Pharmaceutical composition of a programmed death receptor 1 (PD-1) antibody and rHuPH20 or its variant or fragment

Abstract

The present invention provides a pharmaceutical composition comprising an anti-human PD-1 antibody or an antigen-binding fragment thereof containing oxidation of Met105 in the CDRH3 heavy chain region at about 3.0% or less, and rHuPH20 or a variant or fragment thereof. The present invention also provides a pharmaceutical composition comprising an anti-PD-1 antibody main species and its acidic species, and rHuPH20 or a variant or fragment thereof, wherein the amount of the acidic species is about 1.0 to 12.0%. The present invention also provides a formulation comprising an anti-PD-1 antibody and rHuPH20 or a variant or fragment thereof.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 393,111, filed on July 28, 2022, the entire content of which is incorporated herein by reference.

[0002] Reference to Electronically Submitted Sequence Listing The content of the electronic sequence listing (25563 - WO - SEQLIST.xml; size: 22,005 bytes; created on November 28, 2022) is incorporated herein by reference in its entirety.

[0003] The present invention provides a pharmaceutical composition comprising an anti - PD - 1 antibody or an antigen - binding fragment thereof, and rHuPH20 or a variant or fragment thereof. The present invention also provides a pharmaceutical composition comprising an anti - PD - 1 antibody or an antigen - binding fragment thereof having about 3.0% or less oxidation of Met105 and / or about 1.0 - 12.0% acidic species in the CDRH3 heavy - chain region, and rHuPH20 or a variant or fragment thereof.

Background Art

[0004] Immune checkpoint therapies targeting the programmed death receptor-1 (PD-1) axis have led to revolutionary improvements in clinical responses in multiple human cancers (Brahmer et al., N Engl J Med 2012, 366:2455-65; Garon et al, N Engl J Med 2015, 372:2018-28; Hamid et al., N Engl J Med 2013, 369:134-44; Robert et al., Lancet 2014,384:1109-17; Robert et al., N Engl J Med 2015,372:2521-32; Robert et al., N Engl J Med 2015,372:320-30; Topalian et al., N Engl J Med 2012, 366:2443-54; Topalian et al., J Clin Oncol 2014, 32:1020-30; Wolchok et al., N Engl J Med 2013,369:122-33). The interaction between the PD-1 receptor on T cells and its ligands, PD-L1 and PD-L2, on tumors and immune infiltrating cells can regulate T cell-mediated immune responses and play a role in immune evasion by human tumors (Pardoll DM. Nat Rev Cancer 2012,12:252-64). When PD-1 binds to either of its ligands, inhibitory signals are delivered to T cells. Immune therapies targeting the PD-1 axis include monoclonal antibodies directed at the PD-1 receptor (KEYTRUDA™ (pembrolizumab), Merck and Co., Inc., Kenilworth, NJ and OPDIVO™ (nivolumab), Bristol-Myers Squibb, Princeton, NJ), and monoclonal antibodies that bind to the PD-L1 ligand (MPDL3280A; TECENTRIQ™ (atezolizumab), Genentech, San Francisco, CA). Both treatment approaches have demonstrated antitumor effects in numerous cancer types.

[0005] The oxidation of methionine is one of the major degradation pathways in many protein pharmaceuticals. Methionine residues in proteins are prone to oxidation, resulting in the formation of methionine sulfoxide and, under extreme conditions, sulfone. Methionine residues exposed or within the CDRs of antibodies may affect the biological activity of the antibody by oxidation. The major degradation pathways of pembrolizumab include the oxidation of methionine 105 (Met105) and Fc methionine residues in the heavy-chain CDR when exposed to light or peroxide stress. The crystal structure of the pembrolizumab Fab fragment in complex with PD-1 shows that Met105 is part of the paratope and interacts with Lys131 of PD-1. Lee, J.Y. et al. Nat. Commun. 7, 13354 (2016).

[0006] Therefore, it is desirable to obtain a pembrolizumab composition with low oxidation, especially at the Met105 position.

[0007] Deamidation of asparagine residues can lead to succinimide formation, which can then be converted to aspartic acid or isoaspartic acid. Deamidation in antibodies, particularly in the CDR regions, can potentially affect the biological activity of the antibody. The crystal structure of the pembrolizumab Fab fragment in complex with PD-1 shows that N52, N55, and N59 in CDRH2 are part of the paratope. PD-1 residues are involved in water-mediated interactions with N52, N55, and N59. Additionally, PD-1 residues have a direct interaction with N59. Lee, J.Y. et al. Nat. Commun. 7, 13354 (2016). Therefore, it is also desirable to obtain a pembrolizumab composition with fewer deamidated variants.

[0008] Hyaluronidase is an enzyme that degrades hyaluronic acid present in the extracellular matrix. Six types of hyaluronidases, Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20 / SPAM1, are known to exist in humans. PH20 / SPAM1 (hereinafter referred to as PH20) is expressed on the sperm plasma membrane and acrosomal membrane.

[0009] By hydrolyzing hyaluronic acid, hyaluronidase reduces the viscosity of hyaluronic acid in the extracellular matrix and increases its permeability to tissues (skin). The subcutaneous region of the skin has a neutral pH of approximately 7.0 - 7.5. Among various types of hyaluronidases, PH20 is widely used (Bookbinder et al., 2006). In examples where PH20 is used, it is often co-administered with subcutaneously injected antibody therapeutics (Bookbinder et al., 2006). rHuPH20, also known as Hylenex (registered trademark) and approved by the FDA, is shown as an adjuvant for enhancing the dispersion and absorption of other injected drugs.

Prior Art Documents

Non-Patent Documents

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Summary of the Invention

[0011] The present invention provides an anti-human PD-1 antibody or an antigen-binding fragment thereof containing oxidation of Met105 in the CDRH3 heavy chain region at about 3.0% or less, and a pharmaceutical composition containing rHuPH20 or a variant or fragment thereof. The present invention also provides a pharmaceutical composition comprising an anti-human PD-1 antibody main species consisting of two heavy chains and two light chains, each heavy chain consisting of the amino acid sequence of SEQ ID NO: 11, and each light chain consisting of the amino acid sequence of SEQ ID NO: 5, an acidic species of the anti-human PD-1 antibody main species, and rHuPH20 or a variant or fragment thereof, wherein the amount of the acidic species is about 1.0 to 12.0%. The present invention also provides a pharmaceutical composition comprising an anti-human PD-1 antibody main species consisting of two heavy chains and two light chains, each heavy chain consisting of the amino acid sequence of SEQ ID NO: 11, and each light chain consisting of the amino acid sequence of SEQ ID NO: 5, acidic and basic species of the anti-human PD-1 antibody main species, and rHuPH20 or a variant or fragment thereof, wherein the amount of the main species is about 65 to 95%. The present invention also provides a pharmaceutical composition comprising an anti-human PD-1 antibody or an antigen-binding fragment thereof and rHuPH20 or a variant or fragment thereof, the pharmaceutical composition having one or more of (i) oxidation of Met105 in the CDRH3 heavy chain region of the anti-human PD-1 antibody or an antigen-binding fragment thereof at about 3.0% or less, (ii) 1.0 to 12.0% of an acidic species of the anti-human PD-1 antibody or an antigen-binding fragment thereof, and (iii) acidic and basic species of the anti-human PD-1 antibody main species, and the amount of the main species is about 65 to 85%. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment can be produced or obtained by a continuous perfusion process.

[0012] The present invention also provides a formulation of the above pharmaceutical composition comprising: a) an anti-human PD-1 antibody, or an antigen-binding fragment thereof, at about 5 mg / mL to about 175 mg / mL; b) rHuPH20 or a variant or fragment thereof at about 150 to 8000 U / ml; c) a buffer; d) a non-reducing disaccharide; e) a non-ionic surfactant; and optionally f) an antioxidant.

[0013] In one embodiment, the formulation comprises a) an anti-human PD-1 antibody at about 5 mg / mL to about 175 mg / mL, or an antigen-binding fragment thereof, b) rHuPH20 at about 150 U / ml to 8000 U / ml or a variant or fragment thereof, c) a buffer at about 5 mM to about 20 mM, d) a non-reducing disaccharide at about 3% to about 10% weight / volume (w / v) selected from the group consisting of sucrose and trehalose, e) a non-ionic surfactant at about 0.005% to about 0.10%, and optionally, f) an antioxidant at about 1 mM to about 30 mM.

[0014] In certain embodiments of the invention, the rHuPH20 or variant or fragment is rHuPH20 of SEQ ID NO: 18, or rHuPH20 variant fragment 1 shown in the amino acid sequence of SEQ ID NO: 17.

[0015] In another aspect, the invention provides a formulation comprising a) an anti-human PD-1 antibody at about 5 mg / mL to about 165 mg / mL, comprising a light chain variable region comprising three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2 and CDRL3 of SEQ ID NO: 3, and a heavy chain variable region comprising three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7 and CDRH3 of SEQ ID NO: 8 b) about 2000 U / ml of rHuPH20 or variant of SEQ ID NO: 17 or 18 c) a histidine buffer at about 5 mM to about 20 mM, pH about 5.0 to about 6.0 d) a non-reducing disaccharide at about 3% to about 10% w / v f) optionally, about 5 mM to about 20 mM of L-methionine, or a pharmaceutically acceptable salt thereof.

[0016] Surprisingly, under heat stress, in certain embodiments of the co-formulation, the hyaluronidase activity of rHuPH20 or its variant or fragment is increased compared to rHuPH20 or its variant or fragment alone. The formulation can be a liquid formulation or a liquid formulation reconstituted from a lyophilized formulation.

[0017] Also provided herein is a method of treating cancer in a human patient in need of treating cancer, comprising administering to the patient an effective amount of the pharmaceutical composition of the invention.

Brief Description of the Drawings

[0018]

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Mode for Carrying Out the Invention

[0019] I. Definitions and Abbreviations When used throughout this specification and the appended claims, the following abbreviations apply.

[0020] API Active Pharmaceutical Ingredient CDR Complementary Determining Region in the immunoglobulin variable region CHO Chinese Hamster Ovary CI Confidence Interval DS Drug Substance EC50 Concentration that brings about 50% effectiveness or binding ELISA Enzyme-Linked Immunosorbent Assay FFPE Formalin-Fixed Paraffin-Embedded FR Framework Region HC Heavy Chain HNSCC Head and Neck Squamous Cell Carcinoma HP-HIC High-Performance Hydrophobic Interaction Chromatography HP-IEX High-Performance Ion Exchange Chromatography HP-SEC High-Performance Size Exclusion Chromatography IC50 Concentration that Results in 50% Inhibition IgG Immunoglobulin G IHC Immunohistochemistry or Immunohistochemical mAb Monoclonal Antibody NCBI National Center for Biotechnology Information NSCLC Non-Small Cell Lung Cancer PCR Polymerase Chain Reaction PD-1 Programmed Death 1 (Also Known as Programmed Cell Death-1 and Programmed Death Receptor 1) PD-L1 Programmed Cell Death 1 Ligand 1 PD-L2 Programmed Cell Death 1 Ligand 2 PS80 or PS-80 Polysorbate 80 SWFI Sterile Water for Injection TNBC Triple-Negative Breast Cancer V H Variable Region of Immunoglobulin Heavy Chain VK Variable Region of Immunoglobulin Kappa Light Chain V L Variable Region of Immunoglobulin Light Chain v / v Volume / Volume WFI Water for Injection w / v Weight / Volume To make the present invention more readily understandable, specific technical terms and scientific terms are specifically defined below. Unless specifically defined elsewhere in this specification, all other technical terms and scientific terms used in this specification have the meanings generally understood by those skilled in the technical field to which the present invention pertains.

[0021] Throughout this specification and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0022] References to "or" are to be construed as including any and all alternatives or combinations of the listed alternatives, unless the context clearly indicates otherwise. In some cases, "and / or" is used to emphasize either or both possibilities.

