

The term "identity" refers to the percentage of "identity" of two amino acid sequences or two nucleic acid sequences. Percent identity is usually determined by aligning sequences for optimal comparison purposes (eg, gaps may be introduced in the first sequence to best align with the second sequence) and comparing amino acid residues or nucleotides at the appropriate positions. "Best alignment" means the alignment of two sequences that gives the highest percentage of identity. Percent identity is determined by comparing the number of identical amino acid residues or nucleotides in sequences (i.e. % identity=number of identical positions/total number of positions × 100)

Determining the percent identity between two sequences can be done using a mathematical algorithm known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm by Karlin and Altschul (1990) Proc. Natl. Acad. sci. USA 87:2264-2268, with modifications as described in Karlin and Altschul (1993) Proc. Natl. Acad. sci. USA 90:5873-5877. NBLAST and XBLAST programs Altschul, et al (1990) J. Mol. Biol. 215:403-410 include such an algorithm. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. Searches for BLAST proteins can be performed using the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucliec Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used for re-searching that detects distant relationships between molecules (Id.). When using the BLAST, Gapped BLAST and PSI-Blast programs, you can use the default settings of the respective programs (eg XBLAST and NBLAST). See http://www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm used for sequence comparison is the Myers and Miller algorithm, CABIOS (1989). The ALIGN program (version 2.0), which is part of the GCG sequence alignment software package, includes such an algorithm. Other sequence analysis algorithms known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci. 10:3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. sci. 85:2444-8. In FASTA, ktup means a control option that sets the sensitivity and search speed.

In the context of this application, the term "treatment" means an approach to obtain useful or desirable clinical results. For the purposes of the present invention, useful or desirable clinical results include, but are not limited to, one or more of the following: reduced proliferation (or destruction) of neoplastic or cancerous cells, inhibition of neoplastic cell metastasis, shrinkage or reduction in tumor size, remission of cancer, reduction in symptoms caused by cancer, improving the quality of life of people suffering from cancer, reducing the dose of other drugs needed to treat cancer, slowing down the progression of cancer, curing cancer and/or increasing the life expectancy of cancer patients.

In the context of the present application, an "effective dose" or "effective amount" of a drug, compound or pharmaceutical composition is an amount sufficient to achieve any one or more useful or desirable results. In more specific aspects, an effective amount prevents, alleviates or ameliorates the symptoms of a disease and/or prolongs the life of the patient being treated. For prophylactic use, useful or desirable results include the elimination or reduction of risk, reduction in severity or delay in onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications, and intermediate phenotypic manifestations observed during the development of the disease. For therapeutic use, useful or desirable results include clinical results such as reduction in one or more symptoms of a disease such as, for example, cancer, including, for example, without limitation, gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer. , head and neck squamous cell carcinoma, thymus cancer, epithelial cancer, salivary gland cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cavity cancer, skin cancer and melanoma, reduction in the dose of other drugs needed to treat the disease, increased effect of another drug and / or delay cancer progression in patients. An effective dose may be administered in one or more administrations. For the purposes of the present invention, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to effect prophylactic or therapeutic treatment, either directly or indirectly. As understood in a clinical context, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective dose" may be considered in the context of the administration of one or more therapeutic agents, and a single agent is considered to be administered in an effective amount if, in combination with one or more other agents, the desired result can be or is achieved.

In the context of the present application, the term "subject" for purposes of treatment includes any subject, and preferably is a subject in need of treatment of the target pathological condition, such as an autoimmune disease. For prophylactic purposes, a subject is any subject, and preferably a subject, that is at risk or predisposed to developing the target pathological condition. The term "subject" is intended to include living organisms, such as prokaryotes and eukaryotes. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and non-human transgenic animals. In specific embodiments of the invention, the subject is a human.

In the context of this application, the terms "polynucleotide" or "nucleic acid", used interchangeably in this application, means the polymeric form of nucleotides, both ribonucleotides and deoxynucleotides, or a modified form of any type of nucleotide, and may mean a single-stranded or double-stranded form. The sequence of a "polynucleotide" or "nucleic acid" includes its complement unless otherwise indicated. In the context of this application, the term "isolated polynucleotide" or "isolated nucleic acid" means a polynucleotide of genomic, cDNA or synthetic origin, or some combination thereof, and by virtue of its origin or source from which it comes, the isolated polynucleotide has one to three of the following characteristics: (1) is not associated with all or part of a polynucleotide with which the "isolated polynucleotide" occurs naturally, (2) is operably associated with a polynucleotide with which it is not naturally associated, or (3) does not occur naturally as part of a larger large sequence.

In the context of this application, the term "chelating agent" is an excipient that can form at least one bond (eg, covalent, ionic, or otherwise) with a metal ion. The chelating agent is typically a multidentate ligand that can be used in compositions as a stabilizer to complex with species that might otherwise contribute to instability.

In the context of the present application, the term "buffer" refers to an added composition that allows the liquid composition of the antibody to resist changes in pH, typically through the action of the components of its acid-base conjugate. When the concentration of a buffer is indicated, the indicated concentration is understood to be the molar concentration of the free acid form or free base form of the buffer.

"Viscosity" in the context of this application may mean "absolute viscosity" or "kinematic viscosity". "Absolute viscosity", sometimes referred to as dynamic or simple viscosity, is a quantity that describes the resistance of a fluid to flow. "Kinematic viscosity" is the ratio of the absolute viscosity and the density of a liquid. Kinematic viscosity is often determined by characterizing resistive fluid flow using a capillary viscometer. When two liquids of equal volume are placed in identical capillary viscometers and flow under the force of gravity, the viscous liquid takes longer to flow through the capillary than the less viscous liquid. If one fluid takes 200 seconds to completely flow and another fluid takes 400 seconds, the second fluid will be twice as viscous as the first on the kinematic viscosity scale. If both fluids have the same density, the second fluid is twice as viscous as the first on the absolute viscosity scale. The value of the kinematic viscosity is determined in L ² /T, where L is the length and T is the time. The SI units for kinematic viscosity are m ² /sec. Typically, kinematic viscosity is expressed in centistokes, cSt, which is equivalent to mm ² /sec. The value of absolute viscosity is defined in M/L/T, where M is mass and L and T are length and time, respectively. The SI units for absolute viscosity are Pa⋅sec, which is equivalent to kg/m/sec. Absolute viscosity is usually expressed in centipoise, cps, which is equivalent to milliPascal-second, mPa⋅sec.

As used herein, the terms "tonicity regulator" or "tonicity modifier" refer to an excipient that can control the osmotic pressure of a liquid antibody composition. In some embodiments, the tonicity regulator may adjust the osmotic pressure of the liquid antibody composition to isotonic so that the antibody composition is physiologically compatible with tissue cells in the subject's body. In other embodiments, the implementation of the "regulator of tonicity" can help improve the stability of the antibodies described in this application. An "isotonic" composition is a composition that has essentially the same osmotic pressure as human blood. Isotonic compositions typically have an osmotic pressure of about 250 to 350 mOsm. The term "hypotonic" describes a formulation with an osmotic pressure below that of human blood. Accordingly, the term "hypertonic" is used to describe a composition with an osmotic pressure greater than that of human blood. Isotonicity can be measured using a vapor pressure osmometer or a freezing point osmometer, for example. The tonicity regulator may be in enantiomeric (eg L- or D-enantiomer) or racemic form; in the form of isomers such as alpha or beta, including alpha, alpha; or beta, beta; or alpha, beta; or beta, alpha; in the form of free acid or free base; in hydrated form (eg monohydrate) or in anhydrous form.

In the context of the present application, the term "polyol" refers to an excipient with multiple hydroxyl groups and includes sugars (reducing and non-reducing sugars), sugar alcohols and sugar acids.

In the context of this application, the term "surfactant" refers to an excipient that can change the surface tension of the liquid composition of the antibody. In some embodiments, the implementation of the surfactant reduces the surface tension of the liquid composition of the antibody. In other embodiments, the implementation of the "surfactant" can help improve the stability of any antibody in the composition. The surfactant may reduce aggregation of the formulated antibody and/or minimize particle formation in the composition and/or reduce adsorption. The surfactant may also improve the stability of the antibody during and after the freeze/thaw cycle.

In the context of this application, the term "saccharide" refers to a class of molecules that are derivatives of polyhydric alcohols. Saccharides are commonly referred to as carbohydrates and may contain varying amounts of sugar (saccharide) units, such as monosaccharides, disaccharides and polysaccharides.

In the context of this application, the term "reducing sugar" means a sugar containing a hemiacetal group that can reduce metal ions or interact covalently with lysine and other amino groups in proteins, and "non-reducing sugar" is a sugar that does not have these properties of a reducing sugar.

A "lyoprotectant" is a molecule which, in combination with a protein of interest, significantly prevents or reduces the physicochemical instability of the protein during lyophilization and subsequent storage. Examples of lyoprotectants include sugars and their respective sugar alcohols; an amino acid such as monosodium glutamate or histidine; methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric sugar alcohols or higher molecular weight sugar alcohols such as glycerol, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol; Pluronics®; and their combinations. Additional exemplary lyoprotectants include glycerin and gelatin, as well as sugars, mellibiose, melecytose, raffinose, mannotriose, and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other straight chain polyhydric alcohols. Preferred sugar alcohols are monoglycosides, especially those compounds which are obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. The glycosidic side group may be glucosidic or galactosidic. Additional examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. Preferred lyoprotectants are the non-reducing sugars trehalose or sucrose.

The lyoprotectant is added to the pre-lyophilized composition in a "lyoprotective amount", which means that after the protein is lyophilized in the presence of the lyoprotective amount of the lyoprotectant, the protein substantially retains its physicochemical stability during lyophilization and storage.

In the context of the present application, the term "pharmaceutically acceptable carrier" includes any substance which, in combination with the active ingredient, allows the ingredient to retain biological activity and does not interact with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as an oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline, normal (0.9%) saline, or 5% dextrose. Compositions comprising such carriers are prepared by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th ^Ed . Mack Publishing, 2000).

"Reduction in incidence" means any reduction in severity (which may include a reduction in the need and/or amount (e.g., exposure) of other drugs and/or therapies commonly used for that condition. As will be appreciated by those skilled in the art, individuals may differ with regard to their responses to treatment, and thus, for example, a "method to reduce incidence" reflects administration of an anti-PD-1 antibody based on the reasonable expectation that such administration is likely to cause such a reduction in incidence in that particular individual.

"Improvement" means a reduction or improvement in one or more symptoms compared to when no anti-PD-1 antibody is administered. "Improvement" also includes a reduction or reduction in the duration of a symptom.

Reference to "about" a value or parameter in this application includes (and describes) embodiments that are directed to that value or parameter as such. For example, a description referring to "about X" includes a description of "X". Numeric ranges include numbers that define the range.

Where aspects or embodiments of the invention are described in terms of the Markush group or other grouping of alternatives, the present invention covers not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, as well as the main group, where one or more group members are missing. The present invention also provides for the explicit exclusion of one or more members of the group in the claimed invention.

When presenting the elements of the present invention or its preferred embodiments, the articles "a", "an", "the" and the word "specified" are intended to indicate that there is one or more elements. The terms "including", "include", "includes", "including" and "comprising" are intended to be inclusive and mean that there may be additional elements other than those listed. It should be understood that if embodiments are described herein using the term "comprising", similar embodiments described using the terms "consisting of" and/or "consisting essentially of" are also contemplated.

Unless otherwise indicated, all technical and scientific terms used in this application have the same meaning as generally understood by a person skilled in the art to which the present invention belongs. In case of conflict, the present description, including definitions, shall prevail. In the present specification and claims, the word "comprise" or variations such as "comprises" or "comprising" should be understood to mean the inclusion of the specified integer or group of integers, but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms must include the plural, and plural terms must include the singular.

This application describes illustrative methods and materials, however, methods and materials similar or equivalent to those described in this application can also be used in the implementation or testing of the present invention. The materials, methods, and examples are illustrative only and are not intended to be limiting.