[0023] As used herein, "acidic species" refers to anti-PD-1 antibody species that are more acidic than the anti-PD-1 antibody main species (e.g., as measured by cation exchange chromatography). Such acidic species can be detected by various chromatographic purification methods for separating molecular variants by charge, such as ion exchange, e.g., cation exchange chromatography (e.g., the method described in Example 5) or WCX-10 HPLC (weak cation exchange chromatography), followed optionally by mass spectrometry. Generally, acidic species have a lower isoelectric point (pI) than the main species and can have more acidic properties, for example, by methionine oxidation, sialylation of asparagine residues, or deamidation variants of the antibody, or combinations thereof. Examples of acidic species include, but are not limited to, the acidic variants, Acidic 1, and pre-main peak identified in Figures 6 or 7 of the present invention. Any of the acidic species can also have one or more CHO N-linked glycans selected from the group consisting of G0-F, G1-F, G2-F, G0, G1, G2, and Man5, for example, at N297 in the CH2 domain.

[0024] In one embodiment, the anti-PD-1 antibody acidic species is as identified by the peak eluting before the main peak according to the cation exchange method. In another embodiment, the anti-PD-1 antibody acidic species is as identified by the peak eluting before the main peak according to the weak cation exchange method. In the ion exchange chromatography method, "% acidic species" refers to the ratio of the total area of the acidic species peak in the elution chromatogram to the total area of all peaks.

[0025] As used herein, "acidic species 1" refers to an acidic species having one or more of deamidation, succinimide, aspartic acid, or isoaspartic acid formation at one or more of N384, N389, and N390 of the heavy chain of the anti-PD-1 antibody main species. Such an acidic species 1 is detected by various chromatographic purification methods for separating molecular variants by charge, such as ion exchange, e.g., cation exchange chromatography (e.g., the method described in Example 5) or WCX-10 HPLC (weak cation exchange chromatography), followed by mass spectrometry of the acidic species peak.

[0026] In one embodiment, the acidic species 1 of the anti-PD-1 antibody is as identified by the acidic species 1 peak in FIG. 6 or FIG. 7 and elutes according to the cation exchange method described in Example 5. In the ion exchange chromatography method, "% acidic species 1" refers to the total area of the acidic species 1 peak in the elution chromatogram divided by the total area of all peaks.

[0027] As used herein, "acidic variant species" refers to an acidic species having one or more of deamidation, succinimide, aspartic acid, or isoaspartic acid formation at one or more of N31, N52, N55, N59, and N61 of the heavy chain of the anti-PD-1 antibody main species, or the presence of M105 oxidation in the heavy chain, or a combination thereof. Such an acidic variant species is detected by various chromatographic purification methods for separating molecular variants by charge, such as ion exchange, e.g., cation exchange chromatography (e.g., the method described in Example 5) or WCX-10 HPLC (weak cation exchange chromatography), followed by mass spectrometry of the acidic species peak.

[0028] In one embodiment, the anti-PD-1 antibody acidic variant species is an anti-PD-1 antibody species identified by the acidic variant peak in FIG. 6 or FIG. 7 and eluted according to the cation exchange method described in Example 5. In the ion exchange chromatography method, “% acidic variant species” refers to the value obtained by dividing the total area of the acidic variant peak in the elution chromatogram by the total area of all peaks.

[0029] As used herein, “basic species” refers to anti-PD-1 antibody species that are more basic than the anti-PD-1 antibody main species (e.g., as measured by cation exchange chromatography). Such basic species are detected by various chromatographic purification methods for separating molecular variants by charge, such as ion exchange, e.g., cation exchange chromatography (e.g., the method described in Example 5) or WCX-10 HPLC (weak cation exchange chromatography), and mass spectrometry may then be performed in some cases. Generally, basic species have a higher pI than the main species and may have a stronger basic nature due to modifications or differences from the main species including, but not limited to, the presence of a C-terminal lysine residue (SEQ ID NO: 10 or 12), the presence of an N-terminal glutamine residue (SEQ ID NO: 10 or 13), or alpha-amidation of a C-terminal leucine residue (SEQ ID NO: 14 or 15), cleavage of the N-terminal amino acid residue of one or both heavy chains by an amino acid sequence of any one of SEQ ID NOs: 10 to 15, or combinations thereof. Examples of basic species include, but are not limited to, the basic variant A, basic variant B, basic 1, and basic 2 peaks identified in FIGS. 6 or 7 of the present invention. Any of the basic species may also have one or more CHO N-linked glycans selected from the group consisting of G0-F, G1-F, G2-F, G0, G1, G2, and Man5, for example, at N297 in the CH2 domain.

[0030] In one embodiment, the anti-PD-1 antibody basic species is as identified by the peak eluted after the main peak according to the cation exchange method. In another embodiment, the anti-PD-1 antibody basic species is as identified by the peak eluted after the main peak according to the weak cation exchange method. In the ion exchange chromatography method, “% basic species” refers to the total area of the basic species peaks in the elution chromatogram divided by the total area of all peaks.

[0031] As used herein, “main species” refers to an anti-PD-1 antibody species identified as the majority of the antibody species in a mixture with one or more of its acidic or basic species. Such main species are detected by various chromatographic purification methods for separating molecular variants by charge, such as ion exchange, e.g., cation exchange chromatography (e.g., the method described in Example 5) or WCX-10 HPLC (weak cation exchange chromatography), followed optionally by mass spectrometry. The mixture can be, for example, the result of antibody preparations from mammalian cells and their post-translational modifications, upstream and downstream processing, or storage. The main species can also have, for example, one or more CHO N-linked glycans selected from the group consisting of G0-F, G1-F, G2-F, G0, G1, G2, and Man5 at N297 in the CH2 domain.

[0032] In one embodiment, the main species comprises an anti-PD-1 antibody consisting of two heavy chains and two light chains, each heavy chain consisting of the amino acid sequence of SEQ ID NO: 11 and each light chain consisting of the amino acid sequence of SEQ ID NO: 5. In another embodiment, the anti-PD-1 antibody main species is produced from Chinese hamster ovary cells comprising a polynucleotide encoding a light chain consisting of the amino acid sequence of SEQ ID NO: 5 and a polynucleotide encoding a heavy chain consisting of the amino acid sequence of SEQ ID NO: 10, 13, or 15, or a polynucleotide encoding the light chain and the heavy chain.

[0033] In one embodiment, the main species is identified as the main peak by the cation exchange method. In the ion exchange method, “% main species” refers to the value obtained by dividing the total area of the main peak in the elution chromatogram by the total area of all peaks.

[0034] As used herein, “basic species 1” refers to a basic species consisting of two heavy chains and two light chains, wherein one heavy chain consists of the amino acid sequence of SEQ ID NO: 11, one heavy chain consists of the amino acid sequence of SEQ ID NO: 12, and each light chain consists of the amino acid sequence of SEQ ID NO: 5, or a basic species consisting of two heavy chains and two light chains, wherein one heavy chain consists of the amino acid sequence of SEQ ID NO: 11, one heavy chain consists of the amino acid sequence of SEQ ID NO: 14, the C-terminal leucine is alpha-amidated, and each light chain consists of the amino acid sequence of SEQ ID NO: 5, or a combination thereof. Such basic species 1 is detected by various chromatographic purification methods for separating molecular variants by charge, such as ion exchange, for example cation exchange chromatography (e.g., the method described in Example 5) or WCX-10 HPLC (weak cation exchange chromatography), and then mass spectrometry of the basic species peak is performed.

[0035] In one embodiment, the basic species 1 of the anti-PD-1 antibody is as identified by the basic 1 peak in FIG. 6 or FIG. 7 and is eluted according to the cation exchange method described in Example 5. In the ion exchange chromatography method, “% basic species 1” refers to the value obtained by dividing the total area of the basic 1 peak in the elution chromatogram by the total area of all peaks.

[0036] As used herein, “deamidated variant” refers to an antibody in which one or more asparagine residues have been deamidated. The deamidated variant can be in the form of succinimide, aspartic acid or isoaspartic acid, i.e., the neutral amide side chain has been converted to a residue with overall acidic characteristics.

[0037] In one aspect of measuring the main species, acidic species, or basic species, a Thermo Scientific ProPac WCX-10 column is used in the cation exchange method. In another embodiment, the Thermo Scientific ProPac WCX-10 column is used with a mobile phase (A) of 24 mM MES pH 6.1 containing 4% acetonitrile, a mobile phase (B) of 95 mM NaCl pH 8.0 containing 20 mM sodium phosphate and 4% acetonitrile, and a column temperature of 35°C. In one embodiment, a non-linear gradient of 22% - 22% B from 0 - 0.6 minutes, 22% - 29% B from 0.6 - 15.0 minutes, 29% - 70% B from 15.0 - 30.0 minutes, 70% - 100% B from 30.0 - 30.5 minutes, and 100% - 100% B from 30.5 - 33.0 minutes is used. In a further embodiment, the cation exchange method is described in Example 5.

[0038] As used herein, "expressing" and "expression" refer to enabling or causing the manifestation of information in a gene or coding sequence, such as RNA or DNA, for example, producing a protein by activating cell functions involved in the transcription and translation of the corresponding gene. A DNA sequence can be expressed within a cell or by a cell to form an "expression product" such as RNA (e.g., mRNA) or protein. The expression product itself may also be said to be "expressed" by the cell.

[0039] As used herein, an "expression vector" or "expression construct" refers to a vehicle (e.g., a plasmid) by which a polynucleotide containing regulatory sequences operably linked to a coding sequence can be introduced into a host cell, where the coding sequence is expressed using the transcription and translation machinery of the host cell.

[0040] As used herein, an "expression cassette" refers to a polynucleotide that includes, but is not limited to, a promoter operably linked to a gene sequence or operably linked to a multiple cloning site for inserting a gene sequence, and a polyA signal, and includes elements sufficient to control the expression of the gene. In some embodiments, the expression cassette further includes one or more regulatory elements capable of regulating the expression of the gene at the transcriptional level, translational level, and / or chromatin level.

[0041] As used herein, a "promoter" or "promoter sequence" refers to a segment of DNA that includes a regulatory region capable of mobilizing RNA polymerase (e.g., directly or through a protein or substance bound to another promoter) to initiate transcription of a coding sequence. Within the promoter sequence, a transcription start site (conveniently defined, for example, by mapping with nuclease S1) and a protein-binding domain (consensus sequence) that serves to mobilize RNA polymerase can be found.

[0042] As used herein, an "enhancer" or "enhancer sequence" refers to a DNA regulatory region that enhances the transcription of a promoter, independent of its distance, position, or orientation relative to the promoter. In certain embodiments, the enhancer is directly adjacent to the promoter. In some embodiments, the enhancer is distant from the promoter. In other embodiments, the promoter and enhancer are one combined sequence referred to herein as a "combo enhancer / promoter".

[0043] As used herein, "internal ribosome entry site" or "IRES" refers to an RNA element or sequence that enables cap-independent translation initiation by directly recruiting ribosomes. As used herein, the term "internal ribosome entry site" or "IRES" also encompasses a DNA sequence that can be transcribed into an RNA sequence that enables cap-independent translation initiation by directly recruiting ribosomes. An IRES can be of any origin, whether naturally occurring or artificial, and can be a wild-type IRES or a variant or mutant thereof.Examples of IRESs that can be used include, but are not limited to, the nucleotide sequence of the 5' untranslated region of encephalomyocarditis virus (EMCV) (GenBank: M81861.1; Duke et al., Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation. J Virol. 1992 Mar;66(3):1602-9.), the IRES element described by Bochkov & Palmenberg (Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. Biotechniques. 2006 Sep;41(3):283-4), the IRES element from expression vector pInSRT-GFP (GenBank LC417349.1), the IRES element from expression vector pCeMM-CTAP (SG) (GenBank EF467048.1), the IRES element described by Jang & Wimmer (Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein. Genes Dev. 1990 Sep;4(9):1560-72), the IRES element from expression vector pIRESneo3 (Clontech / Takara Bio), the IRES elements described in International Publication No. WO 2015 / 016786, International Publication No. WO 2015 / 021077, International Publication No. WO 2016 / 003368, International Publication No. WO 2016 / 074016 or International Publication No. WO 2013 / 092743, or variants thereof.

[0044] As used herein, "regulatory element", "regulatory region" or "regulatory sequence" refers to a polynucleotide sequence having the ability to regulate (e.g., initiate, activate, enhance, increase, decrease, inhibit, suppress, or silence) the expression of a gene. In some embodiments, regulation is achieved by binding a cytokine to the polynucleotide sequence. In other embodiments, regulation is achieved by interaction between cytokines. Regulation can occur at one or more different levels in the expression process from DNA to protein, including but not limited to transcription, translation, or chromatin levels.

[0045] As used herein, "insulator" refers to a class of DNA elements or sequences having the ability to isolate a proximal DNA region by preventing a position effect from the surrounding chromosomal region. In certain embodiments, when an insulator is located between an enhancer and a promoter, the insulator can block the enhancer. In some embodiments, an insulator can act as a barrier to prevent the progression of nearby condensed chromatin, which may otherwise silence expression. In other embodiments, an insulator can block the enhancer and act as a barrier.

[0046] As used herein, "expression enhancing sequence element" or "EASE" refers to a DNA element or sequence that can increase the expression of a protein when the DNA element or sequence is placed upstream of a promoter that controls the expression of the protein.

[0047] As used herein, "tripartite leader" or "TPL" refers to an RNA element or sequence in the 5' untranslated region of an adenovirus late-expressing mRNA that has the ability to initiate translation of late-expressing mRNAs in a cap-independent manner. As used herein, the term "tripartite leader" or "TPL" also encompasses a DNA sequence that can be transcribed into an RNA sequence in the 5' untranslated region of an adenovirus late-expressing mRNA that has the ability to initiate translation of late-expressing mRNAs in a cap-independent manner.

[0048] As used herein, "inverted terminal repeat" or "ITR" in the context of transposon technology refers to the DNA elements or sequences and their reverse versions at both ends of a transposon that signal where breakage and joining should occur.

[0049] As used herein, "selectable marker" or "selection marker" refers to a protein that enables the specific selection of cells expressing this protein by adding the corresponding selection agent to the culture medium. In certain embodiments, the selectable marker is a eukaryotic selectable marker, which enables the selection of eukaryotic cells expressing the marker protein. In some embodiments, the selectable marker is a bacterial selectable marker, which enables the selection of bacterial cells expressing the marker protein.

[0050] As used herein, "polynucleotide sequence", "nucleic acid sequence" or "nucleotide sequence" refers to a series of nucleotide bases (also referred to as "nucleotides") in a nucleic acid such as DNA or RNA, and means any strand of two or more nucleotides.

[0051] As used herein, "host cell" refers to any cell of any organism that is used for the purpose of producing a recombinant protein encoded by an expression vector or propagating an expression vector introduced into the host cell. "Mammalian recombinant host cell" refers to a mammalian host cell containing a heterologous expression vector, which may or may not be integrated into the host cell chromosome. "Bacterial recombinant host cell" refers to a bacterial host cell containing a heterologous expression vector, which may or may not be integrated into the host cell chromosome.

[0052] As used herein, the term "fed-batch culture" refers to a cell culture method in which additional nutrients are provided to the culture during the culture process. Fed-batch cultures typically are stopped at some point and the cells and / or components in the medium are harvested. The product accumulates and remains in the bioreactor until the end of the run.

[0053] As used herein, "recovering" an antibody or antigen-binding fragment thereof means separating it from particulate matter that may include host cells, cell aggregates, and / or lysed cell fragments into a cell-free fraction that is substantially free of host cells and cell debris, i.e., a cell-free "permeate". Such cells and cell debris are removed from the cell culture broth, for example, by centrifugation, depth filtration, and / or microfiltration. For example, a series of filtration steps such as hollow fiber membranes or depth filtration can be used to produce a cell-free permeate. "Continuously recovering" refers to recovering the cell culture broth while performing antibody production in a bioreactor. The secreted protein product in the bioreactor can be continuously recovered from the cell culture broth by microfiltration during the process of removing the medium through a perfusion system, such that the protein of interest is isolated in the microfilter permeate exiting the perfusion system. The microfilter can be a tangential flow filtration (TFF) unit including a hollow fiber module, or an alternating tangential flow filtration (ATF) unit. Commercially available TFF units include, but are not limited to, the Microza® TFF unit or the KrosFlow® Max. Commercially available hollow fiber modules can be obtained, for example, from Pall or Repligen. In one embodiment, the perfusion bioreactor has a constant permeation rate of the cell culture broth containing the antibody or antigen-binding fragment to maintain a consistent flow rate for the affinity chromatography step.

[0054] As used herein, "cell culture broth" refers to a broth containing host cells, cell debris, cell culture medium, an antibody, or an antigen-binding fragment thereof during the cell growth and antibody production processes.

[0055] As used herein, "recovered cell culture fluid" or "HCCF" refers to a cell culture fluid containing an antibody or an antigen-binding fragment thereof obtained after recovering the cell culture broth that is substantially free of host cells and cell debris. In one embodiment, HCCF is a cell-free permeate.

[0056] As used herein, "fluidly connected", "fluidically", or "fluidly connected to", or "receiving material fluidically from" refers to a process or another process from a separate system in a manufacturing process where the material containing the protein of interest flows between processes or systems by pipes, tubes, or other closed conduits without manual loading or unloading.

[0057] As used herein in the context of HCCF, "continuously purifying" refers to an uninterrupted flow of HCCF to at least one affinity stationary phase in at least the loading step, and optionally an uninterrupted flow in any washing or elution steps.

[0058] As used herein, "perfusion" or "performing perfusion" refers to a method of cell culture in which fresh medium is continuously provided over a period of time during which there is additional fresh medium in the culture (after the start of the culture process), and at the same time the medium is removed while continuously recovering the antibody or antigen-binding fragment from the medium. The fresh medium typically provides nutritional supplementation for the cells that has been depleted during the culture process.

[0059] As used herein, "perfusion rate" refers to the rate at which fresh medium is provided and the cell culture fluid is removed.

[0060] As used herein, "perfusion bioreactor" refers to a bioreactor for culturing cells in which the same amount of culture medium can be added to and simultaneously removed from the reactor. In one embodiment, the cells are retained within the bioreactor. A perfusion bioreactor includes a bioreactor and a perfusion system operably attached thereto that provides a stable source of fresh nutrient medium and removal of cell waste products. The bioreactor and the perfusion system of the perfusion bioreactor can be separate mechanical units that operate in concert. Examples of a number of commercially available ones include, but are not limited to, various Xcellerex® brand single-use bioreactors (SUB; GE Healthcare Life Sciences) and KrosFlo® brand perfusion flow path assemblies and systems (Spectrum; Repligen), and these bioreactors and perfusion systems can be appropriately combined into a perfusion bioreactor by those skilled in the art. Alternatively, the bioreactor and the perfusion system can be assembled into a single mechanical unit, for example, but not limited to, a perfusion bioreactor of the 3D Biotek brand (Sigma-Aldrich).

[0061] The term "surge tank" as used herein refers to a well-mixed (providing sufficient mixing so that the fluid is homogeneous) storage reservoir, mixing tank, feed tank, or collection tank (alternatively, "collection tank") at the downstream end of a conduit, feeder, dam, pipe, or tube for absorbing differences in the flow rates of the operations of two fluid-connected units, for example, the flow rate of the permeate coming from the bioreactor and the flow rate of a first chromatography system under automatic control in a continuous or semi-continuous format process embodiment of the present invention. The surge tank absorbs changes or differences in flow rates between the operations of fluid-connected units by rapidly increasing the volume within a preset volume range limit.

[0062] As used herein, the term "residence time" refers to the average time that a fluid solution spends in a tank. In the case of perfusion, this is the reciprocal of the exchange rate (e.g., 2 VVD has an average residence time of 0.5 days). Sieving of solution components and retention of solution components by a membrane are ignored for residence time.

[0063] As used herein, "M105", "Met105", or "methionine 105" refers to the methionine in the CDRH3 region of the heavy chain of SEQ ID NO: 8 (RDYRFDMGFDY).

[0064] As used herein, "% oxidation of M105" or "% oxidation of Met105" refers to a) the total amount of one or more anti-PD-1 antibody fragments of the invention having oxidized Met105, with or without oxidized Met105, relative to the total amount of one or more anti-PD-1 antibody fragments of the invention, determined by reduced peptide mapping, liquid chromatography, and mass spectrometry, or b) the total amount of an anti-PD-1 antibody of the invention having oxidized Met105, with or without oxidized Met105, relative to the total amount of an anti-PD-1 antibody of the invention, determined by hydrophobic interaction chromatography (HIC).

[0065] "Binding" an antibody or antigen-binding fragment to a solid phase means exposing the antibody or antigen-binding fragment to the solid phase under appropriate conditions (pH and / or conductivity) such that the antibody or antigen-binding fragment reversibly associates with the solid phase by interaction between the antibody or antigen-binding fragment and a ligand immobilized on the solid phase.

[0066] As used herein, the term "equilibration solution" refers to a solution used to equilibrate a solid phase prior to loading an antibody or antigen-binding fragment onto the solid phase. The equilibration solution can contain one or more salts and buffering species. In one embodiment, the equilibration solution is at the same conditions as the loading solution containing the antibody or antigen-binding fragment.

[0067] As used herein, the term "loading solution" refers to a solution used to load a composition containing the antibody or antigen-binding fragment of interest and one or more impurities onto a stationary phase. The loading solution may optionally further contain one or more buffering species and salts.

[0068] As used herein, the term "washing solution" refers to a solution used to re-equilibrate the stationary phase before eluting the antibody or antigen-binding fragment of interest. For washing, the washing solution has a conductivity and / or pH such that impurities are removed from the stationary phase. For re-equilibration, the washing solution and the equilibration solution may be the same, but this is not essential. The washing solution can contain one or more salts and buffering species.

[0069] As used herein, the term "elution solution" refers to a solution used to elute the antibody or antigen-binding fragment of interest from the stationary phase. The elution solution can contain one or more salts or buffering species. The presence, pH, or conductivity of the salt and one or more of the buffering species in the elution solution are such that the antibody or antigen-binding fragment is eluted from the stationary phase.

[0070] As used herein, the term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, the electric current flows by ion transport. Thus, as the amount of ions present in the aqueous solution increases, the conductivity of the solution increases. The unit of measurement of conductivity is mS / cm and can be measured using, for example, a conductivity meter sold within the GE Healthcare Akta™ System. The conductivity of a solution can be altered by changing the concentration of ions therein. For example, the concentration of buffer and / or the concentration of salt (e.g., NaCl or KCl) in the solution can be changed to achieve the desired conductivity. Preferably, the salt concentration of various buffers is changed to achieve the desired conductivity as in the following examples.

[0071] As used herein, "purifying" an antibody or antigen-binding fragment of interest or a "purified composition" refers to increasing the degree of purity of an antibody or antigen-binding fragment in a composition by removing (completely or partially) at least one impurity from the composition. Impurities can be host cell components such as serum, protein or nucleic acid, cell debris, growth medium or antibody aggregates. The term is not intended to mean the complete absence of such biological molecules, or the absence of water, buffer or salt, or to refer to components of a pharmaceutical composition containing an antibody or antigen-binding fragment.

[0072] As used herein, a "continuous multi-column chromatography system" refers to a chromatography system that includes at least two stationary phases having similar impurity separation functions, wherein a sample is loaded onto at least one stationary phase and at least one stationary phase is subjected to a non-loading step (one or more of equilibration, washing, elution and regeneration).