Compositions of anti-PD-1 antibodies

In one aspect, the invention provides a composition comprising an anti-PD-1 antibody that has a viscosity of about 1 centipoise to about 25 centipoise. In another aspect, a method is provided for reducing the viscosity of an anti-PD-1 antibody-containing composition, the method comprising the step of adding to the composition a viscosity-reducing amount of a compound that is capable of reducing the viscosity of an aqueous composition comprising said anti-PD-1 antibody. The composition may be either in aqueous or lyophilized form. In aqueous form, the composition may have a viscosity of no more than about 150 cps, preferably no more than about 120 cps, preferably no more than about 100 cps, preferably no more than about 90 cps, preferably no more than about 80 cps, preferably no more than about 70 cps, preferably no more than about 60 cps, preferably no more than about 50 cps, preferably no more than about 40 cps, preferably no more than about 30 cps, preferably no more than about 20 cps, preferably no more than about 10 centipoise, preferably no more than about 5 centipoise. In some embodiments, a composition comprising an antibody has a viscosity ranging from about 1 cP to about 500 cP, between about 1 cP and 200 cP, between about 1 cP and about 150 cP, between about 1 cP and about 100 cP, between about 1 cP and about 90 cP, between about 1 cP and about 80 cP, between about 1 cP and about 70 cP, between about 1 cP and about 60 cP, between about 1 cP and about 50 cP, between about 1 cP and about 40 centipoise, between about 1 centipoise and about 30 centipoise, between about 1 centipoise and about 20 centipoise, or between about 1 centipoise and about 10 centipoise at 20°C. In some embodiments, the composition has a viscosity of about 120 centipoise, about about 115 centipoise, 110 centipoise, about 105 centipoise, about 100 centipoise, about 95 centipoise, about 90 centipoise, about 85 centipoise, about 80 centipoise, about 75 centipoise, about 70 centipoise , approximately 65 cP, approximately 60 cP, approximately 55 cP, 50 cP, approximately 45 cP, approximately 40 cP, approximately 35 cP, approximately 30 cP, approximately 25 cP, approximately 20 cP, approximately 5 cps. In some embodiments, the composition has a viscosity in the range between 10 cPs and 50 cPs, between about 10 cPs and 100 cPs, between about 20 cPs and 60 cPs, between about 30 cPs and 60 cPs, between about 40 cPs and 60 cPs, or between about 50 cps and 60 cps. In some embodiments, the composition in aqueous form may have a viscosity between about 1 centipoise and 10 centipoise. In some embodiments, the composition in aqueous form may have a viscosity between about 1 centipoise and 15 centipoise. In some embodiments, the composition in aqueous form may have a viscosity between about 1 centipoise and 20 centipoise.

Another aspect of the present invention is directed to a product, including a container containing any of the compositions described in this application.

In some embodiments, the composition includes at least one anti-PD-1 antibody. In some embodiments, more than one antibody may be present. At least one, at least two, at least three, at least four, at least five or more different antibodies may be present. Typically, two or more different antibodies have complementary activities that do not adversely affect each other. Each of the antibodies can also be used in conjunction with other agents that serve to enhance and/or supplement the effectiveness of the antibody. The antibody may be present in the composition at a concentration of from about 0.1 mg/mL to about 300 mg/mL. In some embodiments, the antibody concentration is about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml, about 2.5 mg/ml, about 3 mg/ml, about 3.5 mg/ml, about 4 mg/mL, approximately 4.5 mg/mL, approximately 5 mg/mL, approximately 5.5 mg/mL, approximately 6 mg/mL, approximately 6.5 mg/mL, approximately 7 mg/mL, approximately 7, 5 mg/mL, approximately 8 mg/mL, approximately 8.5 mg/mL, approximately 9 mg/mL, approximately 9.5 mg/mL, approximately 10 mg/mL, approximately 11 mg/mL, approximately 12 mg/mL , approximately 13 mg/mL, approximately 14 mg/mL, approximately 15 mg/mL, approximately 16 mg/mL, approximately 17 mg/mL, approximately 18 mg/mL, approximately 19 mg/mL, approximately 20 mg/mL, approximately 21 mg/mL, approximately 22 mg/mL, approximately 23 mg/mL, approximately 24 mg/mL, approximately 25 mg/mL, approximately 26 mg/mL, approximately 27 mg/mL, approximately 28 mg/mL, approx. positively 29 mg/mL, approximately 30 mg/mL, approximately 31 mg/mL, approximately 32 mg/mL, approximately 33 mg/mL, approximately 34 mg/mL, approximately 35 mg/mL, approximately 36 mg/mL, approximately 37 mg/mL, approximately 38 mg/mL, approximately 39 mg/mL, approximately 40 mg/mL, approximately 41 mg/mL, approximately 42 mg/mL, approximately 43 mg/mL, approximately 44 mg/mL, approximately 45 mg/mL ml, about 46 mg/ml, about 47 mg/ml, about 48 mg/ml, about 49 mg/ml, about 50 mg/ml, about 51 mg/ml, about 52 mg/ml, about 53 mg/ml, approximately 54 mg/mL, approximately 55 mg/mL, approximately 56 mg/mL, approximately 57 mg/mL, approximately 58 mg/mL, approximately 59 mg/mL, approximately 60 mg/mL, approximately 70 mg/mL, approximately 80 mg/mL, approximately 90 mg/mL, approximately 100 mg/mL, approximately 101 mg/mL, approximately 102 mg/mL, approximately 102.5 mg/mL, approximately 103 mg/mL, about 103.5 mg/ml, about 104 mg/ml, about 104.5 mg/ml, about 105 mg/ml, about 105.5 mg/ml, about 106 mg/ml, about 106.5 mg/ml, approximately 107 mg/mL, approximately 107.5 mg/mL, approximately 108 mg/mL, approximately 108.5 mg/mL, approximately 109 mg/mL, approximately 109.5 mg/mL, approximately 110 mg/mL, approximately 111 mg/mL, approximately 112 mg/mL, approximately 113 mg/mL, approximately 114 mg/mL, approximately 115 mg/mL, approximately 116 mg/mL, approximately 117 mg/mL, approximately 118 mg/mL, approximately 119 mg/mL ml, about 120 mg/ml, about 121 mg/ml, about 122 mg/ml, about 123 mg/ml, about 124 mg/ml, about 125 mg/ml, about 126 mg/ml, about 127 mg/ml, approximately 128 mg/mL, approximately 129 mg/mL, approximately 130 mg/mL, approximately 131 mg/mL, approximately 132 mg/mL, approximately 133 mg/mL, approximately 134 mg/m l, about 135 mg/ml, about 136 mg/ml, about 137 mg/ml, about 138 mg/ml, about 139 mg/ml, about 140 mg/ml, about 141 mg/ml, about 142 mg/ml, approximately 143 mg/mL, approximately 144 mg/mL, approximately 145 mg/mL, approximately 146 mg/mL, approximately 147 mg/mL, approximately 148 mg/mL, approximately 149 mg/mL, approximately 150 mg/mL, approximately 151 mg/mL, approximately 152 mg/mL, approximately 153 mg/mL, approximately 154 mg/mL, approximately 155 mg/mL, approximately 156 mg/mL, approximately 157 mg/mL, approximately 158 mg/mL, approximately 159 mg/mL ml, approximately 160 mg/ml, approximately 170 mg/ml, approximately 180 mg/ml, approximately 190 mg/ml, approximately 200 mg/ml, approximately 201 mg/ml, approximately 202 mg/ml, approximately 202.5 mg/ml ml, about 203 mg/ml, about 203.5 mg/ml, about 204 mg/ml, about 204.5 mg/ml, about 205 mg/ml, at about 205.5 mg/ml, about 206 mg/ml, about 206.5 mg/ml, about 207 mg/ml, about 207.5 mg/ml, about 208 mg/ml, about 208.5 mg/ml, about 209 mg/ml, about 209.5 mg/ml, about 210 mg/ml, about 211 mg/ml, about 212 mg/ml, about 213 mg/ml, about 214 mg/ml, about 215 mg/ml, approximately 216 mg/mL, approximately 217 mg/mL, approximately 218 mg/mL, approximately 219 mg/mL, approximately 220 mg/mL, approximately 221 mg/mL, approximately 222 mg/mL, approximately 223 mg/mL, approximately 224 mg/mL, approximately 225 mg/mL, approximately 226 mg/mL, approximately 227 mg/mL, approximately 228 mg/mL, approximately 229 mg/mL, approximately 230 mg/mL, approximately 231 mg/mL, approximately 232 mg/mL ml, approx. 233 mg/ml, approx. 234 mg/ml, approx. 235 mg/ml, approx. 236 mg/ml, approx. 237 mg/ml, approx. 238 mg/ml, approx. 239 mg/mL, approximately 240 mg/mL, approximately 241 mg/mL, approximately 242 mg/mL, approximately 243 mg/mL, approximately 244 mg/mL, approximately 245 mg/mL, approximately 246 mg/mL, approximately 247 mg/mL, approximately 248 mg/mL, approximately 249 mg/mL, approximately 250 mg/mL, approximately 251 mg/mL, approximately 252 mg/mL, approximately 253 mg/mL, approximately 254 mg/mL, approximately 255 mg/mL ml, about 256 mg/ml, about 257 mg/ml, about 258 mg/ml, about 259 mg/ml, about 260 mg/ml, about 270 mg/ml, about 280 mg/ml, about 290 mg/ml approximately 300 mg/ml.

In accordance with some embodiments of the present invention, the pH may be in the range of about pH 4.0-6.0, preferably between about pH 5.0 and any of about pH 4.5, about 4.6, about 4.7 , about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, or about 5.5. More preferably, the pH is within a range chosen between any of approximately pH 4.9, 5.0, or 5.1. In some embodiments, the pH is pH 5.0 +/- 0.5. pH values within these ranges provide a composition with lower viscosity values.

In some embodiments, the implementation of the composition may include arginine. In some embodiments, the arginine is arginine hydrochloride or arginine HCl. [Bryan: although Composition 7 does not contain arginine, we have included it as an alternative. The same applies to other excipients, etc., not present in composition 7, but included in the present description]. The arginine concentration may range from about 0.1 millimoles (mM) to about 200 mM. In some embodiments, the concentration of arginine is from about 10 mM to about 150 mM, from about 50 mM to about 130 mM, from about 80 mM to about 120 mM, or from about 90 mM to about 110 mM. In some embodiments, the arginine concentration is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM , about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, approximately 25 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM, approximately 55 mM, approximately 60 mM, approximately 65 mM, approximately 70 mM, approximately 75 mM, approximately 80 mM , approximately 85 mM, approximately 90 mM, a approximately 95 mM, approximately 100 mM, approximately 105 mM, approximately 110 mM, approx. but 115 mM, approximately 120 mM, approximately 125 mM, approximately 130 mM, approximately 135 mM, approximately 140 mM, approximately 145 mM, approximately 150 mM, approximately 155 mM, approximately 160 mM, approximately 165 mM, approximately 170 mM, approximately 175 mM, approximately 180 mM, approximately 185 mM, approximately 190 mM, approximately 195 mM, or approximately 200 mM. In some embodiments, the arginine concentration is 100 mM.

In some embodiments, the tonicity regulator may include a polyol, a saccharide, a carbohydrate, a salt such as sodium chloride, or mixtures thereof. The polyol may have a molecular weight that is, for example, without limitation, less than about 600 kDa (for example, in the range from about 120 to about 400 kDa) and can be, for example, without limitation, mannitol, trehalose, sorbitol, erythritol, isomalt , lactitol, maltitol, xylitol, glycerin, lactitol, propylene glycol, polyethylene glycol, inositol, or mixtures thereof. The saccharide or carbohydrate may be, for example, without limitation, a monosaccharide, disaccharide or polysaccharide, or mixtures of any of the above. The saccharide or carbohydrate may be, for example, without limitation, fructose, glucose, mannose, sucrose, sorbose, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrins, soluble starch, hydroxyethyl starch, water-soluble glucans, or mixtures thereof. The tonicity regulator may include a saccharide, such as, for example, without limitation, a reducing sugar or a non-reducing sugar, or mixtures thereof. The tonicity regulator may include a saccharide that is a non-reducing sugar, such as, for example, without limitation, sucrose, trehalose, and mixtures thereof.

The concentration of the tonicity regulator in the composition ranges from about 1 mg/mL to about 300 mg/mL, from about 1 mg/mL to about 200 mg/mL, or from about 1 mg/mL to about 100 mg/mL. Preferably, the concentration of the tonicity regulator in the composition is about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml, about 2.5 mg/ml, about 3 mg/ml, about 3.5 mg/ml, about 4 mg/mL, approximately 4.5 mg/mL, approximately 5 mg/mL, approximately 5.5 mg/mL, approximately 6 mg/mL, approximately 6.5 mg/mL, approximately 7 mg/mL, approximately 7, 5 mg/mL, approximately 8 mg/mL, approximately 8.5 mg/mL, approximately 9 mg/mL, approximately 9.5 mg/mL, approximately 10 mg/mL, approximately 11 mg/mL, approximately 12 mg/mL , approximately 13 mg/mL, approximately 14 mg/mL, approximately 15 mg/mL, approximately 16 mg/mL, approximately 17 mg/mL, approximately 18 mg/mL, approximately 19 mg/mL, approximately 20 mg/mL, approximately 21 mg/mL, approximately 22 mg/mL, approximately 23 mg/mL, approximately 24 mg/mL, approximately 25 mg/mL, approximately 26 mg/mL, approximately 27 mg/mL, approximately 28 mg/mL, approximately 29 mg/mL, approximately 30 mg/mL, approximately 31 mg/mL, approximately 32 mg/mL, approximately 33 mg/mL, approximately 34 mg/mL, approximately 35 mg/mL, approximately 36 mg/mL, approximately 37 mg/mL, approximately 38 mg/mL, approximately 39 mg/mL, approximately 40 mg/mL, approximately 41 mg/mL, approximately 42 mg/mL, approximately 43 mg/mL, approximately 44 mg/mL, approximately 45 mg/mL ml, about 46 mg/ml, about 47 mg/ml, about 48 mg/ml, about 49 mg/ml, about 50 mg/ml, about 51 mg/ml, about 52 mg/ml, about 53 mg/ml, approximately 54 mg/mL, approximately 55 mg/mL, approximately 56 mg/mL, approximately 57 mg/mL, approximately 58 mg/mL, approximately 59 mg/mL, approximately 60 mg/mL, approximately 65 mg/mL, approximately 70 mg/mL, approximately 75 mg/mL, approximately 80 mg/mL, approximately 81 mg/mL, approximately 82 mg/mL, approximately 83 mg/mL, approximately 84 mg/mL, approximately 85 mg/mL, approximately 86 mg/mL, approximately 87 mg/mL, approximately 88 mg/mL, approximately 89 mg/mL, approximately 90 mg/mL, approximately 91 mg/mL, approximately 92 mg/mL, approximately 93 mg/mL, approximately 94 mg/mL, approximately 95 mg/mL, approximately 96 mg/mL, approximately 97 mg/mL, approximately 98 mg/mL, approximately 99 mg/mL, approximately 100 mg/mL, approximately 101 mg/mL ml, about 102 mg/ml, about 103 mg/ml, about 104 mg/ml, about 105 mg/ml, about 106 mg/ml, about 107 mg/ml, about 108 mg/ml, about 109 mg/ml, approximately 110 mg/mL, approximately 111 mg/mL, approximately 112 mg/mL, approximately 113 mg/mL, approximately 114 mg/mL, approximately 115 mg/mL, approximately 116 mg/mL, approximately 117 mg/mL, approximately 118 mg/mL, approximately 119 mg/mL, approximately 120 mg/mL, approximately 121 mg/mL, approximately 122 mg/mL, approximately 123 mg/mL, n approximately 124 mg/mL, approximately 125 mg/mL, approximately 126 mg/mL, approximately 127 mg/mL, approximately 128 mg/mL, approximately 129 mg/mL, approximately 130 mg/mL, approximately 131 mg/mL, approximately 132 mg/mL, approximately 133 mg/mL, approximately 134 mg/mL, approximately 135 mg/mL, approximately 136 mg/mL, approximately 137 mg/mL, approximately 138 mg/mL, approximately 139 mg/mL, approximately 140 mg/mL ml, about 141 mg/ml, about 142 mg/ml, about 143 mg/ml, about 144 mg/ml, about 145 mg/ml, about 146 mg/ml, about 147 mg/ml, about 148 mg/ml, about 149 mg/ml or about 150 mg/ml.