[0073] As used herein, a "stationary phase" refers to any surface onto which one or more ligands can be immobilized. The stationary phase may be a suspension, a discontinuous phase of individual particles, a plate, a sensor, a chip, a capsule, a cartridge, a resin, beads, a monolith, a gel, a membrane, or a membrane adsorber, etc. The stationary phase may also be packed (e.g., with resin beads) into a purification column. Examples of materials for forming the stationary phase include mechanically stable matrices such as porous or non-porous beads, inorganic materials (e.g., porous silica, controlled pore glass (CPG) and hydroxyapatite), synthetic organic polymers (e.g., polyacrylamide, polymethyl methacrylate, polystyrene-divinylbenzene, poly(styrene divinyl)benzene, polyacrylamide, ceramic particles and derivatives of any of the above) and polysaccharides (e.g., cellulose, agarose and dextran). See Jansson, J.C.; Ryden, L. Protein Purification; Wiley: New York, 1998.

[0074] As used herein, "impurity" refers to a substance different from the desired antibody or antigen-binding fragment. Impurities can be host cell proteins (HCPs), host cell DNA (HC-DNA), protein aggregates or clippings, and other undesired protein modifications (i.e., oxidized species, acid variant species).

[0075] As used herein, "treating" cancer or "treatment" of cancer refers to administering a composition of the invention to a subject having an immune or cancerous condition, or diagnosed with cancer or a pathogenic infection (e.g., virus, bacterium, fungus), to achieve at least one positive therapeutic effect, e.g., a decrease in the number of cancer cells, a reduction in tumor size, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth. "Treatment" includes one or more of the following: inducing / increasing an anti-tumor immune response; stimulating an immune response against a pathogen, toxin, and / or self-antigen; stimulating an immune response against a viral infection; decreasing the number of one or more tumor markers; halting or delaying the progression of a tumor or blood cancer, or a disease associated with the binding of PD-1 to its ligands PD-L1 and / or PD-L2 (a "PD-1 associated disease"), e.g., cancer; stabilizing a PD-1 associated disease; inhibiting the growth or survival of tumor cells; eliminating or reducing the size of one or more cancerous lesions or tumors; decreasing the level of one or more tumor markers; improving the clinical symptoms of a PD-1 associated disease; suppressing, decreasing the severity or duration of the clinical symptoms of a PD-1 associated disease such as cancer; extending the patient's survival period compared to the expected survival period of patients not receiving similar treatment; and inducing complete or partial remission of a cancerous condition or other PD-1 associated disease.

[0076] "Immune status" or "immune disorder" includes, for example, pathological inflammation, inflammatory disorders, and autoimmune disorders or diseases. "Immune status" also refers to cancers, tumors, and angiogenesis, including infectious diseases, persistent infectious diseases, and proliferative states, such as infectious diseases, tumors, and cancers that resist eradication by the immune system. "Cancerous states" include, for example, precancerous states such as cancer, cancer cells, tumors, angiogenesis, and dysplasia.

[0077] The positive therapeutic effect in cancer can be measured in several ways (see W.A. Weber, J. Nucl. Med. 50:1S-10S (2009)). For example, with respect to tumor growth inhibition, according to the NCI criteria, T / C ≤ 42% is the minimum level of antitumor activity. T / C < 10% is considered a high level of antitumor activity, and T / C(%) = median of the treated tumor volume / median of the control tumor volume × 100. In some embodiments, the treatment achieved by administration of the compositions of the present invention is any of progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). PFS, also referred to as "time to tumor progression," indicates the length of time during and after treatment that the cancer does not grow, including the length of time the patient experiences a complete or partial response, and the length of time the patient experiences disease stability. DFS refers to the length of time during and after treatment that the patient remains disease-free. OS refers to the extension of the average life span compared to naive or untreated individuals or patients. Embodiments of the compositions, treatment methods, and uses of the present invention may not be effective in achieving a positive therapeutic effect in all subjects, but it should be so in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, Mann-Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0078] As used herein, the term "patient" (alternatively referred to herein as "subject" or "individual") refers to a mammal (e.g., rat, mouse, dog, cat, rabbit) that can be treated with the compositions of the present invention or the compositions, most preferably a human. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a pediatric patient. "Those in need of treatment" include patients who can benefit from treatment with the compositions of the present invention or the compositions, such as patients suffering from cancer or an immune condition.

[0079] As used herein, the term "antibody" refers to any form of an antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized fully human antibodies, and chimeric antibodies.

[0080] Generally, the basic antibody structural unit contains a tetramer. Each tetramer contains two identical pairs of polypeptide chains, and each pair has one "light" chain (about 25 kDa) and one "heavy" chain (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 - 110 or more amino acids that is mainly responsible for antigen recognition. The variable regions of each light chain / heavy chain pair form the antibody binding site. Thus, generally, a complete antibody has two binding sites. The carboxy-terminal portion of the heavy chain may define a constant region that is mainly responsible for effector functions. Typically, human light chains are classified as kappa light chains and lambda light chains. Further, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the isotypes of the antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 or more amino acids. Generally, see Fundamental Immunology Ch.7 (Paul, W., ed. 2nd ed. Raven Press, N.Y. (1989)).

[0081] Typically, the variable domains of both the heavy and light chains contain three hypervariable regions, also called complementarity-determining regions (CDRs), which are located within relatively conserved framework regions (FRs). The CDRs are usually aligned by the framework regions, enabling binding to a particular epitope. Generally, proceeding from the N-terminus to the C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The amino acid assignments to each domain generally follow the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th edition; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J. Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

[0082] As used herein, the terms “pharmaceutically effective amount” or “effective amount” refer to an amount of a therapeutic composition or composition introduced into a patient that is sufficient to treat a disease or condition. One of ordinary skill in the art recognizes that this level may vary depending on characteristics of the patient such as age, weight, etc.

[0083] The term "about" when modifying the amount of a substance or composition (e.g., mM or M), the ratio of components of a composition (v / v or w / v), the pH of a solution / composition, or the value of a parameter characterizing a step of a method, refers to the variability in numerical quantities that can occur due to typical measurement, handling, and sampling procedures involved in the preparation, characterization, and / or use of the substance or composition, by instrumental error in these procedures, differences in the manufacture, source, or purity of the components used to make or use the composition or to carry out the procedure, and otherwise. In certain embodiments, "about" can mean a variation of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% of the value.

[0084] As used herein, the terms "cancer," "cancerous," or "malignant" refer to or describe a physiological state in a mammal that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinomas, lymphomas, leukemias, blastomas, and sarcomas. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin lymphoma, non-Hodgkin lymphoma, gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer.

[0085] As used herein, the terms "PD-1 binding fragment", "antigen-binding fragment thereof", and "binding fragment thereof" include fragments or derivatives of an antibody that bind to an antigen (human PD-1) and still substantially retain the biological activity of inhibiting its activity (e.g., blocking the binding of PD-1 to PDL1 and PDL2). Thus, the term "antibody fragment" or PD-1 binding fragment refers to a part of a full-length antibody, generally its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments. Typically, the binding fragment or derivative retains at least 10% of its PD-1 inhibitory activity. In some embodiments, the binding fragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its PD-1 inhibitory activity, but any binding fragment having sufficient affinity to exert the desired biological effect is useful. In some embodiments, the antigen-binding fragment binds to its antigen with an affinity that is at least 2-fold greater, preferably at least 10-fold greater, more preferably at least 20-fold greater, and most preferably at least 100-fold greater than its affinity for an irrelevant antigen. In one embodiment, the antibody has an affinity of greater than about 10 9 liters / mol as determined, for example, by Scatchard analysis. Munsen et al. (1980) Analyt. Biochem. 107:220-239. It is also contemplated that the PD-1 binding fragment can include variants having conservative amino acid substitutions that do not substantially alter its biological activity.

[0086] As used herein, "humanized antibody" refers to an antibody form that contains sequences from non-human (e.g., mouse) antibodies and human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of human immunoglobulin sequence. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin. Humanized forms of rodent antibodies generally contain the same CDR sequences as the parental rodent antibodies, but may contain specific amino acid substitutions for reasons such as enhancing affinity, enhancing the stability of the humanized antibody, or other reasons.

[0087] The antibodies of the invention also include antibodies having an Fc region modified (or blocked) to provide altered effector function. See, e.g., U.S. Patent No. 5,624,821, International Publication No. WO 2003 / 086310, International Publication No. WO 2005 / 120571, International Publication No. WO 2006 / 0057702, Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Such modifications can be used to enhance or suppress various responses of the immune system and can have beneficial effects in diagnosis and treatment. Modifications of the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and addition of multiple Fcs. Modifications of Fc can also change the half-life of an antibody in a therapeutic antibody, and a longer half-life results in a decrease in the frequency of administration, which in turn results in increased convenience and decreased use of materials. See Presta (2005) J. Allergy Clin Immunol. 116:734-35 at 731.

[0088] As used herein, "conservatively modified variant" or "conservative substitution" refers to an amino acid substitution that is known to those of ordinary skill in the art and that can generally be made even in an essential region of a polypeptide without changing the biological activity of the resulting molecule. Such exemplary substitutions are preferably made in accordance with those shown in Table 1 below. [Table 1]

[0089] In addition, those of ordinary skill in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially change its biological activity. See, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th ed.).

[0090] The phrases "consists essentially of", "consist essentially of", or "consisting essentially of", as used throughout the specification and claims, are variations such as any recited element or group of elements, and any inclusion of other elements of the same or different nature that do not substantially change the basic or novel characteristics of the specified dosage regimen, method, or composition. By way of non-limiting example, a binding compound consisting essentially of the recited amino acid sequence may also contain one or more amino acids including substitutions of one or more amino acid residues that do not substantially affect the properties of the binding compound.

[0091] The terms "comprising", "comprise", "comprises", or "comprised of" and variations thereof are used throughout this specification and the claims in an inclusive sense, that is, specifying the presence of the stated features but not excluding the presence or addition of further features that may substantially enhance the operation or utility of any embodiment of the invention, except where the context dictates otherwise by express language or necessary implication.

[0092] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a substantially homogeneous population of antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically contain a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their CDRs, and are specific for many different epitopes. The modifier "monoclonal" indicates the characteristics of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J. Mol. Biol. 222:581-597. See also Presta (2005) J. Allergy Clin Immunol. 116:731.

[0093] A "tumor" applicable to a subject diagnosed with cancer or suspected of having cancer refers to a neoplasm or tissue mass of any size that is malignant or potentially malignant, including primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or tissue mass that usually does not contain cysts or fluid areas. The various types of solid tumors are named after the cell types that form them. Examples of solid tumors are sarcoma, carcinoma, and lymphoma. Leukemia (cancer of the blood) generally does not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

[0094] The term "tumor size" refers to the overall size of a tumor that can be measured as the length and width of the tumor. Tumor size can be determined by various methods known in the art, for example, by measuring the dimensions of the tumor at the time of excision from the subject, for example, using calipers, or by measuring in vivo using imaging techniques such as bone scans, ultrasounds, CT or MRI scans.

[0095] "Tumor Proportion Score (TPS)" refers to the proportion of tumor cells expressing PD-L1 on the cell membrane at any intensity (weak, medium, strong). Linear partial or complete cell membrane staining is interpreted as positive for PD-L1.

[0096] "Mononuclear Inflammatory Density Score (MIDS)" refers to the ratio of the number of PD-L1-expressing mononuclear inflammatory cells (MICs) (small and large lymphocytes, monocytes, and macrophages in tumor nests and adjacent supporting stroma) infiltrating or adjacent to a tumor compared to the total number of tumor cells. MIDS is on a scale of 0 - 4, where 0 = none, 1 = present but <1 MIC per 100 tumor cells (<1%), 2 = ≥1 MIC per 100 tumor cells but <1 MIC per 10 tumor cells (1 - 9%), 3 = ≥1 MIC per 10 tumor cells but fewer MICs than tumor cells (10 - 99%), 4 = ≥ the same number of MICs as tumor cells (≥100%) are recorded.