The surfactant may be, for example, without limitation, polysorbate, poloxamer, triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glycoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl Sarcosine, Linoleyl Sarcosine, Stearyl Sarcosine, Linoleyl Betaine, Myristyl Betaine, Cetyl Betaine, Lauroamidopropyl Betaine, Cocamidopropyl Betaine, Linoleamidopropyl Betaine, Myristamidopropyl Betaine, Palmidopropyl Betaine, Isostearamidopropyl Betaine, Myristamidopropyl Dimethylamine, palmidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl taurate, disodium methyl oleoyl taurate, dihydroxypropyl PEG 5 linoleammonium chloride, polyethylene glycol, polypropylene glycol and mixtures thereof. The surfactant may be, for example, without limitation, Polysorbate 20, Polysorbate 21, Polysorbate 40, Polysorbate 60, Polysorbate 61, Polysorbate 65, Polysorbate 80, Polysorbate 81, Polysorbate 85, PEG3350, and mixtures thereof. In some embodiments, the surfactant is polysorbate 80 (PS80).

The surfactant concentration typically ranges from about 0.01 mg/mL to about 10 mg/mL, from about 0.01 mg/mL to about 5.0 mg/mL, from about 0.01 mg /ml to about 2.0 mg/ml, from about 0.01 mg/ml to about 1.5 mg/ml, from about 0.01 mg/ml to about 1.0 mg/ml, from about 0.01 mg/mL to about 0.5 mg/mL, from about 0.01 mg/mL to about 0.4 mg/mL, from about 0.01 mg/mL to about 0.3 mg/mL, from about 0, 01 mg/mL to about 0.2 mg/mL, about 0.01 mg/mL to about 0.15 mg/mL, about 0.01 mg/mL to about 0.1 mg/mL, or about 0 .01 mg/ml to about 0.05 mg/ml. More preferably, the surfactant concentration is about 0.5 mg/ml, about 0.05 mg/ml about 0.06 mg/ml about 0.07 mg/ml about 0.08 mg/ml, about 0.09 mg /ml approximately 0.1 mg/ml approximately 0.11 mg/ml approximately 0.12 mg/ml approximately 0.13 mg/ml approximately 0.14 mg/ml approximately 0.15 mg/ml approximately 0.16 mg/ ml about 0.17 mg/ml about 0.18 mg/ml about 0.19 mg/ml, about 0.2 mg/ml. In some embodiments, the surfactant concentration is 0.2 mg/mL.

The buffer may be, for example, without limitation, acetate, histidine, succinate, gluconate, citrate, acetic acid, phosphate, phosphoric acid, ascorbate, tartaric acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, bicarbonate, and carbonic acid. acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetate, acetate, malate, imidazole, tris, phosphate, and mixtures thereof. In some embodiments, the buffer is a histidine buffer, where the histidine may include either L-histidine or D-histidine, a solvated form of histidine, a hydrated form (e.g., monohydrate) of histidine, a salt of histidine (e.g., histidine hydrochloride), or an anhydrous form of histidine, or their mixture. Preferably the buffer is an acetate buffer, where the acetate may include sodium acetate, acetic acid, or a mixture thereof.

The buffer concentration may range from about 0.1 millimoles (mM) to about 100 mM. Preferably, the buffer concentration is from about 0.5 mM to about 50 mM, more preferably from about 1 mM to about 30 mM, more preferably from about 1 mM to about 18 mM, even more preferably from about 1 mM to about 15 mM. Preferably, the buffer concentration is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, approximately 13 mM, approximately 14 mM, approximately 15 mM, approximately 16 mM, approximately 17 mM, approximately 18 mM, approximately 19 mM, approximately 20 mM, approximately 21 mM, approximately 22 mM, approximately 23 mM, approximately 24 mM , approximately 25 mm, approximately 30 mm, approximately 35 mm, approximately 40 mm, approximately 45 mm or approximately 50 mm. In some embodiments, the buffer concentration is about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290, about 300 mM, about 310 mM, or about 320 mM.

In some embodiments, the chelating agent may be selected from the group consisting of aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycines, 2-(2-amino-2-oxoctyl)aminoethanesulfonic acid (BES), deferoxamine (DEF), citric acid, niacinamide and deoxycholates and mixtures thereof. In some embodiments, the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid 5 (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)glycolic ether, N,N,N',N'-tetraacetic acid (EGTA), trans-diaminocyclohexanetetraacetic acid (DCTA), glutamic acid and aspartic acid, N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bis-hydroxyethylglycine (bicine) and N-(trishydroxymethylmethyl) 10 glycine (tricine), glycylglycine, sodium deoxycholate, ethylenediamine; propylenediamine; diethylenetriamine; triethylenetetraamine (triene), ethylenediaminetetraaceto EDTA; disodium EDTA, EDTA, calcium EDTA of oxalic acid, malate, citric acid, citric acid monohydrate and trisodium citrate dihydrate, 8-hydroxyquinolate, amino acids, histidine, cysteine, methionine, peptides, polypeptides and proteins and mixtures thereof. In some embodiments, the chelating agent is selected from the group consisting of EDTA salts including dipotassium edetate, disodium edetate, calcium disodium edetate, sodium edetate, trisodium edetate, and potassium edetate; and a suitable salt of deferoxamine (DEF) is deferoxamine mesylate (DFM), or mixtures thereof. The chelating agents used in the invention may be present, whenever possible, in the form of the free acid or free base, or in the form of a salt of the compound, also in the form of an anhydrous, solvated or hydrated form of the compound, or the corresponding salt. After being dissolved in a solution at a given pH and at the same concentration of ethylenediaminetetraacetic acid (EDTA), various salt forms, free acid forms, hydrated or anhydrous forms of EDTA containing sodium and water are considered equivalent chelating agents. For example, 0.0500 g/L disodium EDTA dihydrate (FW: 372.3), 0.0393 g/L EDTA (FW: 292.3), 0.0452 g/L disodium EDTA (FW: 336.2), 0.0481 g/l trisodium EDTA (FW: 358.2), 0.0511 g/l tetrasodium EDTA (FW: 380.2), 0.0422 g/l Sodium EDTA (FW: 314.2) and 0. 0446 g/l sodium EDTA hydrate (FW: 332.3), which all contain 134.4 µM EDTA, are considered equivalent chelating agents.

The most preferred chelating agent is EDTA, such as but not limited to disodium EDTA, calcium EDTA, or disodium EDTA dihydrate.

Particularly preferred is disodium EDTA because it provides a composition with improved antibody stability and/or resistance to aggregation.

The chelating agent concentration typically ranges from about 0.01 mg/mL to about 50 mg/mL, from about 1 mg/mL to about 10.0 mg/mL, from about 5 mg/mL to about 15, 0 mg/ml, about 0.01 mg/ml to about 1.0 mg/ml, or about 0.03 mg/ml to about 0.5 mg/ml. More preferably, the concentration of the chelating agent typically ranges from about 0.01 mM to about 2.0 mM, from about 0.01 mM to about 1.5 mM, from about 0.01 mM to about 0.5 mM, from about 0.01 mM to about 0.4 mM, from about 0.01 mM to about 0.3 mM, from about 0.01 mM to about 0.2 mM, from about 0.01 mM to about 0 .15 mM, from about 0.01 mM to about 0.1 mM, from about 0.01 mM to about 0.09 mM, from about 0.01 mM to about 0.08 mM, from about 0.01 mM to about 0.07 mM, from about 0.01 mM to about 0.06 mM, from about 0.01 mM to about 0.05 mM, from about 0.01 mM to about 0.04 mM, from about 0.01 mM to about 0.03 mM, from about 0.01 mM to about 0.02 mM, or from about 0.05 mM to about 0.01 mM. Preferably, the concentration of the chelating agent may be about 0.01 mg/ml, 0.02 mg/ml, 0.03 mg/ml, about 0.04 mg/ml, about 0.05 mg/ml, about 0.06 mg/ml ml, about 0.07 mg/ml, about 0.10 mg/ml, about 0.20 mg/ml. More preferably, the concentration of the chelating agent is about 0.04 mg/ml, about 0.041 mg/ml, about 0.042 mg/ml, about 0.043 mg/ml, about 0.044 mg/ml, about 0.045 mg/ml, about 0.046 mg/ml, about 0.047 mg/ml, about 0.048 mg/ml, about 0.049 mg/ml, about 0.05 mg/ml, about 0.051 mg/ml, about 0.052 mg/ml, about 0.053 mg/ml, about 0.054 mg/ml, about 0.055 mg/ml or about 0.056 mg/ml. Most preferably, the concentration of the chelating agent is about 0.05 mg/ml.

Chelating agents can reduce the formation of reduced oxygen species, reduce the formation of acidic forms (eg, deamidation), reduce antibody aggregation and/or reduce antibody fragmentation and/or reduce antibody oxidation in the compositions of the present invention. Such chelating agents can reduce or prevent degradation of an antibody that is formulated as compared to a disclosed antibody without chelating agent protection.

Unless otherwise indicated, the concentrations given are those at ambient conditions, i.e. at 25°C and atmospheric pressure.

In preferred embodiments, the composition does not contain an antioxidant. For example, preferred embodiments of the present compositions do not contain methionine, sodium thiosulfate, catalase, or platinum.

In some embodiments, the implementation of the composition may contain an antioxidant. In some embodiments, the antioxidant is selected from the group consisting of methionine, sodium thiosulfate, catalase, and platinum.

The concentration of the antioxidant typically ranges from about 0.01 mg/mL to about 50 mg/mL, from about 0.01 mg/mL to about 10.0 mg/mL, from about 0.01 mg/mL to about 5.0 mg/ml, about 0.01 mg/ml to about 1.0 mg/ml, or about 0.01 mg/ml to about 0.02 mg/ml. Preferably, the antioxidant concentration may be about 0.01 mg/ml, 0.02 mg/ml, 0.03 mg/ml, about 0.04 mg/ml, about 0.05 mg/ml, about 0.06 mg/ml , approximately 0.07 mg/ml, 0.08 mg/ml, 0.09 mg/ml, approximately 0.10 mg/ml, 0.11 mg/ml, 0.12 mg/ml, 0.13 mg/ml ml, approximately 0.14 mg/ml, approximately 0.15 mg/ml, approximately 0.16 mg/ml, approximately 0.17 mg/ml, 0.18 mg/ml, 0.19 mg/ml approximately 0, 20 mg/ml, approximately 0.25 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0, 8 mg/ml, 0.9 mg/ml, 1.0 mg/ml. Most preferably, the concentration of the antioxidant is about 0.01 mg/ml.

In some embodiments, the composition may include a preservative. Preferably the preservative is selected from Phenol, m-cresol, benzyl alcohol, benzalkonium chloride, benetonium chloride, phenoxyethanol and methyl paraben.

The concentration of preservative, as a rule, is in the range from about 0.001 mg/ml to about 50 mg/ml, from about 0.005 mg/ml to about 15.0 mg/ml, from about 0.008 mg/ml to about 12.0 mg/ml. ml or from about 0.01 mg/ml to about 10.0 mg/ml. Preferably, the preservative concentration may be about 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, about 0.4 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml. ml, approximately 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml approximately 1.0 mg/ml, 2.0 mg/ml, 3.0 mg/ml, approximately 4.0 mg /ml, about 5.0 mg/ml, about 6.0 mg/ml, about 7.0 mg/ml, 8.0 mg/ml, 9.0 mg/ml about 9.1 mg/ml, about 9 .2 mg/ml, 9.3 mg/ml, 9.4 mg/ml, 9.5 mg/ml, 9.6 mg/ml, 9.7 mg/ml, 9.8 mg/ml, 9, 9 mg/ml, 10.0 mg/ml. Most preferably, the concentration of the preservative is about 0.1 mg/ml or 9.0 mg/ml.

In some embodiments, the composition does not contain an antioxidant.

In some embodiments, the composition does not contain a preservative.

In some embodiments, the antibody comprises a heavy chain constant region, such as, for example, an IgG, IgM, IgD, IgA, and IgE constant region; and any isotypes such as IgG1, lgG2, IgG3 and IgG4. Preferably, the antibody is an IgG2 or IgG4 antibody. In some embodiments, the antibody comprises an IgG4 constant region comprising the following mutations (Armour et al., 2003, Molecular Immunology 40 585-593): E233F234L235 to P233V234A235 (IgG4 _Δ c) E233F234L235, where the numbering is according to the IgG4 wild type. In yet another embodiment, the Fc is human IgG4 comprising the E233F234L235 to P233V234A235 mutation with a G236 deletion (IgG4 _Δb ). In some embodiments, the Fc is any human IgG4 Fc (IgG4, IgG4 _Δb , or IgG4 _Δc ) containing the hinge-stabilizing mutation S228 to P228 (Aalberse et al., 2002, Immunology 105, 9-19).

In some embodiments, the antibody may be selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, ScFv etc.), chimeric antibodies, bispecific antibodies , heteroconjugated antibodies, single-chain (ScFv), their mutants, fusion proteins that include part of an antibody (for example, a domain antibody), humanized antibodies, human antibodies and any other modified configuration of the immunoglobulin molecule that includes an antigen recognition site of the desired specificity, including variants of antibody glycosylation , antibody amino acid sequence variants, and covalently modified antibodies. The antibody may be of murine, rat, human or any other origin (including chimeric or humanized antibodies). In some embodiments, the antibody may be human, but more preferably humanized. Preferably the antibody is isolated, more preferably it is substantially pure. When an antibody is an antibody fragment, it preferably retains the functional characteristics of the original antibody, i. ligand-binding and/or antagonistic or agonistic activity.

Exemplary anti-PD-1 antibodies include, but are not limited to, for example: nivolumab ( ^OPDIVO® , ONO-4538, BMS-936558, MDX1106, Bristol-Myers Squibb Company), pembrolizumab ( ^KEYTRUDA® , MK-3475, lambrolizumab, Merck & Co., Inc.), BCD100 (BIOCAD Biopharmaceutical Company), BGB-A317 (BeiGene Ltd./Celgene Corporation), CBT-501 (CBT Pharmaceuticals), GLS-010 (Harbin Gloria Pharmaceuticals Co., Ltd.), IBI308 (Innovent Biologics, Inc.), AMP-224 (GlaxoSmithKline plc), BI 754091 (Boehringer Ingelheim GmbH), PDR001 (Novartis AG), MEDI0680 (AstraZeneca PLC), PDR001 (Novartis AG), PF-06801591 (aka RN888) ( Pfizer Inc.) described as mAb7 in International Patent Publication No. WO2016/092419, the disclosure of which is hereby incorporated by reference in its entirety, REGN2810 (Regeneron Pharmaceuticals, Inc.), SHR-1210 (Incyte Corporation), TSR-042 ( Tesaro, Inc.), AGEN2034 (Agenus Inc.), JNJ-63723283 (Johnson & Johnson), MGD013 (MacroGenics, Inc.), ANA011 (AnaptysBio, Inc.), ANB011 (An aptysBio, Inc.), AUNP-12 (Pierre Fabre Medicament SA), ENUM 244C8 (Enumeral Biomedical Holdings, Inc.), hAb21 (Stainwei Biotech, Inc.), J43 (Transgene SA), JTX-4014 (Jounce Therapeutics, Inc. .), MCLA-134 (Merus BV), PRS-332 (Pieris AG), SHR-1316 (Atridia Pty Ltd.), STI-A1010 (Sorrento Therapeutics, Inc.), STI-A1110 (Les Laboratoires Servier), and XmAb20717 (Xencor Inc.).

BGB-A317 (trislelizumab), under development by BeiGene Ltd., is a humanized IgG4 monoclonal antibody having an Fc engineered region (ie where the ability to bind to the Fc receptor gamma I is specifically omitted). BGB-A317 binds to PD-1 and inhibits PD-1 binding to PD-L1 and PD-L2.

In one or more embodiments, the PD-1 axis binding antagonist is selected from PF-06801591, nivolumab, pembrolizumab, spartalizumab, and BGB-A317.

In a specific aspect, the anti-PD-1 antibody is PF-06801591 (Pfizer Inc., CAS Accession Number 2029210-61-3). In another specific aspect, the anti-PD-1 antibody is nivolumab ( ^OPDIVO® , MDX-1106, CAS accession number 946414-94-4). In another specific aspect, the anti-PD-1 antibody is cemiplimab ( ^LIBTAYO® , also known as REGN2810, Regeneron Pharmaceuticals, Inc. and sanofi-aventis US LLC, CAS Accession Number 1801342-60-8). In another specific aspect, the anti-PD-1 antibody is nivolumab ( ^OPDIVO® , MDX-1106, CAS accession number 946414-94-4). In another specific aspect, the anti-PD-1 antibody is spartalizumab (PDR001, Novartis, CAS accession number 1935694-88-4). In another specific aspect, a PD-1 binding antagonist is an antibody comprising a VH region produced by expression vector ATCC PTA-121 183 and having a VL region produced by expression vector ATCC PTA-1 21 182, also known as as mAb7 or mAb15 (Rinat Neuroscience, Pfizer Inc.) as described herein.

In a specific aspect, the anti-PD-1 antibody comprises a heavy chain variable region comprising:

(a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4 (GYTFTSYWIN);

(b) a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5 (NIYPGSSLTNYNEKFKN); and

(c) a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6 (LSTGTFAY).

In some embodiments, the antibody may be an anti-PD-1 antibody comprising a light chain variable region comprising:

(a) a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7 (KSSQSLWDSGNQKNFLT);

(b) a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8 (WTSYRES); and

(c) a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9 (QNDYFYPHT).

In some embodiments, the antibody may be an anti-PD-1 antibody comprising three CDRs from a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 2.

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSLTNYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTLVTVSS (SEQ ID NO: 2)

In some embodiments, the antibody may be an anti-PD-1 antibody comprising three CDRs from a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 3.

DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKPGQPPKLLIYWTSYRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPHTFGGGTKVEIK (SEQ ID NO: 3)

In some embodiments, the anti-PD-1 antibody may include a heavy chain variable region comprising an amino acid sequence of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, including the amino acid sequence shown in SEQ ID NO. 2 and/or a light chain variable region comprising an amino acid sequence of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% identical to the amino acid sequence, including the amino acid sequence shown in SEQ ID NO. 3, where the antibody specifically binds to human PD-1.

In some embodiments, the anti-PD-1 antibody may comprise a heavy chain variable region comprising an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and/or may comprise a light chain variable region comprising an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 3.

In some embodiments, the anti-PD-1 antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOs: 2 and 3.

In some embodiments, an anti-PD-1 antibody may include a heavy chain region comprising an amino acid sequence of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99% identical to an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 10 and/or a light chain region comprising an amino acid sequence of at least about 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, comprising the amino acid sequence shown in SEQ ID NO: 11, wherein the antibody specifically binds to human PD -one.

Heavy chain sequence

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSLTNYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10)

Light chain sequence

DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKPGQPPKLLIYWTSYRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPHTFGGGTKVEIKRGTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11)

In some embodiments, the anti-PD-1 antibody may include a heavy chain region comprising an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 10 and/or may include a light chain region comprising an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the C-terminal lysine of the heavy chain sequence shown in SEQ ID NO: 10 may be cleaved; those. the heavy chain sequence may lack a C-terminal lysine.

In some embodiments, the anti-PD-1 antibody may be an antibody comprising the amino acid sequences shown in SEQ ID NOS: 10 and 11.

In some embodiments, an anti-PD-1 antibody can compete for PD-1 binding with an anti-PD-1 antibody as defined herein. An anti-PD-1 antibody can compete for PD-1 binding with an antibody comprising a heavy chain variable region comprising an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2 and/or a light chain variable region comprising the amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 3.

In some embodiments, the anti-PD-1 antibody may be a monoclonal antibody, mAb7, that specifically binds to human PD-1. The mAb7 antibody is described in WO2016/092419, the contents of which are hereby incorporated by reference in their entirety. The amino acid sequences of the mAb7 heavy chain and light chain variable regions are shown in SEQ ID NOs: 2 and 3, respectively. The CDRs of the mAb7 antibody portion (including the Chothia and Kabat CDRs) are shown schematically in Table 1 of WO2016/092419. The mAb7 antibody is highly effective in blocking the biological activity of PD-1.

In some embodiments, the anti-PD-1 antibody may also include a fragment or region of the mAb7 antibody. In one embodiment, the fragment is a mAb7 light chain comprising the amino acid sequence shown in SEQ ID NO: 11 herein. In another embodiment, the fragment is a mAb7 antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO: 10 herein. In yet another embodiment, the fragment contains one or more variable regions from the light chain and/or heavy chain of the mAb7 antibody. In yet another embodiment, the fragment contains one or more CDRs from the light chain and/or heavy chain of the mAb7 antibody, comprising the amino acid sequences shown in SEQ ID NOs: 11 and 10, respectively, herein.

In some embodiments, the antibody may include one or more (one, two, three, four, five, or six) CDRs derived from the mAb7 antibody. In some embodiments, the CDRs may be Kabat CDRs, Chothia CDRs, or a combination of Kabat and Chothia CDRs (referred to herein as "extended" or "combined" CDRs). In some embodiments, the implementation of the polypeptides include any of the CDR configurations (including combinations, variants, etc.) described in this application.

In some embodiments, the C-terminal heavy chain lysine of any of the anti-PD-1 antibodies described herein is deleted. In various cases, the heavy and/or light chain of anti-PD-1 antibodies described in this application may optionally include a signal sequence.

In other embodiments, the antibody may be selected from an anti-PD-1 antibody known in the art, such as those described, for example, without limitation, in any of the following US Patent Nos. , 8779105, 8952136 and 8709416. An antibody may bind to the same epitope as an anti-PD-1 antibody known in the art and/or may compete for binding to PD-1 with such an antibody.

In accordance with another aspect of the present invention, there is provided a composition comprising or consisting essentially of:

from about 140 mg/ml to about 160 mg/ml antibody,

from about 10.0 mm to about 30.0 mm histidine buffer,

from about 40 mg/ml to about 100 mg/ml trehalose,

from about 0.01 to about 0.3 mg/ml polysorbate 80 (PS80) and

about 0.01 to about 0.1 mg/ml disodium EDTA,

where the specified composition has a pH selected from the range between about pH 4.5 and any of about pH 5.5, or, alternatively, from the range between about pH 4.5 and any of the pH values of about 4.6, 4.7 , 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9

According to a preferred embodiment, the composition comprises or essentially consists of any amount of about 90 mg/ml, about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml ml or approximately 150 mg/ml antibody,

approximately 20 mM histidine buffer,

approximately 84 mg/ml trehalose,

approximately 0.2 mg/mL PS80 and

approximately 0.05 mg/ml disodium EDTA,

where the specified composition has a pH selected from the range between approximately pH 5.0 and any of the pH values of approximately 5.0, 5.2, 5.5 or 5.8, or, alternatively, from the range between approximately pH 4, 5 and any one of pH values approximately 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5 , and wherein said antibody comprises a heavy chain variable region sequence comprising the amino acid sequence shown in SEQ ID NO. 2 and a light chain variable region sequence comprising the amino acid sequence shown in SEQ ID NO. 3.

According to a preferred embodiment, the composition comprises or essentially consists of any amount of about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml ml or approximately 180 mg/ml antibody,

approximately 20 mM histidine buffer,

approximately 84 mg/ml trehalose,

approximately 0.2 mg/mL PS80 and

approximately 0.05 mg/ml disodium EDTA,

wherein said composition has a pH of approximately pH 5.0, +/- 0.5, and wherein said antibody comprises a heavy chain variable region sequence comprising the amino acid sequence shown in SEQ ID NO. 2 and a light chain variable region sequence comprising the amino acid sequence shown in SEQ ID NO. 3. In some embodiments, the volume of dose used is about 0.5 ml, about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, approximately 10 ml, approximately 11 ml, approximately 12 ml, approximately 13 ml, approximately 14 ml, approximately 15 ml, approximately 16 ml, approximately 17 ml, approximately 18 ml, approximately 19 ml, approximately 20 ml, approximately 21 ml, approx 22 ml approx 23 ml approx 24 ml approx 25 ml approx 26 ml approx 27 ml approx 28 ml approx 29 ml approx 30 ml approx 31 ml approx 32 ml approx 33 ml approx 34 ml, approx. 35 ml, approx. 36 ml, approx. 37 ml, approx. 38 ml, approx. 39 ml, approx. 40 ml, approx. 41 ml, approx. 42 ml, approximately 43 ml, approximately 44 ml, approximately 45 ml, approximately 46 ml, approximately 47 ml, approximately 48 ml, approximately 49 ml or approximately 50 ml.