[0097] "Combined Positive Score (CPS)" refers to the ratio of the number of PD-L1 positive tumor cells and PD-L1 positive mononuclear inflammatory cells (MIC) in tumor nests and adjacent supporting stroma (numerator) compared to the total number of tumor cells (denominator; i.e., the number of PD-L1 positive cells and PD-L1 negative tumor cells). PD-L1 expression at any intensity, i.e., weak (1+), moderate (2+), or strong (3+), is considered positive.

[0098] "PD-L1 expression positive" means that the Tumor Proportion Score, Mononuclear Inflammatory Density Score, or Combined Positive Score is at least 1%, the AIS is ≧5, or the PD-L1 expression (protein and / or mRNA) level is increased by malignant cells and / or infiltrating immune cells within the tumor compared to an appropriate control.

[0099] "Microsatellite instability (MSI)" refers to a form of genomic instability associated with incomplete DNA mismatch repair in tumors. See Boland et al., Cancer Research 58,5258-5257,1998. In one embodiment, MSI analysis can be performed using five National Cancer Institute (NCI)-recommended microsatellite markers: BAT25 (GenBank accession number 9834508), BAT26 (GenBank accession number 9834505), D5S346 (GenBank accession number 181171), D2S123 (GenBank accession number 187953), D17S250 (GenBank accession number 177030). Additional markers, such as BAT40, BAT34C4, TGF-β-RII, and ACTC, can be used. Commercially available MSI analysis kits include, for example, the Promega MSI multiplex PCR assay, FoundationOne® CDx (F1CDx) next-generation sequencing, based on in vitro diagnostic devices that use DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens.

[0100] "High-frequency microsatellite instability" or "microsatellite instability-high (MSI-H)" refers to the situation where two or more of the above five NCI markers show instability, or 30-40% or more of all markers show instability (i.e., have insertion / deletion mutations).

[0101] As used herein, "non-MSI-H cancer" refers to microsatellite stable (MSS) and low-frequency MSI (MSI-L) cancers.

[0102] "Microsatellite stability (MSS)" refers to the situation where none of the above five NCI markers show instability (i.e., have insertion / deletion mutations).

[0103] "Normal mismatch repair function (pMMR) cancer" refers to the normal expression of MMR proteins (MLH1, PMS2, MSH2, and MSH6) in tumor specimens by IHC. Commercially available kits for MMR analysis include the Ventana MMR IHC assay.

[0104] "Deficient mismatch repair (dMMR) cancer" refers to the low expression of one or more of the MMR proteins (MLH1, PMS2, MSH2, and MSH6) in tumor specimens by IHC.

[0105] As used herein, "variable region" or "V region" means the segment of the IgG chain whose sequence is variable between different antibodies. This extends from residue 109 of the light chain and residue 113 of the heavy chain.

[0106] As used herein, “pembrolizumab” (previously known as MK-3475, SCH900475, and lambrolizumab) is a humanized IgG4 mAb having the structure described in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013), and includes the heavy and light chain amino acid sequences and CDRs set forth in Table 2. Pembrolizumab is approved by the US FDA as described in Prescribing Information for KEYTRUDA® (Merck & Co., Inc., Whitehouse Station, NJ, USA; first US approval 2014).

[0107] As used herein, “pembrolizumab variant” means a monoclonal antibody comprising heavy and light chain sequences that are substantially identical to those of pembrolizumab, but having 3, 2, or 1 conservative amino acid substitutions at positions located outside the light chain CDRs, and 6, 5, 4, 3, 2, or 1 conservative amino acid substitutions at positions located outside the heavy chain CDRs; for example, the variant positions are located in the FR regions or the constant regions and may have a deletion of the C-terminal lysine residue of the heavy chain. In other words, pembrolizumab and pembrolizumab variants differ from each other by having conservative amino acid substitutions at no more than 3 or no more than 6 other positions in their respective full-length light and heavy chain sequences, while containing the same CDR sequences. Pembrolizumab variants are substantially the same as pembrolizumab with respect to the following properties: binding affinity for PD-1, and the ability to block the binding of each of PD-L1 and PD-L2 to PD-1.

[0108] The term “buffer” includes agents that maintain the solution pH of the compositions of the invention within an acceptable range.

[0109] The terms “pharmaceutical composition”, “formulation” or “pharmaceutical formulation” refer to a preparation having pharmaceutically acceptable excipients, in a form that enables the active ingredient to be effective and that does not contain additional ingredients that are toxic to the subject to which the composition is administered.

[0110] The terms "lyophilization", "lyophilized", and "freeze-dried" refer to a process in which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. In order to enhance the stability of the lyophilized product during storage, excipients may be included in the pre-lyophilized formulation.

[0111] "Pharmaceutically acceptable" refers to excipients (vehicles, additives) and compositions that can be reasonably administered to a subject to provide an effective amount of the active ingredient used and are "generally regarded as safe", for example, physiologically acceptable and typically do not cause allergic reactions or similar adverse reactions, such as stomach upsets, when administered to humans. In another embodiment, this term refers to a molecular entity or composition that has been approved by a federal or state government regulatory agency or is described in the United States Pharmacopeia or another generally recognized pharmacopeia for use in animals, more specifically in humans.

[0112] A "reconstituted" formulation is prepared by dissolving a lyophilized protein formulation in a diluent such that the protein is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration, for example, parenterally or intravenously, and may in some cases be suitable for subcutaneous administration.

[0113] "Reconstitution time" is the time required to rehydrate a lyophilized formulation into a clear solution free of particles with a solution.

[0114] The "stable" formulation is a formulation in which the protein therein essentially retains its physical stability and / or chemical stability and / or biological activity. Various analytical techniques for measuring protein stability are available in the art and are outlined in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured over a selected period at a selected temperature. For example, in one embodiment, a stable formulation is a formulation in which no significant change is observed for at least 12 months at refrigeration temperature (2-8°C). In another embodiment, a stable formulation is a formulation in which no significant change is observed for at least 18 months at refrigeration temperature (2-8°C). In another embodiment, a stable formulation is a formulation in which no significant change is observed for at least 3 months at room temperature (23-27°C). In another embodiment, a stable formulation is a formulation in which no significant change is observed for at least 6 months at room temperature (23-27°C). In another embodiment, a stable formulation is a formulation in which no significant change is observed for at least 12 months at room temperature (23-27°C). In another embodiment, a stable formulation is a formulation in which no significant change is observed for at least 18 months at room temperature (23-27°C). The criteria for the stability of an antibody formulation are as follows. Typically, as measured by SEC-HPLC, the degradation of the antibody monomer is 10% or less, preferably 5% or less. Typically, the formulation is colorless or slightly milky from transparent by visual analysis. Typically, the changes in the concentration, pH and osmolality of the formulation are + / −10% or less. Typically, the potency is within 60-140%, preferably 80-120% of the control or reference. Typically, clipping of 10% or less, preferably 5% of the antibody, i.e., the % low molecular weight species determined, for example, by HP-SEC is observed. Typically, aggregation of 10% or less, preferably 5% or less of the antibody, i.e., the % high molecular weight species determined, for example, by HP-SEC is observed.

[0115] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering. Changes in protein conformation can be evaluated by fluorescence spectroscopy, which determines the tertiary structure of the protein, and FTIR spectroscopy, which determines the secondary structure of the protein.

[0116] An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be evaluated by detecting and quantifying the chemically altered forms of the protein. Degradation processes that often change the chemical structure of the protein include hydrolysis or clipping (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing electrophoresis, peptide mapping, and measurement of isoaspartic acid), and isomerization (evaluated by measurement of isoaspartic acid content, peptide mapping, etc.).

[0117] An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time point is within a predetermined range of the biological activity shown at the time the pharmaceutical formulation was prepared. The biological activity of an antibody can be determined, for example, by an antigen-binding assay. The formulations of the present invention include antibodies and their fragments that are biologically active when reconstituted or in liquid form.

[0118] The term "isotonic" means that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 270 - 328 mOsm. Slightly lower osmotic pressures are 250 - 269, and slightly higher osmotic pressures are 328 - 350 mOsm. Osmotic pressure can be measured, for example, using a vapor pressure or cryoscopic osmometer.

[0119] A "non - reducing disaccharide" is a disaccharide that does not contain a free aldehyde or ketone group or cannot be converted to contain one, and thus cannot act as a reducing agent. Examples of non - reducing disaccharides include, but are not limited to, disaccharides such as sucrose and trehalose.

[0120] "PH20" refers to the wild - type PH20 hyaluronidase of SEQ ID NO: 16.

[0121] "Unit" or "U" refers to one unit of hyaluronidase activity, i.e., the amount of rHuPH20 or its variant or fragment calculated according to a calibration curve using an activity standard that causes a change in the optical density at 600 nm under conditions suitable for the reaction of hyaluronic acid with the enzyme. Examples of assays are described in Example 4 of U.S. Patent No. 2022 / 0089738 and Example 6 below. Hyaluronic acid (HA) binds to albumin, and the albumin - HA complex causes turbidity. When HA is hydrolyzed by hyaluronidase, the turbidity of the albumin - HA complex decreases. Thus, this assay measures turbidity to determine the hyaluronidase enzyme activity of rHuPH20 or its variant or fragment. Hyaluronidase activity is based on the following reaction Hyaluronic acid ------------> Disaccharides and monosaccharides + smaller hyaluronic acid fragments. Those skilled in the art understand that the hyaluronidase activity in units per milligram of hyaluronidase can vary depending on the purity of the hyaluronidase, the manufacturing process, etc.

[0122] Anti - PD - 1 antibody and its antigen - binding fragment In some embodiments, the anti-human PD-1 antibody or antigen-binding fragment thereof for use in the compositions of the present invention comprises a light chain variable region comprising three light chain CDRs of CDRL1, CDRL2 and CDRL3, and a heavy chain variable region comprising three heavy chain CDRs of CDRH1, CDRH2 and CDRH3.

[0123] In one embodiment of the present invention, CDRL1 is SEQ ID NO: 1, CDRL2 is SEQ ID NO: 2, and CDRL3 is SEQ ID NO: 3. In one embodiment, CDRH1 is SEQ ID NO: 6, CDRH2 is SEQ ID NO: 7, and CDRH3 is SEQ ID NO: 8. In one embodiment, the three light chain CDRs are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the three heavy chain CDRs are SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0124] The anti-PD-1 binding fragment of the composition of the present invention comprises a light chain variable region and a heavy chain variable region. In one embodiment of the composition of the present invention, the antibody or antigen-binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO: 4 and a heavy chain variable region comprising or consisting of SEQ ID NO: 9.

[0125] In another embodiment, the composition of the present invention is at least 95%, 90%, 85%, 80%, 75% sequence homologous to one of the above V L domain or V H domain and has a V L domain and / or V H domain and comprises an antibody or antigen-binding fragment that exhibits specific binding to PD-1. In another embodiment, the antibody or antigen-binding fragment of the composition of the present invention has a V L domain and a V H domain with a maximum of 1, 2, 3, 4 or 5 or more amino acid substitutions and exhibits specific binding to PD-1.

[0126] In any of the above embodiments, the anti-PD-1 antibody can be a full-length anti-PD-1 antibody that specifically binds to human PD-1. In certain embodiments, the full-length anti-PD-1 antibody is selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, the antibody is an IgG antibody. Any isotype of IgG, including IgG1, IgG2, IgG3, and IgG4, can be used. The V L region and V H region may be appended with different constant domains. For example, if a particular intended use of the antibody (or fragment) of the present invention requires modification of effector function, a heavy chain constant domain other than IgG1 can be used. IgG1 antibodies provide a long half-life and effector functions, such as complement activation and antibody-dependent cytotoxicity, but such activities may not be desirable for all uses of the antibody. In such cases, for example, an IgG4 constant domain can be used.