In some embodiments, a composition is provided that is lyophilized and/or that has been lyophilized. In some embodiments, a composition is provided that is not lyophilized and has not been lyophilized.

In some embodiments, the antibody concentration is any concentration of about 100 mg/ml, about 105 mg/ml, about 110 mg/ml, about 115 mg/ml, about 120 mg/ml, about 125 mg/ml, about 130 mg/ml ml, about 135 mg/ml, about 140 mg/ml, about 145 mg/ml, about 150 mg/ml, about 155 mg/ml, or about 160 mg/ml.

According to a more preferred aspect of the present invention, there is provided a composition according to any of the above aspects or embodiments for the preparation of a medicament for the treatment of a hyperproliferative disorder such as cancer in a subject.

In some embodiments, the cancer is selected from one or more of gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, head and neck squamous cell carcinoma, thymus cancer, epithelial cancer, salivary gland cancer, liver cancer, gastric cancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer cavity, skin cancer and melanoma.

In accordance with yet another embodiment of the invention, there is provided a composition according to any of the above-described aspects or embodiments for the preparation of a medicament for the treatment of a hyperproliferative disorder, such as cancer, in a subject.

According to a preferred embodiment, the composition can be administered directly into the bloodstream, into muscle, into tissue, into adipose tissue, or into an internal organ. Suitable parenteral routes include subcutaneous, intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intraosseous, and intradermal administration. Suitable devices for parenteral administration include needles (including microneedles, microinjections, dissolvable needles, and other microporation techniques), injectors, needleless injectors, and infusion techniques.

In some embodiments, the drug regimen includes administering a dose of drug once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once at seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every fifteen weeks, once every twenty weeks, once every twenty-five weeks, or once every twenty-six weeks. In some embodiments, the anti-PD-1 antibody is administered once a month, once every two months, once every three months, once every four months, once every five months, or once every six months. In some embodiments, the drug regimen comprises administering a dose of drug once every four or eight weeks.

In some embodiments, the dose volume is less than or equal to about 3 ml, about 2.5 ml, about 2 ml, about 1.5 ml, about 1 ml, about 0.75 ml, about 0.5 ml, about 0.25 ml or approximately 0.1 ml.

In some embodiments, the dose volume is about 20 ml, about 19 ml, about 18 ml, about 17 ml, about 16 ml, about 15 ml, about 14 ml, about 13 ml, about 12 ml, about 11 ml, about 10 ml , about 9 ml, about 8 ml, about 7 ml, about 6 ml, about 5 ml, about 4 ml, about 3 ml, about 2 ml, or about 1 ml. Alternatively, the dose volume is about 20.5 ml, about 19.5 ml, about 18.5 ml, about 17.5 ml, about 16.5 ml, about 15.5 ml, about 14.5 ml, about 13. 5 ml, approximately 12.5 ml, approximately 11.5 ml, approximately 10.5 ml, approximately 9.5 ml, approximately 8.5 ml, approximately 7.5 ml, approximately 6.5 ml, approximately 5.5 ml , about 4.5 ml, about 3.5 ml, about 2.5 ml, about 1.5 ml, or about 0.5 ml. Alternatively, the dose volume is about 900 microliters, about 800 microliters, about 700 microliters, about 600 microliters, about 500 microliters, about 400 microliters, about 300 microliters, about 200 microliters, or about 100 microliters, alternatively about 950 microliters, about 850 microliters, about 750 microliters, about 650 microliters, about 550 microliters, about 450 microliters, about 350 microliters, about 250 microliters, about 150 microliters, or about 50 microliters. In some embodiments, the dose volume is less than or equal to about 2.0 ml.

According to a preferred embodiment, the antibody concentration may range from about 0.1 to about 200 mg/mL. Preferably, the antibody concentration is about 100 mg/ml, about 101 mg/ml, about 102 mg/ml, about 103 mg/ml, about 104 mg/ml, about 105 mg/ml, about 106 mg/ml, about 107 mg /ml, about 108 mg/ml, about 109 mg/ml or about 110 mg/ml, about 111 mg/ml, about 112 mg/ml, about 113 mg/ml, about 114 mg/ml, about 115 mg/ml , approximately 116 mg/mL, approximately 117 mg/mL, approximately 118 mg/mL, approximately 119 mg/mL, approximately 120 mg/mL, approximately 121 mg/mL, approximately 122 mg/mL, approximately 123 mg/mL, approximately 124 mg/mL, approximately 125 mg/mL, approximately 126 mg/mL, approximately 127 mg/mL, approximately 128 mg/mL, approximately 129 mg/mL, approximately 130 mg/mL, approximately 131 mg/mL, approximately 132 mg /ml, approximately 133 mg/ml, approximately 134 mg/ml, approximately 135 mg/ml, approx. no 136 mg/mL, approximately 137 mg/mL, approximately 138 mg/mL, approximately 139 mg/mL, approximately 140 mg/mL, approximately 141 mg/mL, approximately 142 mg/mL, approximately 143 mg/mL, approximately 144 mg/ml, about 145 mg/ml, about 146 mg/ml, about 147 mg/ml, about 148 mg/ml, about 149 mg/ml, or about 150 mg/ml. Most preferably, the antibody concentration is from about 100 mg/ml to about 180 mg/ml and can be selected from the group consisting of about 100 mg/ml, about 105 mg/ml, about 110 mg/ml, about 115 mg/ml, approximately 120 mg/mL, approximately 125 mg/mL, approximately 130 mg/mL, approximately 135 mg/mL, approximately 140 mg/mL, approximately 145 mg/mL, approximately 150 mg/mL, approximately 155 mg/mL, approximately 160 mg/ml, about 165 mg/ml, about 170 mg/ml, about 175 mg/ml, or about 180 mg/ml.

According to a preferred embodiment, the dose contains an amount of antibody less than or equal to about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg , approximately 59 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 110 mg, approximately 120 mg, approximately 130 mg, approximately 140 mg, approximately 150 mg, approximately 160 mg, approximately 170 mg, approximately 180 mg, approximately 190 mg, approximately 200 mg, approximately 210 mg, approximately 220 mg, approximately 230 mg, approximately 240 mg, approximately 250 mg, approximately 260 mg, approximately 270 mg, approximately 280 mg, approximately 290 mg , approximately 300 mg, approximately 310 mg, approximately 320 mg, approximately 330 mg, approximately 340 mg, etc. and about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, approximately 480 mg, approximately 490 mg, approximately 500 mg, approximately 510 mg, approximately 520 mg, approximately 530 mg, approximately 540 mg, approximately 550 mg, approximately 560 mg, approximately 570 mg, approximately 580 mg, approximately 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, approximately 730 mg, approximately 740 mg, approximately 750 mg, approximately 760 mg, approximately 770 mg, approximately about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 850 mg, about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg, about 900 mg, about 910 mg, about 920 mg, about 930 mg, about 940 mg, about 950 mg, about 960 mg, about 970 mg, about 980 mg, about 990 mg, or about 1000 mg.

In some embodiments, the dose contains an amount of antibody that is about 1 µg/kg, about 10 µg/kg, about 20 µg/kg, about 25 µg/kg, about 50 µg/kg, about 100 µg/kg, approximately 200 mcg/kg, approximately 250 mcg/kg, approximately 500 mcg/kg, approximately 1 mg/kg, approximately 2 mg/kg, approximately 3 mg/kg, approximately 4 mg/kg, approximately 5 mg/kg, approximately 6 mg/kg, approximately 7 mg/kg, approximately 8 mg/kg, approximately 9 mg/kg, approximately 10 mg/kg, or approximately 11 mg/kg (body weight of the subject for whom this dose is intended). In some embodiments, the dose contains about 20 µg/kg, about 25 µg/kg, about 50 µg/kg, about 100 µg/kg, about 200 µg/kg, about 250 µg/kg, 1 mg/kg, about 2 mg /kg, approximately 3 mg/kg, approximately 4 mg/kg, approximately 5 mg/kg, approximately 6 mg/kg, approximately 7 mg/kg, approximately 8 mg/kg, approximately 9 mg/kg or approximately 10 mg/kg .

Dosing regimens may depend on the nature of the pharmacokinetic decay that the practitioner wishes to achieve. For example, in some embodiments, administration is contemplated from one to four times a week. Even less frequent administration can be used. In some embodiments, the dose is administered once every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 15 weeks, every 20 weeks, every 25 weeks or less. In some embodiments, the dose is administered monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or less frequently. The progress of this therapy is easily monitored using conventional methods and assays. Dosing regimen may change over time.

For the purposes of the present invention, the appropriate dosage of the drug will depend on the antibody used, the type and severity of the disease being treated, whether the drug is being administered prophylactically or therapeutically, previous therapy, the patient's medical history, and the drug response and judgment of the attending physician. . Typically, the physician will administer the drug until a dosage is reached that achieves the desired result. Dosages can be determined empirically. For example, increasing doses are administered to subjects to evaluate the effectiveness of the drug.

The dose and/or frequency of administration may vary during treatment. Empirical considerations, such as the half-life of the antibody, will usually influence dosage determination. The frequency of administration may be determined and adjusted over the course of treatment and is generally, but not necessarily, dependent on treatment and/or suppression and/or improvement and/or delay of one or more of the symptoms of the hyperproliferative disease. Some subjects may require more than one dose. The frequency of administration can be determined and adjusted during the course of therapy. For example, without limitation, with regard to repeated administrations for several days or longer, depending on the disease and its severity, treatment is continued until the desired suppression of symptoms occurs or until sufficient therapeutic levels for cancer treatment are achieved.

Administration of the drug comprising the composition may be continuous or intermittent, depending on, for example, the physical condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of the drug comprising the composition may be substantially continuous over a preselected period of time, or may be administered as a series of doses administered at predetermined intervals.

Preferably the administration is parenteral, preferably selected from intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intraosseous, intradermal and subcutaneous administration. Preferably, the drug is in a sterile parenteral dosage form.

The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention in addition to those shown and described in this application will be apparent to those skilled in the art based on the foregoing description and fall within the scope of the appended claims. Examples in WO 2016/092419 are provided to illustrate antibodies for use in the present invention. The entire content of WO 2016/092419 is incorporated into this application by reference.

EXAMPLES

Methods

This section on methods briefly describes the methods used in the following Examples 1-6.

The viscosity of the antibody compositions was measured on an m-VROC device on a chip, where the pressure difference correlates with the dynamic viscosity of the solution. Measurements were taken at various flow rates and shear rates. The sample size was approximately 70-100 μl. Aliquots were loaded into a 100 μl Hamilton syringe and connected to the m-VROC chip. The measurements were carried out in triplicate at 20°C.

To determine freeze/thaw stability, anti-PD-1 antibody at 100-150 mg/ml was subjected to -20°C to 2-8°C for 5 cycles. Antibody stability was assessed based on pH, concentration and SEC.

To determine the agitation stability, anti-PD-1 antibody at high concentration was evaluated after 24 hours of agitation at 300 rpm and ambient temperature and under plumb light. Stability was assessed based on pH, concentration and SEC.

pH Stability of highly concentrated antibody formulations was measured according to USP Method <791> and Pharmacopoeial Method EP 2.2.3 using a suitable, properly standardized potentiometric device (Thermo Scientific Orion Star A111 pH meter) capable of reproducing pH values down to 0.02 pH units using an indicator electrode sensitive to hydrogen ion activity, a glass electrode, and a suitable reference electrode (Thermo Scientific Orion™ PerpHecT™ ROSS™ Combined pH Microelectrode). A two-point or three-point calibration at 22.5-25.4°C was performed on the day of use to check the standardization of the pH meter, samples were equilibrated at ambient temperature for at least 30 minutes, and values down to 0.01 pH units were recorded after stabilization of the pH reading for at least 60 seconds.

Total protein concentrations were measured spectroscopically using a SoloVPE device (C Technologies Inc.) based on variable path length and the Beer-Lambert law (A=clε where A=absorbance, c=concentration, l=pathlength and ε=extinction coefficient/molar absorption coefficient in mg ml ^-1 cm ^-1 ). For the anti-PD-1 antibody mAb7, the value of ε used is 1.62 mg mg mg ml ^-1 cm ^-1 . For nivolumab, the ε value is 1.68 mg mg mg ml ^-1 cm ^-1 . For pembrolizumab, the ε value used is 1.42 mg mg ml ^-1 cm ^-1 . The samples were equilibrated to ambient temperature, added to a sample vial (C Tech Inc. # OC0009-1-P50) and inserted into the vial holder on the platform of the detection window. For each sample, a clean fiber (#OC0002-P50) was placed in the fiber seat and the tilt angle was read at 280 nm (scatter correction at 320 mm).