[0127] In embodiments of the present invention, the anti-PD-1 antibody comprises a light chain comprising or consisting of the amino acid residue sequence shown in SEQ ID NO: 5 and a heavy chain comprising or consisting of the amino acid residue sequence shown in SEQ ID NO: 10. In another embodiment, the anti-human PD-1 antibody consists of two light chains and two heavy chains, the two light chains consisting of the amino acid sequence shown in SEQ ID NO: 5, and the two heavy chains consisting of the amino acid sequence shown in any one of SEQ ID NOs: 10-15, or a combination thereof. In some compositions of the present invention, the anti-PD-1 antibody is pembrolizumab or a variant of pembrolizumab.

[0128] Generally, amino acid sequence variants of the anti-PD-1 antibodies and antigen-binding fragments of the present invention are reference antibodies or antigen-binding fragments (e.g., heavy chain, light chain, V H , V Lor an amino acid sequence having at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95, 98, or 99% amino acid sequence identity to the amino acid sequence of the humanized sequence). Identity or homology with respect to a sequence is, if necessary, the percentage of amino acid residues in a candidate sequence that are identical to the anti-PD-1 residues after aligning the sequences and introducing gaps to achieve maximum percent sequence identity, and any conservative substitutions are not considered as part of sequence identity. Neither internal extensions, deletions, nor insertions to the N-terminus, C-terminus, or antibody sequence should be construed as affecting sequence identity or homology.

[0129] Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two arrays are optimally aligned. Sequence identity can be determined using the BLAST algorithm, and the parameters of the algorithm are selected to give the maximum match between the respective sequences over the full length of each reference sequence. The following references relate to the BLAST algorithm often used in sequence analysis: BLAST algorithm: Altschul, S.F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, T.L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S.F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, J.C., et al., (1993) Comput. Chem. 17:149-163; Hancock, J.M. et al., (1994) Comput. Appl. Biosci. 10:67-70; Alignment scoring system: Dayhoff, M.O., et al., "A model of evolutionary change in proteins." (Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3) M.O. Dayhoff (ed.), pp. 345-352,; Natl. Biomed. Res. Found., Washington, DC; Schwartz, R.M., et al., "Matrices for detecting distant relationships." (In Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3), M.O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, S.F., (1991) J. Mol. Biol.219:555-565; States, D.J., et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, S.F., et al., (1993) J. Mol. Evol. 36:290-300; Alignment statistics: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S.F. "Evaluating the statistical significance of multiple distinct local alignments." (In Theoretical and Computational Methods in Genome Research) (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York.

[0130] Similarly, any class of light chains can be used in the compositions and methods of the present specification. Specifically, kappa, lambda, or variants thereof are useful in the compositions and methods of the present invention.

Table 2

[0131] TIFF2025524964000004.tif176153

[0132] rHuPH20 and its fragments rHuPH20, also known as Hylenex®, consists of the amino acid sequence set forth in SEQ ID NO: 17, which is amino acid residues 36 to 482 of wild-type human PH20 set forth in SEQ ID NO: 16 (amino acid residues 1 to 36 are the signal peptide sequence). In one embodiment, rHuPH20 or a variant or fragment thereof is amino acid residues 36 to 464, 36 to 465, 36 to 466, 36 to 467, 36 to 468, 36 to 469, 36 to 470, 36 to 471, 36 to 472, 36 to 473, 36 to 474, 36 to 475, 36 to 476, 36 to 477, 36 to 478, 36 to 479, 36 to 480, 36 to 481, 36 to 482, or 36 to 483 of SEQ ID NO: 16; amino acid residues 37 to 464, 37 to 465, 37 to 466, 37 to 467, 37 to 468, 37 to 469, 37 to 470, 37 to 471, 37 to 472, 37 to 473, 37 to 474, 37 to 475, 37 to 476, 37 to 477, 37 to 478, 37 to 479, 37 to 480, 37 to 481, 37 to 482 or 37 to 483 of SEQ ID NO: 16; amino acid residues 38 to 464, 38 to 465, 38 to 466, 38 to 467, 38 to 468, 38 to 469, 38 to 470, 38 to 471, 38 to 472, 38 to 473, 38 to 474, 38 to 475, 38 to 476, 38 to 477, 38 to 478, 38 to 479, 38 to 480, 38 to 481, 38 to 482 or 38 to 483 of SEQ ID NO: 16; amino acid residues 39 - 464, 39 - 465, 39 - 466, 39 - 467, 39 - 468, 39 - 469, 39 - 470, 39 - 471, 39 - 472, 39 - 473, 39 - 474, 39 - 475, 39 - 476, 39 - 477, 39 - 478, 39 - 479, 39 - 480, 39 - 481, 39 - 482 or 39 - 483 of SEQ ID NO: 16; amino acid residues 40 - 464, 40 - 465, 40 - 466, 40 - 467, 40 - 468, 40 - 469, 40 - 470, 40 - 471, 40 - 472, 40 - 473, 40 - 474, 40 - 475, 40 - 476, 40 - 477, 40 - 478, 40 - 479, 40 - 480, 40 - 481, 340 - 482 or 40 - 483 of SEQ ID NO: 16; amino acid residues 41 - 464, 41 - 465, 41 - 466, 41 - 467, 41 - 468, 41 - 469, 41 - 470, 41 - 471, 41 - 472, 41 - 473,41-474, 41-475, 41-476, 41-477, 41-478, 41-479, 41-480, 41-481, 41-482 or 41-483, or amino acid residues 42-464, 42-465, 42-466, 42-467, 42-468, 42-469, 42-470, 42-471, 42-472, 42-473, 42-474, 42-475, 42-476, 42-477, 42-478, 42-479, 42-480, 42-481, 42-482 or 42-483 of SEQ ID NO: 16. In a preferred embodiment, the rHuPH20 variant consists of amino acid residues 36-483 of SEQ ID NO: 16 (which is SEQ ID NO: 17). In a preferred embodiment, the rHuPH20 fragment consists of amino acid residues 36-477 of SEQ ID NO: 16. In a preferred embodiment, the rHuPH20 fragment consists of amino acid residues 36-478 of SEQ ID NO: 16. In a preferred embodiment, the rHuPH20 fragment consists of amino acid residues 36-479 of SEQ ID NO: 16. In a preferred embodiment, the rHuPH20 fragment consists of amino acid residues 36-480 of SEQ ID NO: 16. In a preferred embodiment, the rHuPH20 fragment consists of amino acid residues 36-481 of SEQ ID NO: 16. In a further embodiment, the rHuPH20 variant or fragment is one disclosed in U.S. Patent No. 7,767,429, which is hereby incorporated by reference in its entirety., [Table 3]

[0133] A composition comprising an anti-PD-1 or antigen-binding fragment and rHuPH20 or a variant or fragment The present invention provides an anti-human PD-1 antibody or an antigen-binding fragment thereof having less than about 3.0% oxidation of methionine 105, the anti-human PD-1 antibody or antigen-binding fragment thereof comprising a light chain variable region comprising three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a heavy chain variable region comprising three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8, and a composition comprising rHuPH20 or a variant or fragment thereof. In one embodiment, the anti-human PD-1 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9. In another embodiment, at least a portion of the anti-human PD-1 antibody comprises a light chain comprising the amino acid sequence shown in SEQ ID NO: 5 and a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 10. In a further embodiment, the anti-human PD-1 antibody consists of two light chains and two heavy chains, and at least a portion of the anti-human PD-1 antibody is such that the two light chains consist of the amino acid sequence shown in SEQ ID NO: 5, and the two heavy chains consist of the amino acid sequence shown in any one of SEQ ID NOs: 10 to 15, or a combination thereof. In a further embodiment, the anti-human PD-1 antibody consists of two light chains and two heavy chains, and at least a portion of the anti-human PD-1 antibody is such that the two light chains consist of the amino acid sequence shown in SEQ ID NO: 5, and the two heavy chains consist of the amino acid sequence shown in SEQ ID NO: 11.

[0134] In one embodiment, the oxidation of methionine 105 is about 0.1 to 3.0%. In one embodiment, the oxidation of methionine 105 is 0.1 to 3.0%. In one embodiment, the oxidation of methionine 105 is about 0.2 to 3.0%. In one embodiment, the oxidation of methionine 105 is 0.2 to 3.0%. In another embodiment, the oxidation of methionine 105 is about 0.5 to 3.0%. In another embodiment, the oxidation of methionine 105 is 0.5 to 3.0%. In another embodiment, the oxidation of methionine 105 is about 0.5 to 2.5%. In another embodiment, the oxidation of methionine 105 is 0.5 to 2.5%. In another embodiment, the oxidation of methionine 105 is about 0.5 to 2.0%. In another embodiment, the oxidation of methionine 105 is 0.5 to 2.0%. In another embodiment, the oxidation of methionine 105 is about 0.5 to 1.5%. In another embodiment, the oxidation of methionine 105 is 0.5 to 1.5%.

[0135] In one embodiment, the methionine oxidation % is measured by reduced peptide mapping followed by liquid chromatography and mass spectrometry. In one embodiment, reduced peptide mapping includes the steps of denaturing and reducing 100 μg of anti-PD-1 antibody in 100 μL of 6M guanidine-HCl, 50 μM Tris-HCl, 50 μM EDTA and 200 μM DTT, incubating the mixed sample at 37° C. for 30 minutes in a thermomixer while shaking at 300 rpm, alkylating with 5 μL of 1M iodoacetamide protected from light at 25° C. for 30 minutes, blocking unreacted iodoacetamide with 5 μL of 200 μM DTT, adding lysyl endopeptidase (Lys-C) (Wako, 125-05061) enzyme (1:10 (wt:wt)) into 500 μL, incubating the digest at 37° C. for 60 minutes in a thermomixer, and quenching the digest with 15 μL of 20% TFA.

[0136] In one embodiment, liquid chromatography and mass spectrometry include the steps of injecting 20 μL of a digested sample at 5°C into a Waters Acquity liquid chromatography column (UPLC HSS T3 100 Å, 1.8 μm, 2.1 mm × 150 mm, P / N: 186003540) with a gradient of mobile phase A of 0.02% TFA in water and mobile phase B of 0.02% TFA in acetonitrile, where mobile phase B is 0.1% from 0 to 5 minutes, 0.1% to 10% from 5 to 7 minutes, and then linearly increased to 35% over the next 38 minutes, and collecting MS1 data with a Q Exactive Orbitrap MS (Thermo Fisher Scientific).

[0137] In another embodiment, the % methionine oxidation is measured by hydrophobic interaction chromatography (HIC). In one embodiment, the HIC method is performed by HPLC using a Tosoh Phenyl-5PW column with a mobile phase containing the following components (mobile phase A: 5 mM sodium phosphate in 2% acetonitrile, pH 7.0; mobile phase B: 400 mM ammonium sulfate, 5 mM sodium phosphate in 2% acetonitrile, pH 6.9) with a gradient where mobile phase A is 0% from 0 to 2 minutes and 0 to 100% from 2 to 52 minutes. The % Met105 oxidation is determined by the percentage of the pre-peak (peak before the main peak) containing Met105 oxidation relative to the sum of the pre-peak, main peak (main species), and post-peak (peak after the main peak).

[0138] In a further aspect, the present invention provides a composition comprising a main species of anti-human PD-1 antibody comprising an antibody consisting of two heavy chains and two light chains, wherein each light chain comprises a light chain variable region comprising three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and each heavy chain comprises a heavy chain variable region comprising three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8, an acidic species and a basic species of the main species of anti-human PD-1 antibody, and rHuPH20 or a variant or fragment thereof, and the amount of the main species is 65 to 95%. In one embodiment, the main species of anti-human PD-1 antibody comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9.

[0139] In a further aspect, the present invention provides a composition comprising a main species of anti-human PD-1 antibody comprising an antibody consisting of two heavy chains and two light chains, wherein each light chain comprises a light chain variable region comprising three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and each heavy chain comprises a heavy chain variable region comprising three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8, an acidic species of the main species of anti-human PD-1 antibody, and rHuPH20 or a variant or fragment thereof, and the amount of the acidic species is 1.0 to 12.0%. In one embodiment, the main species of anti-human PD-1 antibody comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9.

[0140] In a further aspect, provided is a composition comprising an anti-PD-1 antibody main species consisting of two heavy chains and two light chains, wherein each heavy chain consists of the amino acid sequence of SEQ ID NO: 11, each light chain consists of the amino acid sequence of SEQ ID NO: 5, an acidic species of the anti-PD-1 antibody main species, and rHuPH20 or a variant or a fragment, and the amount of the acidic species is about 1.0 to 12.0%. In another aspect, provided is a composition comprising an anti-PD-1 antibody main species consisting of two heavy chains and two light chains, wherein each heavy chain consists of the amino acid sequence of SEQ ID NO: 11, each light chain consists of the amino acid sequence of SEQ ID NO: 5, acidic and basic species of the anti-PD-1 antibody main species, and rHuPH20 or a variant or a fragment, and the amount of the main species is about 65 to 95%. In a further aspect, the present invention provides a composition comprising an anti-PD-1 antibody main species produced from Chinese hamster ovary cells, the composition comprising a polynucleotide encoding a light chain and a polynucleotide encoding a heavy chain, or a polynucleotide encoding a light chain and a heavy chain, wherein the heavy chain consists of the amino acid sequence of SEQ ID NO: 10, 13 or 15, the light chain consists of the amino acid sequence of SEQ ID NO: 5, an acidic species of the anti-PD-1 antibody main species, and rHuPH20 or a variant or a fragment, and the amount of the acidic species is 1.0 to 12.0%. In one embodiment, the amount of the main species is about 65 to 95%. In one embodiment, the amount of the main species is about 65 to 85%. In one embodiment, the amount of the main species is 65 to 80%. In another embodiment, the amount of the main species is about 70 to 85%. In a further embodiment, the amount of the main species is about 70 to 75%. In a further embodiment, the amount of the main species is at least about 65%.

[0141] In one embodiment, the amount of acidic species is about 6 - 10%. In one embodiment, the amount of acidic species is about 7 - 9%. In another embodiment, the amount of acidic species 1 is about 1 - 4%. In another embodiment, the amount of acidic species 1 is about 2 - 4%. In another embodiment, the amount of acidic species 1 is about 2 - 3%. In a further embodiment, the amount of acidic variant species is about 1 - 5%. In a further embodiment, the amount of acidic variant species is about 2 - 5%. In a further embodiment, the amount of acidic variant species is about 2 - 4%. In yet another embodiment, the composition further comprises about 12 - 27% basic species. In yet another embodiment, the composition further comprises about 15 - 20% basic species. In still a further embodiment, the amount of basic species 1 is about 4 - 12%. In still a further embodiment, the amount of basic species 1 is about 8 - 12%.

[0142] In one embodiment, the amount of acidic species is 6 - 10%. In one embodiment, the amount of acidic species is 7 - 9%. In another embodiment, the amount of acidic species 1 is 1 - 4%. In another embodiment, the amount of acidic species 1 is 2 - 4%. In another embodiment, the amount of acidic species 1 is 2 - 3%. In a further embodiment, the amount of acidic variant species is 1 - 5%. In a further embodiment, the amount of acidic variant species is 2 - 5%. In a further embodiment, the amount of acidic variant species is 2 - 4%. In yet another embodiment, the composition further comprises 12 - 27% basic species. In yet another embodiment, the composition further comprises 15 - 20% basic species. In still a further embodiment, the amount of basic species 1 is 4 - 12%. In still a further embodiment, the amount of basic species 1 is 8 - 12%.

[0143] In one aspect of measuring the main species, acidic species, or basic species, cation exchange chromatography is used. In one embodiment, the cation exchange column is ProPac WCX-10, Sepax Proteomix WCX-NP1.7, Thermo MAbPac SCX-10G, or Thermo MAbPac SCX50G. Those skilled in the art will understand that any industrial equivalent of the aforementioned columns can be used. In another embodiment, a weak cation exchange column using a carboxylate functional group is used. In a further embodiment, the weak cation exchange column using a carboxylate functional group has a particle size of 10 μm, a diameter of 4 mm, and a length of 250 mm. In one embodiment, a Thermo Scientific ProPac WCX-10 column is used for the cation exchange method. In another embodiment, the Thermo Scientific ProPac WCX-10 column is used at 35 °C with a mobile phase (A) of 24 mM MES pH 6.1 containing 4% acetonitrile and a mobile phase (B) of 95 mM NaCl pH 8.0 containing 20 mM sodium phosphate and 4% acetonitrile, and the chromatogram is generated using detection at 280 nm. In one embodiment, a non-linear gradient of 22% - 22% B from 0 to 0.6 minutes, 22% - 29% B from 0.6 to 15.0 minutes, 29% - 70% B from 15.0 to 30.0 minutes, 70% - 100% B from 30.0 to 30.5 minutes, and 100% - 100% B from 30.5 to 33.0 minutes is used. In a further embodiment, the cation exchange method is described in Example 5. In another aspect of measuring the main species, acidic species, or basic species, anion exchange chromatography is used.

[0144] In one embodiment, the antibody is produced from mammalian cells.

[0145] In another embodiment, the antibody is produced from CHO cells.

[0146] In another aspect of the above embodiment, the composition comprises an antibody at about 5 - 200 mg / ml. In one embodiment, the composition comprises an antibody at about 25 - 165 mg / ml. In one embodiment, the composition comprises an antibody at about 25 mg / ml. In one embodiment, the composition comprises an antibody at about 120 mg / ml. In one embodiment, the composition comprises an antibody at about 130 mg / ml. In one embodiment, the composition comprises an antibody at about 165 mg / ml. In another embodiment, the composition comprises an antibody at 200 - 800 mg / ml. In another embodiment, the composition comprises an antibody at 200 mg / ml. In another embodiment, the composition comprises an antibody at 400 mg / ml.

[0147] In another aspect, the present invention provides a pharmaceutical composition or formulation disclosed herein, comprising an anti-human PD-1 antibody at about 165 mg / mL, about 10 mM histidine buffer, about 10 mM L-methionine, or a pharmaceutically acceptable salt thereof, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and rHuPH20 or a variant or fragment thereof. In one embodiment, the pharmaceutical composition comprises an anti-human PD-1 antibody at about 130 mg / mL, about 10 mM histidine buffer, about 10 mM L-methionine, or a pharmaceutically acceptable salt thereof, about 7% w / v sucrose, about 0.02% w / v polysorbate 80, and rHuPH20 or a variant or fragment thereof.

[0148] In some embodiments of the formulations of the present invention, rHuPH20 or a variant or fragment thereof is present at a concentration of about 1000 U / ml. In another embodiment, the concentration of rHuPH20 or a variant or fragment thereof is about 1500 U / ml. In another embodiment, the concentration of rHuPH20 or a variant or fragment thereof is about 2000 U / ml. In another embodiment, the concentration of rHuPH20 or a variant or fragment thereof is about 3000 U / ml. In another embodiment, the concentration of rHuPH20 or a variant or fragment thereof is about 4000 U / ml. In another embodiment, the concentration of rHuPH20 or a variant or fragment thereof is about 5000 U / ml. In another embodiment, the concentration of rHuPH20 or a variant or fragment thereof is about 6000 U / ml. In further embodiments, the concentration of rHuPH20 or a variant or fragment thereof is about 1000 - 6000 U / ml. In further embodiments, the concentration of rHuPH20 or a variant or fragment thereof is about 2000 - 5000 U / ml.

[0149] In some embodiments of the formulations of the present invention, rHuPH20 or a variant or fragment thereof is present at a concentration of about 150 U / ml. In another embodiment, the concentration of rHuPH20 or a variant or fragment thereof is about 300 U / ml. In another embodiment, the concentration of rHuPH20 or a variant or fragment thereof is about 600 U / ml. In another embodiment, the concentration of rHuPH20 or a variant or fragment thereof is about 750 U / ml. In further embodiments, the concentration of rHuPH20 or a variant or fragment thereof is about 150 - 5000 U / ml.

[0150] Specific embodiments and forms of the pharmaceutical composition In one embodiment, the pharmaceutical composition comprises a) an anti-human PD-1 antibody or an antigen-binding fragment thereof at about 5 mg / mL to about 175 mg / mL b) rHuPH20 at about 2000 U / ml or a variant or fragment thereof c) A histidine buffer of about 5 mM to about 20 mM at a pH of about 5.0 to about 6.0 d) Sucrose at about 6% to about 8% w / v e) Optionally, contains about 5 mM to about 20 mM of L-methionine, or a pharmaceutically acceptable salt thereof.

[0151] In another embodiment, the pharmaceutical composition is a) An anti-human PD-1 antibody at about 5 mg / mL to about 175 mg / mL or an antigen-binding fragment thereof b) rHuPH20 at about 2000 U / ml or a variant or fragment thereof c) A histidine buffer of about 5 mM to about 20 mM at a pH of about 5.0 to about 6.0 d) Sucrose at about 6% to about 8% w / v e) About 0.01% to about 0.04% w / v of polysorbate 80 or 20, and f) Optionally, contains about 5 mM to about 20 mM of L-methionine, or a pharmaceutically acceptable salt thereof.

[0152] In another embodiment, the pharmaceutical composition is a) An anti-human PD-1 antibody at about 5 mg / mL to about 175 mg / mL or an antigen-binding fragment thereof b) rHuPH20 at about 2000 U / ml or a variant or fragment thereof c) A histidine buffer of about 10 mM at a pH of about 5.5 d) Sucrose at about 7% w / v e) Optionally, contains about 10 mM of L-methionine, or a pharmaceutically acceptable salt thereof.

[0153] In a further embodiment, the pharmaceutical composition is a) An anti-human PD-1 antibody at about 5 mg / mL to about 175 mg / mL or an antigen-binding fragment thereof b) rHuPH20 at about 2000 U / ml or a variant or fragment thereof c) A histidine buffer of about 10 mM at a pH of about 5.5 d) About 7% w / v of sucrose e) About 0.02% w / v of polysorbate 80, and f) Optionally, about 5 mM to about 20 mM of L-methionine, or a pharmaceutically acceptable salt thereof.

[0154] In one embodiment, the present invention is a) An anti-human PD-1 antibody or an antigen-binding fragment thereof at about 50 mg / mL to about 175 mg / mL b) About 2000 U / ml of rHuPH20 or a variant or fragment thereof c) A histidine buffer of about 5 mM to about 20 mM d) About 6% to about 8% w / v of sucrose e) About 0.01% to about 0.04% w / v of polysorbate 80, and f) About 5 mM to about 20 mM of L-methionine, or a pharmaceutically acceptable salt thereof, and provides a pharmaceutical composition.

[0155] In one embodiment, the present invention is a) An anti-human PD-1 antibody or an antigen-binding fragment thereof at about 50 mg / mL to about 175 mg / mL b) About 2000 U / ml of rHuPH20 or a variant or fragment thereof c) A histidine buffer of about 5 mM to about 20 mM, and d) About 6% to about 8% w / v of sucrose, and provides a pharmaceutical composition.

[0156] In one embodiment, the present invention is a) An anti-human PD-1 antibody or an antigen-binding fragment thereof at about 50 mg / mL to about 175 mg / mL b) About 2000 U / ml of rHuPH20 or a variant or fragment thereof c) A histidine buffer of about 5 mM to about 20 mM d) About 6% to about 8% w / v of sucrose, and e) A pharmaceutical composition comprising about 0.01% to about 0.04% w / v of polysorbate 80.

[0157] In one embodiment, the present invention a) An anti-human PD-1 antibody or an antigen-binding fragment thereof at about 50 mg / mL to about 175 mg / mL b) rHuPH20 or a variant or fragment thereof at about 2000 U / ml c) A histidine buffer at about 5 mM to about 20 mM d) About 6% to about 8% w / v of sucrose, and e) Optionally, about 5 mM to about 20 mM of L-methionine or a pharmaceutically acceptable salt thereof, and provides a pharmaceutical composition.