The formation of higher molecular weight compounds (HMMS) was analyzed by Size Exclusion Chromatography (SEC) on an Agilent HPLC system. 50 µg of protein was separated based on hydrodynamic volume on a YMC-Pack Diol-200 column (Waters, cat. no. DL20S053008WT) maintained at 30±2°C using an isocratic gradient (20 mM sodium phosphate, 400 mM sodium chloride, pH 7, 2) at 0.75 ml/min. Species with different molecular weights were eluted and determined by UV absorbance at 280 nm.

Charge heterogeneity and antibody stability at high concentration to deamidation and fragmentation was quantified using visualized capillary isoelectric focusing (iCE), which separates protein species based on their charge difference (pI value) in a pH gradient. Samples were diluted into the mixture to a final concentration of 0.3 mg/mL protein, 0.01 mg/mL marker pI 6.14 (Protein Simple, part no. 102220), 0.01 mg/mL marker pI 9/50 (Protein Simple, part no. 101996), 4% Pharmalyte pH 3-10 (GE part no. 17-0456-01), 0.25% methylcellulose (Protein Simple, part no. 101876)) and 2 M urea (Sigma-Aldrich, part no. .U4883). Samples were injected into a fluorocarbon-coated cIEF cartridge column (Protein Simple, part no. 101701) installed in a Protein simple iCE3 device, focused in a capillary column at high voltage (≤3000 V) and monitored on the column's Real Time Video Detection (WCID) system. at a fixed wavelength of 280 nm. The resulting electrophoregrams were analyzed using appropriate software to determine pI values and peak areas for each species.

Particles invisible to the naked eye were analyzed using microflow imaging (MFI). Prior to any sample analysis, a 15 µm Duke latex standard was used to test system suitability. Washing with water was used between each analysis. In addition, a blank water analysis was performed prior to analysis to ensure background values were suitable for testing. Data are presented as mean cumulative amounts per ml. In addition, channels of several sizes were tracked to provide information on the number of particles in each size range.

Example 1 Effect of Buffer and pH on Viscosity

This example illustrates the effect of pH on viscosity in a highly concentrated anti-PD-1 antibody formulation.

To prepare the antibody composition, the anti-PD-1 antibody mAb7 was buffer exchanged in 20 mM Histidine pH 5.5, 20 mM Histidine pH 6.0, 20 mM Histidine pH 6.5, 20 mM Histidine pH 7.0, 20 mM Acetate pH 5.0 and 20 mM Acetate pH 6.0 by continually repeating the buffer dilution procedure followed by concentration to approximately 160 mg/mL until buffer exchange is complete. The target final concentration of all samples was 160 mg/ml.

Protein concentration, viscosity and pH were measured for all samples. The results are presented in Table 1 and in FIG. 1. The results demonstrate that lower pH formulations have significantly lower viscosities. In addition, at the same pH, histidine and acetate compositions have the same viscosity.

Table 1 Composition (target pH value) anti-PD-1 antibody concentration
(mg/ml) Viscosity at 20°C (cP) Measured pH value 20 mM Histidine pH 5.5 159.7 35.2 5.75 20 mM Histidine pH 6.0 161.2 85.2 6.24 20 mM Histidine pH 6.5 160.9 230.9 6.64 20 mM Histidine pH 7.0 152.6 279.1 7.04 20 mM Acetate pH 5.0 156.4 27.9 5.44 20 mM Acetate pH 6.0 162.5 88.6 6.14

Example 2 Arginine Evaluation

This example illustrates the effect of varying concentrations of arginine on the viscosity of an anti-PD-1 antibody.

To evaluate the effect of arginine on the viscosity of the anti-PD-1 antibody, mAb7 was formulated with 20 mM Acetate pH 5.0, 50 g/l sucrose, 0.05 g/l Na ₂ EDTA dihydrate, and 0.2 g/l PS80 by dialysis (without PS80) using 20 kDa MWCO dialysis cassettes, concentrating anti-PD-1 antibody to approximately 200 mg/mL using Amicon 50 kDa centrifuge filters, and injecting a high concentration of PS80. Compositions with varying concentrations of arginine were prepared by adding a highly concentrated solution of arginine hydrochloride (HCl).

Protein concentration and pH were measured for all samples. The results are presented in Table 2 and in FIG. 2. The viscosity of anti-PD-1 antibody compositions with 0, 50, 100, 150, 200 and 250 mM arginine is shown over a range of anti-PD-1 antibody concentrations (Table 2, Fig. 2). The results demonstrate that the addition of 50 mM arginine reduces the viscosity of highly concentrated anti-PD-1 antibody formulations. In addition, a gradual increase in the concentration of arginine to 250 mm further reduces the viscosity.

table 2 Arginine (mM) Anti-PD-1 antibody concentration
(mg/ml) Viscosity at 20°C (cP) 0 153 17.0 178 38.6 205 97.0 fifty 150 15.0 174 30.1 200 64.1 100 151 14.5 176 23.7 196 47.3 150 151 11.7 179 25.0 195 42.0 165 21.7 200 148 11.2 171 18.9 192 32.5 250 154 11.0 171 18.5 187 27.8

Example 3 Evaluation of Arginine-Containing Compositions

This example illustrates the effect of additional excipients on the viscosity and stability of an anti-PD-1 antibody.

To prepare arginine-containing anti-PD-1 antibody formulations, mAb7 was formulated into each of formulations 1-6 (Table 3) by incorporating mAb7 into each formulation by dialysis (without PS80) using 20 kDa MWCO dialysis cassettes, concentrating to 150-200 mg/mL anti-PD-1 antibody using 50 kDa Amicon centrifuge filters and injecting a high concentration of PS80.

Table 3 Song number Buffer/pH sucrose _Na2EDTA dihydrate PS80 Arginine HCl Proline one 20 mM Acetate pH 4.5 50 g/l 0.05 g/l 0.2 g/l 150 mm Not 2 20 mM Acetate pH 5.0 50 g/l 0.05 g/l 0.2 g/l 150 mm Not 3 20 mM Acetate pH 5.5 50 g/l 0.05 g/l 0.2 g/l 150 mm Not four No pH 5.0 Not Not 0.2 g/l 100 mM 200 mm 5 20 mM Acetate pH 5.0 50 g/l 0.05 g/l 0.2 g/l 100 mM 100 mM 6 20 mM Acetate pH 5.0 50 g/l 0.05 g/l 0.2 g/l 100 mM Not

Compositions 1, 2 and 3 were evaluated to determine the effect of pH in arginine-containing compositions. Table 4 and Fig. 3 represent the viscosity of compositions 1, 2 and 3 over a range of anti-PD-1 concentrations. The results demonstrate that in formulations containing 150 mM arginine, lowering the pH from 5.5 to 4.5 significantly reduces the viscosity.

Table 4 Composition Antibody concentration
(mg/ml) Viscosity at 20°C (cP) one 154.6 10.5 193.3 26.8 2 151 11.7 179 25 195 42 165.4 21.7 3 149 15.6 163.5 28.9

Compositions 4, 5 and 6 were evaluated to determine the effect of adding proline to compositions containing 100 mm Arginine at pH 5.0. Table 5 and FIG. 4 represent the viscosity of formulations 4, 5 and 6 over a range of anti-PD-1 antibody concentrations. The results demonstrate that there is no significant difference in viscosity between highly concentrated anti-PD-1 antibody formulations containing 0, 100 or 200 mM proline.

Table 5 Composition Antibody concentration
(mg/ml) Viscosity at 20°C (cP) four 151 11.6 171 21.7 197 41.4 215 60.9 5 149 12.6 165 18.0 183 29.8 199 42.0 230 104.0 6 151 14.5 176 23.7 196 47.3

The stability of formulations 1, 2, 3, 4 and 5 at 150 mg/ml anti-PD-1 antibody was determined by placing the samples under stability conditions at 5°C, 25°C and 40°C. Protein stability was assessed based on aggregation (SEC), charged isoforms (iCE), concentration and pH. Tables 6, 7, 8, 9 and 10 present the thermal stability of compositions 1, 2, 3, 4 and 5, respectively.

Table 6
Thermal stability of composition 1 T=0 5°C 25°C 40°C Trial 4W 10W 2W 4W 10W 2W 4W 10W Anti-PD-1 antibody concentration (mg/ml) 154.6 156.0 154.1 NS 155.0 156.7 NS 153.6 162.9 pH 4.5 4.5 4.5 4.5 4.5 4.5 4.6 4.6 4.7 SEC: Total content of HMMS (%) 0.6 0.6 0.6 NS 0.8 1.0 NS 2.6 1.9 ICE: Acid (%) 25.5 23.4 23.8 26.0 24.5 28.5 20.8 19.3 11.6 ICE: Alkaline (%) 12.9 13.4 16.6 14.4 14.5 17.3 40.3 49.4 68.3 ICE: Main view (%) 61.6 62.4 59.6 59.6 61.0 54.2 39.0 31.2 16.2

Table 7
Thermal stability of composition 2 T=0 5°C 25°C 40°C Trial 4W 10W 2W 4W 10W 2W 4W 10W Anti-PD-1 antibody concentration (mg/ml) 151.0 149.7 148.6 NS 150.3 151.7 NS 151.0 152.6 pH 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 SEC: Total content of HMMS (%) 0.6 0.6 0.6 NS 0.7 0.8 NS 2.3 2.4 ICE: Acid (%) 24.4 22.9 24.3 26.7 23.6 27.9 29.1 31.3 40.6 ICE: Alkaline (%) 13.1 13.8 14.5 13.0 14.3 17.0 17.6 16.3 22.2 ICE: Main view (%) 62.5 63.4 61.2 60.3 62.0 55.1 53.3 52.4 37.2

Table 8
Thermal stability of composition 3 T=0 5°C 25°C 40°C Trial 4W 10W 2W 4W 10W 2W 4W 10W Anti-PD-1 antibody concentration (mg/ml) 149.0 153.4 149.9 NS 148.7 147.6 NS 148.4 156.0 pH 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 SEC: Total content of HMMS (%) 0.6 0.6 0.6 NS 0.7 0.7 NS 1.3 2.2 ICE: Acid (%) 27.1 23.9 24.5 25.8 23.2 24.9 30.3 33.7 46.0 ICE: Alkaline (%) 13.9 13.5 14.6 14.3 14.2 16.4 15.8 14.0 13.1 ICE: Main view (%) 59.0 62.6 60.9 59.9 62.6 58.7 54.0 52.3 41.0

Table 9
Thermal stability of composition 4 Trial T=0 5°C 25°C 40°C 4W 2W 4W 2W 4W Anti-PD-1 antibody concentration (mg/ml) 151.5 154.2 NS 150.6 NS 154.0 pH 5.1 5.0 5.1 5.0 5.1 5.1 SEC: Total content of HMMS (%) 1.0 0.9 NS 1.0 NS 2.5 ICE: Acid (%) 26.5 23.1 25.8 22.4 29.1 31.2 ICE: Alkaline (%) 12.9 14.0 14.1 14.5 17.5 16.9 ICE: Main view (%) 60.6 62.9 60.1 63.1 53.4 51.8

Table 10
Thermal stability of composition 5 Trial T=0 5°C 25°C 40°C 4W 2W 4W 2W 4W Anti-PD-1 antibody concentration (mg/ml) 148.8 148.1 NS 145.9 NS 148.6 pH 5.0 5.0 5.1 5.0 5.0 5.0 SEC: Total content of HMMS (%) 0.6 0.6 NS 0.7 NS 2.0 ICE: Acid (%) 21.9 23.1 25.4 23.7 29.6 32.5 ICE: Alkaline (%) 12.3 13.8 15.2 14.4 16.7 17.5 ICE: Main view (%) 65.8 63.0 59.4 61.8 53.7 50.0

For all compositions 1-5 after 10 weeks (10W) (4 weeks (4W) for compositions 4 and 5), as for charge species and aggregates (HMMS), no significant change at 5°C and 25°C did not have. At 40°C composition 1 (pH 4.5) had a significant increase in alkaline species, while compositions 2, 3, 4 and 5 (pH > 4.5) had an increase in acid species. The increase in aggregates (HMMS) was similar for all compositions after storage at 40°C, however, this increase is considered acceptable for liquid compositions with expected storage conditions of 5°C.

Example 4 Evaluation of Trehalose-Containing Compositions

This example illustrates the effect of trehalose on the stability and viscosity of anti-PD-1 antibody formulations.

Trehalose dihydrate was evaluated as a stabilizer in arginine-free formulations and in arginine-containing formulations (Table 11).

Table 11 Song number Buffer/pH Trehalose dihydrate _Na2EDTA
dihydrate PS80 Arginine
HCl 7 20 mM Histidine pH 5.0 84 g/l 0.05 g/l 0.2 g/l Not eight 20 mM Histidine pH 5.0 50 g/l 0.05 g/l 0.2 g/l 100 mM

To prepare trehalose-containing anti-PD-1 antibody formulations, mAb7 was buffer exchanged in 20 mM histidine and 50 g/l trehalose dihydrate using tangential flow filtration using 50 kDa ultrafiltration cartridges. The concentration of anti-PD-1 antibody mAb7 was increased using ultrafiltration, followed by the addition of excipients using highly concentrated solutions and dilution if necessary. The anti-PD-1 antibody mAb7 was formulated into composition 7 at the following concentrations: 104 mg/ml, 154 mg/ml, 174 mg/ml and 200 mg/ml. The anti-PD-1 antibody mAb7 was formulated into composition 8 at the following concentrations: 150 mg/ml, 170 mg/ml and 200 mg/ml. Viscosity was measured as described above. The results are presented in Table 12 and in FIG. 5.

Table 12
Viscosity of trehalose-containing compositions Composition Antibody concentration
(mg/ml) Viscosity at 20°C (cP) 7 104 5.3 154 16.9 174 32.6 200 67.6 eight 150 14.1 170 25.0 200 54.4

The viscosities of formulation 7 containing 104 mg/mL, 154 mg/mL, 174 mg/mL and 200 mg/mL mAb7 are 5.3, 16.9, 32.5 and 67.6 cps, respectively, at 20°C . The viscosity of composition 8 containing 150 mg/ml, 170 mg/ml and 200 mg/ml mAb7 is 14.1, 25.0 and 54.4 cps, respectively, at 20°C.

The results demonstrate that formulation 8 (containing arginine) has a lower viscosity than formulation 7 at anti-PD-1 antibody concentrations ≥ 175 g/l. The viscosities of compositions 7 and 8 at about 150 mg/ml were the same (FIG. 5).

The stability of the compositions of anti-PD-1 antibodies 7 and 8 was evaluated at 5°C, 25°C and 40°C. Composition 7 was tested using 100 mg/ml and 150 mg/ml anti-PD-1 antibody, and composition 8 using 150 mg/ml anti-PD-1 antibody. Samples were placed under conditions of 5°C, 25°C and 40°C and stability was assessed by measuring aggregation (SEC), isoforms differing in charge (iCE), concentration, pH and particles invisible to the naked eye (MFI) as described above . The results are presented in Tables 13-21 (NT=not tested).

Tables 13, 14 and 15 present the thermal stability for composition 7 at 150 mg/ml anti-PD-1 at 5°C, 25°C and 40°C, respectively.

Table 13
Thermal stability of composition 7 (150 mg/ml) at 5°C Trial T0 T2W T4W T6W T10W T14W Anti-PD-1 antibody concentration (mg/ml) 154.2 NT 153.5 156.5 153.8 154.1 pH 5.1 NT 5.0 5.0 5.0 5.0 SEC: Total content of HMMS (%) 0.6 NT 0.6 0.6 0.6 0.6 ICE: Acid (%) 23.0 NT 24.2 23.8 26.4 26.2 ICE: Alkaline (%) 13.1 NT 12.5 13.9 12.2 13.3 ICE: Main view (%) 63.9 NT 63.3 62.3 61.3 60.5 MFI: Total content ≥ 10µm (particles/ml) 330 NT 38 eight 57 eleven MFI: Total content ≥ 25µm (particles/ml) 83 NT 0 four eleven eight

Table 14
Thermal stability of composition 7 (150 mg/ml) at 25°C Trial T0 T2W T4W T6W T10W T14W Anti-PD-1 antibody concentration (mg/ml) 154.2 150.4 154.1 156.0 155.1 153.9 pH 5.1 5.1 5.0 5.0 5.1 5.0 SEC: Total content of HMMS (%) 0.6 0.6 0.7 0.7 0.8 0.9 ICE: Acid (%) 23.0 22.7 24.5 24.9 29.9 29.6 ICE: Alkaline (%) 13.1 15.5 12.9 13.8 13.5 13.9 ICE: Main view (%) 63.9 61.8 62.5 61.3 56.6 56.5 MFI: Total content ≥ 10µm (particles/ml) 330 23 eleven 76 92 31 MFI: Total content ≥ 25µm (particles/ml) 83 four four 0 23 eight

Table 15
Thermal stability of composition 7 (150 mg/ml) at 40°C Trial T0 T2W T4W T6W T10W T14W Anti-PD-1 antibody concentration (mg/ml) 154.2 152.6 156.7 154.2 154.6 NT pH 5.1 5.1 5.1 5.1 5.1 NT SEC: Total content of HMMS (%) 0.6 1.1 1.8 2.2 3.4 NT ICE: Acid (%) 23.0 28.5 35.0 39.0 49.5 NT ICE: Alkaline (%) 13.1 15.9 15.8 15.6 13.4 NT ICE: Main view (%) 63.9 55.7 49.2 45.3 37.1 NT MFI: Total content ≥ 10µm (particles/ml) 330 23 157 23 88 NT MFI: Total content ≥ 25µm (particles/ml) 83 0 four 0 eleven NT

Tables 16, 17 and 18 present the thermal stability for composition 7 at 10 mg/ml anti-PD-1 at 5°C, 25°C and 40°C, respectively.

Table 16
Thermal stability of composition 7 (100 mg/ml) at 5°C Trial T0 T2W T4W T6W T10W T14W Anti-PD-1 antibody concentration (mg/ml) 104.4 NT 104.7 104.2 107.0 104.0 pH 5.0 NT 5.0 5.1 5.0 5.0 SEC: Total content of HMMS (%) 0.6 NT 0.6 0.6 0.6 0.6 ICE: Acid (%) 23.2 NT 28.2 25.0 27.7 26.4 ICE: Alkaline (%) 13.0 NT 12.3 13.5 13.1 13.6 ICE: Main view (%) 63.8 NT 59.5 61.5 59.1 60.0 MFI: Total content ≥ 10µm (particles/ml) 84 NT 126 19 19 fifteen MFI: Total content ≥ 25µm (particles/ml) four NT eleven 0 eight eight

Table 17
Thermal stability of composition 7 (100 mg/ml) at 25°C Trial T0 T2W T4W T6W T10W T14W Anti-PD-1 antibody concentration (mg/ml) 104.4 103.4 104.0 104.5 104.0 104.6 pH 5.0 5.1 5.0 5.0 5.0 5.0 SEC: Total content of HMMS (%) 0.6 0.6 0.6 0.7 0.7 0.7 ICE: Acid (%) 23.2 23.1 27.2 25.6 30.7 29.7 ICE: Alkaline (%) 13.0 14.8 12.6 13.7 13.3 13.6 ICE: Main view (%) 63.8 62.2 60.2 60.7 56.0 56.7 MFI: Total content ≥ 10µm (particles/ml) 84 eleven 0 23 183 23 MFI: Total content ≥ 25µm (particles/ml) four four 0 eight eleven four

Table 18
Thermal stability of composition 7 (100 mg/ml) at 40°C Trial T0 T2W T4W T6W T10W T14W Anti-PD-1 antibody concentration (mg/ml) 104.4 104.3 105.7 NT NT NT pH 5.0 5.0 5.0 5.0 5.0 NT SEC: Total content of HMMS (%) 0.6 1.0 1.5 1.9 3.2 NT ICE: Acid (%) 23.2 27.6 36.3 39.5 49.1 NT ICE: Alkaline (%) 13.0 18.5 14.7 15.8 13.5 NT ICE: Main view (%) 63.8 53.9 49.0 44.6 37.5 NT MFI: Total content ≥ 10µm (particles/ml) 84 31 643 27 fifty NT MFI: Total content ≥ 25µm (particles/ml) four four 46 0 eight NT

Tables 19, 20 and 21 present the thermal stability for composition 8 at 150 mg/ml anti-PD-1 at 5°C, 25°C and 40°C, respectively.

Table 19
Thermal stability of composition 8 (150 mg/ml) at 5°C Trial T0 T2W T4W T6W T10W T14W Anti-PD-1 antibody concentration (mg/ml) 148.8 NT 151.0 150.4 NT NT pH 5.1 NT 5.1 5.1 NT NT SEC: Total content of HMMS (%) 0.6 NT 0.6 0.6 NT NT ICE: Acid (%) 22.8 NT 27.3 24.8 NT NT ICE: Alkaline (%) 12.8 NT 12.6 13.2 NT NT ICE: Main view (%) 64.4 NT 60.1 62.0 NT NT MFI: Total content ≥ 10µm (particles/ml) 508 NT 61 23 NT NT MFI: Total content ≥ 25µm (particles/ml) 37 NT eight eight NT NT

Table 20
Thermal stability of composition 8 (150 mg/ml) at 25°C Trial T0 T2W T4W T6W T10W T14W Anti-PD-1 antibody concentration (mg/ml) 148.8 152.0 153.8 152.0 NT NT pH 5.1 5.1 5.1 5.1 NT NT SEC: Total content of HMMS (%) 0.6 0.6 0.7 0.7 NT NT ICE: Acid (%) 22.8 24.1 25.1 24.6 NT NT ICE: Alkaline (%) 12.8 14.9 13.7 13.7 NT NT ICE: Main view (%) 64.4 61.1 60.1 61.7 NT NT MFI: Total content ≥ 10µm (particles/ml) 508 332 2637 134 NT NT MFI: Total content ≥ 25µm (particles/ml) 37 four 573 eleven NT NT

Table 21
Thermal stability of composition 8 (150 mg/ml) at 40°C Trial T0 T2W T4W T6W T10W T14W Anti-PD-1 antibody concentration (mg/ml) 148.8 149.0 152.8 153.0 NT NT pH 5.1 5.1 5.0 5.1 NT NT SEC: Total content of HMMS (%) 0.6 1.1 2.0 1.9 NT NT ICE: Acid (%) 22.8 27.9 NT 37.8 NT NT ICE: Alkaline (%) 12.8 18.0 NT 16.1 NT NT ICE: Main view (%) 64.4 54.1 NT 46.2 NT NT MFI:
Total content ≥ 10µm (particles/ml) 508 four 27 65 NT NT MFI:
Total content ≥ 25µm (particles/ml) 37 0 eight eight NT NT

No significant changes in terms of protein concentration, pH, or particles invisible to the naked eye were observed for all compositions under all test conditions (Tables 13-21). In addition, no significant changes in aggregation or species differing in charge were observed at 5°C or 25°C. The number of aggregates (HMMS) and acid species increased for all compositions after storage at 40°C, however, the increase is the same in all compositions, and the relative increase is considered to be acceptable for liquid compositions with assumed storage conditions of 5°C.

These results demonstrate that composition 7, containing 150 mg/ml anti-PD-1 antibody mAb7, 20 mM histidine, 84 mg/ml trehalose dihydrate, 0.05 mg/ml disodium EDTA dihydrate, and 0.2 mg/ml PS80, at pH 5.0-5.1 is stable after 14 weeks (14W) of storage at 5°C or 25°C (Tables 13 and 14). These results also demonstrate that composition 7 containing 100 mg/ml anti-PD-1 antibody mAb7, 20 mM histidine, 84 mg/ml trehalose dihydrate, 0.05 mg/ml disodium EDTA dihydrate, and 0.2 mg/ml PS80 , at pH 5.0-5.1 is stable after 14 weeks of storage at 5°C or 25°C (Tables 16 and 17). These results also demonstrate that composition 8, containing 150 mg/ml anti-PD-1 antibody mAb7, 20 mM histidine, 100 mM arginine, 50 mg/ml trehalose dihydrate, 0.05 mg/ml disodium EDTA dihydrate, and 0.2 mg/ml PS80, at pH 5.0 is stable after 6 weeks (6W) of storage at 5°C or 25°C (Tables 19 and 20).

Freeze-thaw and shake stability of composition 7 at 100 and 150 mg/ml anti-PD-1 and composition 8 at 150 mg/ml anti-PD-1 was determined by subjecting the compositions to stress conditions or 5 cycles of freeze/thaw (FT), or shaking (AG) for 24 hours. The results are presented in Table 22.

Table 22
Stability of composition 7 (100 mg/ml and 150 mg/ml) and composition 8 after freezing/thawing and shaking Trial Composition 7
(150 mg/ml) Composition 7
(100 mg/ml) Composition 8 (150 mg/ml) Initial value AG FT Initial value AG FT Initial value AG FT Anti-PD-1 antibody concentration (mg/ml) 154.2 152.9 152.1 104.4 104.4 102.8 148.8 148.7 151.7 pH 5.1 5.0 5.1 5.0 5.0 5.0 5.1 5.1 5.1 SEC: Total content of HMMS (%) 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6

Similar low levels of aggregate formation after exposure to stress conditions either by freeze/thaw or shaking were observed for composition 7 and composition 8, i. 0.6%, (Table 22).

These results demonstrate that composition 7 containing 100 or 150 mg/ml of anti-PD-1 antibody mAb7, 20 mM histidine, 84 mg/ml trehalose dihydrate, 0.05 mg/ml disodium EDTA dihydrate, and 0.2 mg/ml PS80, at pH 5.0-5.1 is stable after 5 freeze/thaw cycles or 24 hours of shaking. Composition 8 containing 150 mg/ml anti-PD-1 antibody mAb7, 20 mM histidine, 100 mM arginine HCl, 50 mg/ml trehalose dihydrate, 0.05 mg/ml disodium EDTA dihydrate, 0.2 mg/ml PS80, pH 5.0) is also stable after 5 freeze/thaw cycles or 24 hours of shaking.

Example 5 Glycosylation pattern of anti-PD-1 antibody mAb7

This example illustrates the glycosylation pattern of the anti-PD-1 antibody mAb7.

Peptide mapping by LC/MS confirmed one N-glycosylation site located on the heavy chain peptide containing the N ²⁹⁴ ST consensus sequence. N ²⁹⁴ ST the consensus sequence is essentially completely occupied. The profile of N-linked oligosaccharides observed for mAb7 shows two major N-glycans, G0F and G1F, which are both complex-type bianthenary structures with a fucosylated core. In addition, less abundant N-glycans corresponding to truncated and/or afucosylated complex-type bianthenary structures, Man5 high-mannose structures, and sialylated, fucosylated-core bianthenary complex-type oligosaccharides have also been defined and identified.

Profiling of N-linked oligosaccharides mAb7 included 2-aminobenzamide (2-AB)-labeling of N-linked oligosaccharides released by peptide-N-glycosidase F (PNGaseF). 2-AB-labeled N-linked oligosaccharides were separated by hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection and structure determination by mass spectrometry. Glycan heterogeneity in mAb7 is plotted in FIG. 6.

Example 6 Evaluation of Anti-PD-1 Antibodies in Formulation 7

This example evaluates the possibility of using composition 7 with ^KEYTRUDA® (pembrolizumab) and ^OPDIVO® (nivolumab).

Pembrolizumab and nivolumab were each formulated in 20 mM histidine buffer, 84 mg/ml trehalose dihydrate, pH 5.0. Highly concentrated polysorbate 80 and disodium EDTA dihydrate were added to the samples to prepare the final composition of 20 mM histidine, 84 mg/ml trehalose dihydrate, 0.05 mg/ml disodium EDTA dihydrate and 0.2 mg/ml PS80, at pH 5.0 (composition 7). All compositions were then filtered using 0.22 µm PES filters and diluted to the required concentrations using a solution of protein-free composition 7.

Pembrolizumab was formulated into formulation 7 at 129, 150, 175 and 193 mg/ml. Nivolumab was formulated into formulation 7 at 125, 148 and 179 mg/ml. Viscosity at 20°C was measured as described above. The results are presented in Table 23.

Table 23
Viscosity of anti-PD-1 antibodies in composition 7 Antibody Antibody Concentration (mg/ml) Viscosity at 20°C (cP) pembrolizumab 129 7.1 150 11.7 175 24.2 193 41.4 Nivolumab 125 7.1 148 13.9 179 35.1

The results demonstrate that the viscosities as a function of protein concentration of pembrolizumab and nivolumab in formulation 7 are comparable to those of anti-PD-1 antibody mAb7 in formulation 7 (Table 12).

The thermal stability of pembrolizumab, nivolumab and the anti-PD-1 antibody mAb7 in formulation 7 was assessed by measuring the melting point by differential scanning calorimetry. All three antibodies were diluted to 1 mg/mL using a protein-free formulation 7 solution prior to analysis. Thermal scanning was performed from 10°C to 110°C at a temperature change rate of 100°C/hour. Thermograms are shown in Fig. 7, and the start of thermal expansion (T _start ) and melting temperature (T _m1, m _{, 2} ) are shown in Table 24.

Table 24
Thermal properties of anti-PD-1 antibodies in composition 7 Antibody T _start (°C) _Tm1 (°C) _Tm2 (°C) pembrolizumab 52.2 62.5 74.1 Nivolumab 52.7 61.4 69.0 anti-PD-1 antibody mAb7 51.8 60.5 71.9

The thermal stability results demonstrate that pembrolizumab, nivolumab, and the anti-PD-1 antibody mAb7 in formulation 7 have comparable thermal profiles, melt onset temperatures, and melting points.

The stability of pembrolizumab and nivolumab at 150 mg/ml in formulation 7 was assessed at 40° C. for 2 weeks by determination of aggregation (SEC), isoform charge differences (iCE), concentration, purity by reductive capillary gel electrophoresis (rCGE), and pH . The results are presented in Tables 25 and 26.

Table 25
Thermal stability of composition 7 (150 mg/ml pembrolizumab) at 40°C Trial T0 T2W Pembrolizumab antibody concentration (mg/ml) 154.0 150.7 pH 5.0 5.1 SEC: Total content of HMMS (%) 0.3 1.5 ICE: Acid (%) 28.3 26.9 ICE: Alkaline (%) 3.4 16.4 ICE: Main view (%) 53.6 54.6 rCGE: Purity (%) 99.6 99.2

Table 26
Thermal stability of composition 7 (150 mg/ml nivolumab) at 40°C Trial T0 T2W Nivolumab antibody concentration (mg/ml) 150.2 153.3 pH 5.1 5.2 SEC: Total content of HMMS (%) 0.7 1.9 ICE: Acid (%) 36.9 38.3 ICE: Alkaline (%) 5.1 12.7 ICE: Main view (%) 54.7 47.9 rCGE: Purity (%) 99.0 99.1

These results demonstrate that composition 7, containing 150 mg/ml pembrolizumab, 20 mM histidine, 84 mg/ml trehalose dihydrate, 0.05 mg/ml disodium EDTA dihydrate, and 0.2 mg/ml PS80, at pH 5.0- 5.1 is stable after 2 weeks of storage at 40°C. These results also demonstrate that composition 7 containing 150 mg/ml nivolumab, 20 mM histidine, 84 mg/ml trehalose dihydrate, 0.05 mg/ml disodium EDTA dihydrate and 0.2 mg/ml PS80, at pH 5.1 -5.2 is stable after 2 weeks at 40°C.

All references cited in this application, including patents, patent applications, articles, reference books, etc., as well as references cited therein, are incorporated into this application by reference in their entirety. In the event that one or more of the included literature and similar materials differ from or contradict the present application, including, but not limited to, certain terms, use of terms, methods described, and the like, the present application takes precedence.

The above description and examples describe in detail some specific embodiments of the invention and describe the best method envisaged by the inventors. However, it should be understood that no matter how detailed the foregoing may be in the text, the invention may be practiced in a variety of ways and the invention should be read in accordance with the appended claims and any equivalents thereof.

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SEQUENCE LIST

<110> Pfizer Inc.

<120> ANTI-PD-1 ANTIBODY COMPOSITIONS

<130> PC72395A

<150> 62/807.912

<151> 2019-02-20

<150> 62/639.587

<151> 2018-03-07

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<223> mouse affinity matured CDR sequence

<400> 4

Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn

1 5 10

<210> 5

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> mouse affinity matured CDR sequence

<400> 5

Asn Ile Tyr Pro Gly Ser Ser Leu Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 6

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> mouse affinity matured CDR sequence

<400> 6

Leu Ser Thr Gly Thr Phe Ala Tyr

fifteen

<210> 7

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> mouse affinity matured CDR sequence

<400> 7

Lys Ser Ser Gln Ser Leu Trp Asp Ser Gly Asn Gln Lys Asn Phe Leu

1 5 10 15

Thr

<210> 8

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> mouse affinity matured CDR sequence

<400> 8

Trp Thr Ser Tyr Arg Glu Ser

fifteen

<210> 9

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> mouse affinity matured CDR sequence

<400> 9

Gln Asn Asp Tyr Phe Tyr Pro His Thr

fifteen

<210> 10

<211> 444

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized antibody sequence

<400> 10

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Gly Ser Ser Leu Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Ser Thr Gly Thr Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 11

<211> 221

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized antibody sequence

<400> 11

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Trp Asp Ser

20 25 30

Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Thr Ser Tyr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro His Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Gly Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

115 120 125

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

130 135 140

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

145 150 155 160

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

165 170 175

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

180 185 190

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

195 200 205

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<---

Claims (40)

1. Pharmaceutical composition for the treatment of cancer, comprising or consisting of: about 150 mg/ml anti-PD-1 antibody; approximately 20 mm histidine buffer; approximately 84 mg/ml trehalose; about 0.2 mg/ml PS80; and about 0.045 or about 0.05 mg/ml EDTA, where the specified pharmaceutical composition has a pH of 5.0 +/- 0.5. 2. The pharmaceutical composition of claim 1, wherein the viscosity of the composition is between about 10 centipoise and about 18 centipoise at 20°C. 3. Pharmaceutical composition according to claim 2, wherein the viscosity of the composition is about 15 centipoise at 20°C. 4. The pharmaceutical composition of claim 1, wherein 0.045 mg/mL EDTA is calculated based on the molecular weight of Na2EDTA dihydrate and 0.050 mg/mL EDTA is calculated based on the molecular weight of Na2EDTA, each corresponding to 0.13 nM EDTA. 5. A pharmaceutical composition according to claim 4, wherein the viscosity of the composition is between about 10 centipoise and about 18 centipoise at 20°C. 6. The pharmaceutical composition according to claim 5, wherein the viscosity of the composition is about 15 centipoise at 20°C. 7. Pharmaceutical composition according to any one of paragraphs. 1-6, where EDTA includes disodium EDTA, disodium EDTA dihydrate, or a combination of disodium EDTA and disodium EDTA dihydrate. 8. Pharmaceutical composition according to any one of paragraphs. 1-7, where the antibody is a human or humanized monoclonal antibody. 9. Pharmaceutical composition according to any one of paragraphs. 1-8, where the antibody is an IgG4 antibody. 10. The pharmaceutical composition according to claim 9, wherein the antibody is an IgG4 S228P antibody. 11. Pharmaceutical composition according to any one of paragraphs. 1-10, wherein the antibody comprises a heavy chain variable region (VH) comprising the first VH complementarity determining region (CDR1), VH CDR2, and VH CDR3 of the VH sequence shown in SEQ ID NO: 2; and/or a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 of the VL sequence shown in SEQ ID NO: 3. 12. The pharmaceutical composition of claim 11, wherein VH CDR1 includes the amino acid sequence shown in SEQ ID NO: 4, VH CDR2 includes the amino acid sequence shown in SEQ ID NO: 5, and VH CDR3 includes the amino acid sequence shown in SEQ ID NO: : 6, VL CDR1 includes the amino acid sequence shown in SEQ ID NO: 7, VL CDR2 includes the amino acid sequence shown in SEQ ID NO: 8, and VL CDR3 includes the amino acid sequence shown in SEQ ID NO: 9. 13. Pharmaceutical composition according to any one of paragraphs. 1-10, wherein the antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 2 and an amino acid sequence that is at least 90% identical to the amino acid sequence of the light chain variable region. chain shown in SEQ ID NO: 3. 14. Pharmaceutical composition according to any one of paragraphs. 1-10, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence shown in SEQ ID NO: 2, or a variant with one or more conservative amino acid substitutions at residues that are not in the CDR, and/or a variable region light chain (VL) comprising the amino acid sequence shown in SEQ ID NO: 3, or a variant thereof with one or more amino acid substitutions in amino acids that are not in the CDR. 15. Pharmaceutical composition according to any one of paragraphs. 1-14, where the antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 10, with or without the C-terminal lysine of SEQ ID NO: 10; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 11. 16. Pharmaceutical composition according to claim 15, wherein the antibody exhibits glycosylation at Asn294, including G0F and G1F as the main types of glycans. 17. The pharmaceutical composition according to claim 16, wherein the glycosylation further comprises, as minor glycan species, truncated and/or afucosylated complex-type bianthenary structures, a high-mannose Man5 structure, and sialylated complex-type core-fucosylated bianthenary oligosaccharides. 18. Pharmaceutical composition according to any one of paragraphs. 1-17, where the pharmaceutical composition is lyophilized or non-lyophilized. 19. Pharmaceutical composition according to any one of paragraphs. 1, 4 or 8-18, where the pharmaceutical composition has a viscosity of from about 10 to about 18 centipoise at 20°C. 20. Pharmaceutical composition according to any one of paragraphs. 1-8, where the antibody is PF-06801591, nivolumab, pembrolizumab, cemiplimab, or spartalizumab, or an antigen-binding portion of any of the above. 21. Pharmaceutical composition for the treatment of cancer, comprising: 150 mg/ml of an anti-PD-1 antibody, wherein the antibody comprises a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 10, with or without the C-terminal lysine of SEQ ID NO: 10; and a light chain comprising the amino acid sequence shown in SEQ ID NO: 11; 20 mM histidine buffer; 84 mg/ml trehalose; 0.2 mg/ml PS80; and 0.045 or 0.05 mg/ml EDTA, where the specified pharmaceutical composition has a pH of 5.0 +/- 0.1 and has a viscosity of 10 to 18 centipoise at 20°C. 22. The pharmaceutical composition of claim 21, wherein 0.045 mg/mL EDTA is calculated based on the molecular weight of Na2EDTA dihydrate and 0.050 mg/mL EDTA is calculated based on the molecular weight of Na2EDTA, each corresponding to 0.13 nM EDTA. 23. The pharmaceutical composition of claim 21 or 22, wherein the EDTA includes disodium EDTA, disodium EDTA dihydrate, or a combination of disodium EDTA and disodium EDTA dihydrate. 24. Pharmaceutical composition according to any one of paragraphs. 21-23, where trehalose is trehalose dihydrate. 25. Pharmaceutical composition according to any one of paragraphs. 21-24, where the pharmaceutical composition does not include an antioxidant. 26. The pharmaceutical composition according to claim 25, wherein the antioxidant is L-methionine or a pharmaceutically acceptable salt thereof. 27. Pharmaceutical composition according to any one of paragraphs. 21-24, where the pharmaceutical composition does not include methionine. 28. Pharmaceutical composition according to any one of paragraphs. 21-27, where the pharmaceutical composition does not include arginine.

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