[0158] In one embodiment, the present invention a) An anti-human PD-1 antibody or an antigen-binding fragment thereof at about 50 mg / mL to about 175 mg / mL b) rHuPH20 or a variant or fragment thereof at about 2000 U / ml c) A histidine buffer at about 10 mM d) About 10 mM of L-methionine or a pharmaceutically acceptable salt thereof e) About 7% w / v of sucrose, and f) About 0.02% w / v of polysorbate 80, and provides a pharmaceutical composition.

[0159] In one embodiment, the present invention a) An anti-human PD-1 antibody or an antigen-binding fragment thereof at about 50 mg / mL to about 175 mg / mL b) rHuPH20 or a variant or fragment thereof at about 2000 U / ml c) A histidine buffer at about 10 mM, and d) About 7% w / v of sucrose, and provides a pharmaceutical composition.

[0160] In one embodiment, the present invention a) An anti-human PD-1 antibody or an antigen-binding fragment thereof at about 50 mg / mL to about 175 mg / mL b) rHuPH20 or a variant or fragment thereof at about 2000 U / ml c) A histidine buffer at about 10 mM d) About 7% w / v sucrose, and e) About 0.02% w / v polysorbate 80, and provides a pharmaceutical composition comprising the same.

[0161] In one embodiment, the present invention a) An anti-human PD-1 antibody or an antigen-binding fragment thereof at about 50 mg / mL to about 175 mg / mL b) rHuPH20 or a variant or fragment thereof at about 2000 U / ml c) A histidine buffer at about 10 mM d) About 7% w / v sucrose, and e) About 10 mM L-methionine, or a pharmaceutically acceptable salt thereof, and provides a pharmaceutical composition comprising the same.

[0162] In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 5 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 50 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 75 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 75 to 175 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 100 to 175 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 100 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 130 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 150 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 130 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 165 mg / ml.

[0163] In any of the above specific aspects and embodiments, one of the anti-PD-1 antibodies or antigen-binding fragments described in the section "Compositions Comprising an Anti-PD-1 Antibody or Antigen-Binding Fragment and rHuPH20 or Variant or Fragment" is used.

[0164] In any of the following specific aspects and embodiments, one of the anti-PD-1 antibodies or antigen-binding fragments described in the section "Anti-PD-1 Antibody or Antigen-Binding Fragment" is used.

[0165] In another aspect, the present invention a) An anti-human PD-1 antibody from about 5 mg / mL to about 165 mg / mL, comprising a light chain variable region containing three light chain CDRs where the antibody contains CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a heavy chain variable region containing three heavy chain CDRs where the antibody contains CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8, or an antigen-binding fragment thereof b) rHuPH20 of SEQ ID NO: 17 or 18 or a variant thereof at about 2000 U / ml c) A histidine buffer from about 5 mM to about 20 mM, pH about 5.0 to about 6.0 d) A non-reducing disaccharide from about 3% to about 10% w / v e) Optionally, a non-ionic surfactant from about 0.005% to about 0.4% w / v f) Optionally, L-methionine from about 5 mM to about 20 mM, or a pharmaceutically acceptable salt thereof, to provide a pharmaceutical composition

[0166] In one embodiment, the non-reducing disaccharide is selected from the group consisting of sucrose and trehalose at 6% - 8% w / v

[0167] In yet another aspect, the present invention a) An anti-human PD-1 antibody from about 5 mg / mL to about 165 mg / mL, comprising a light chain variable region containing three light chain CDRs where the antibody contains CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a heavy chain variable region containing three heavy chain CDRs where the antibody contains CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8, or an antigen-binding fragment thereof b) rHuPH20 of SEQ ID NO: 17 or 18 or a variant thereof at about 2000 U / ml c) A histidine buffer from about 5 mM to about 20 mM, pH about 5.0 to about 6.0 d) Sucrose at 6% - 8% w / v f) Optionally, L-methionine from about 5 mM to about 20 mM, or a pharmaceutically acceptable salt thereof, to provide a pharmaceutical composition

[0168] In another aspect, the present invention a) an anti-human PD-1 antibody or an antigen-binding fragment thereof at about 5 mg / mL to about 165 mg / mL b) rHuPH20 or a variant thereof at about 2000 U / ml c) a histidine buffer at about 5 mM to about 20 mM with a pH of about 5.0 to about 6.0 d) sucrose at about 6% to about 8% w / v e) polysorbate 80 or 20 at about 0.01% to about 0.04% w / v, and f) optionally, L-methionine at about 5 mM to about 20 mM, or a pharmaceutically acceptable salt thereof, to provide a pharmaceutical composition.

[0169] In another aspect, the present invention a) an anti-human PD-1 antibody or an antigen-binding fragment thereof at about 5 mg / mL to about 165 mg / mL b) rHuPH20 or a variant thereof at about 2000 U / ml c) a histidine buffer at about 10 mM with a pH of about 5.5 d) sucrose at about 7% w / v, and f) optionally, L-methionine at about 10 mM, or a pharmaceutically acceptable salt thereof, to provide a pharmaceutical composition.

[0170] In another aspect, the present invention a) an anti-human PD-1 antibody or an antigen-binding fragment thereof at about 5 mg / mL to about 165 mg / mL b) rHuPH20 or a variant thereof at about 2000 U / ml c) a histidine buffer at about 10 mM with a pH of about 5.5 d) sucrose at about 7% w / v, e) polysorbate 80 at about 0.02% w / v, and f) optionally, L-methionine at about 10 mM, or a pharmaceutically acceptable salt thereof, to provide a pharmaceutical composition.

[0171] In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 50 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 100 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 130 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 150 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 130 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 165 mg / ml.

[0172] In yet another aspect, the present invention a) an anti-human PD-1 antibody from about 50 mg / mL to about 165 mg / mL, the antibody comprising a light chain variable region comprising three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a heavy chain variable region comprising three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8, or an antigen-binding fragment thereof b) rHuPH20 or variant of SEQ ID NO: 17 or 18 at about 2000 U / ml c) a buffer having a pH of about 5.0 to about 6.0, d) a non-reducing disaccharide from about 3% to about 10% w / v e) a non-ionic surfactant from about 0.005% to about 0.4% w / v f) optionally, a pharmaceutical composition comprising from about 5 mM to about 20 mM of L-methionine, or a pharmaceutically acceptable salt thereof.

[0173] In yet another aspect, the present invention a) An anti-human PD-1 antibody at about 50 mg / mL to about 165 mg / mL, comprising a light chain variable region containing three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a heavy chain variable region containing three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8, or an antigen-binding fragment thereof b) rHuPH20 or variant of SEQ ID NO: 17 or 18 at about 2000 U / ml c) A histidine or acetate buffer with a pH of about 5.0 to about 6.0 d) Sucrose or trehalose at about 6% to about 8% w / v e) Optionally, a non-ionic surfactant at about 0.02% to about 0.2% w / v f) Optionally, a pharmaceutical composition comprising L-methionine at about 5 mM to about 20 mM, or a pharmaceutically acceptable salt thereof.

[0174] In yet another aspect, the present invention a) An anti-human PD-1 antibody at about 50 mg / mL to about 165 mg / mL, comprising a light chain variable region containing three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a heavy chain variable region containing three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8, or an antigen-binding fragment thereof b) rHuPH20 or variant of SEQ ID NO: 17 or 18 at about 2000 U / ml c) A histidine or acetate buffer at about 1 mM to about 30 mM with a pH of about 5.0 to about 6.0 d) Sucrose or trehalose at about 6% to about 8% w / v e) Optionally, polysorbate 80 at about 0.01% to about 0.05% w / v f) Optionally, a pharmaceutical composition comprising L-methionine at about 5 mM to about 20 mM, or a pharmaceutically acceptable salt thereof.

[0175] In yet another aspect, the present invention a) An anti-human PD-1 antibody at about 50 mg / mL to about 165 mg / mL, comprising a light chain variable region containing three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a heavy chain variable region containing three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8, or an antigen-binding fragment thereof b) rHuPH20 of SEQ ID NO: 17 or 18 or a variant thereof at about 2000 U / ml c) A histidine or acetate buffer at about 1 mM to about 30 mM with a pH of about 5.0 to about 6.0 d) Sucrose or trehalose at about 6% to about 8% w / v e) Optionally, a poloxamer at about 0.1% to about 0.4% w / v, preferably poloxamer 188 or 407 at 0.1% to about 0.2% w / v f) Optionally, L-methionine at about 5 mM to about 20 mM, or a pharmaceutically acceptable salt thereof, to provide a pharmaceutical composition

[0176] In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is at about 100 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is at about 130 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is at about 150 to 165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is at about 130 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is at about 165 mg / ml.

[0177] In any of the above specific aspects and embodiments, for example, one of rHuPH20 or a variant or fragment thereof as described in the section entitled "rHuPH20 or a Variant or Fragment Thereof" is used.

[0178] In some embodiments of the present invention, any of the formulations described herein are in an aqueous solution. In alternative embodiments, the present invention provides a lyophilized formulation produced by lyophilizing an aqueous formulation, as described more fully below, to provide a reconstituted formulation of the present invention.

[0179] Formulation excipient In embodiments of the present invention, the non-reducing disaccharide is sucrose. In further embodiments, the non-reducing disaccharide is trehalose.

[0180] In some embodiments, the non-reducing disaccharide is sucrose at about 6% to about 8% w / v. In some embodiments, the non-reducing disaccharide is trehalose at about 6% to about 8% w / v.

[0181] In still further embodiments, sucrose and trehalose are present in amounts of about 6% w / v, about 6.25% w / v, about 6.5% w / v, about 6.75% w / v, about 7% w / v, about 7.25% w / v, about 7.5% w / v, about 7.75% w / v or about 8% w / v.

[0182] In addition to the anti-PD-1 antibody or antigen-binding fragment thereof, and rHuPH20 or variant or fragment thereof, and non-reducing disaccharide in the above amounts / concentrations, the formulations of the present invention may also contain a buffer. In some embodiments, the buffer is present in an amount of about 5 mM to about 20 mM. In further embodiments, the buffer has a pH in the range of about 5.0 to about 6.0. In still further embodiments, the pH is about 5.3 to about 5.8. In other embodiments, the pH is about 6.0 to about 6.4.

[0183] In certain embodiments, the buffer has a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.2 or about 6.4. In certain embodiments of the invention, the buffer is a histidine or acetate having a pH of about 5.0 to about 6.0. In some embodiments, the buffer is an L-histidine buffer or an acetate buffer. In embodiments where the formulation is lyophilized, since the acetate buffer system is not compatible with the lyophilization process, it is preferred that the buffer is not an acetate.

[0184] When ranges of pH values are recited, such as "a pH between 5.5 and 6.0", the range is intended to include the recited values. Unless otherwise indicated, for lyophilized formulations, pH refers to the pH after reconstitution of the lyophilized formulation of the invention. pH is typically measured at 25 °C using a standard glass bulb pH meter. As used herein, a solution containing "a histidine buffer at pH X" refers to a solution at pH X containing a histidine buffer, i.e., pH is intended to refer to the pH of the solution.

[0185] In addition to the amounts / concentrations of the anti-PD-1 antibody or antigen-binding fragment thereof, and rHuPH20 or variant or fragment thereof, non-reducing disaccharide, and buffer described above, the formulations of the invention may also contain an antioxidant. In embodiments of the invention, the antioxidant is methionine. In embodiments of the invention, the antioxidant is L-methionine or a pharmaceutically acceptable salt thereof. In further embodiments, the methionine is L-methionine. In other embodiments, the antioxidant is L-methionine HCl.

[0186] In some embodiments, an antioxidant (e.g., L-methionine) is present in the formulations of the present invention in an amount of 1 mM to about 20 mM. In another embodiment, the antioxidant is present in an amount of about 5 mM to about 20 mM. In a further embodiment, the antioxidant is present at about 5 mM to about 15 mM. In a further embodiment, the antioxidant is present at about 5 mM to about 10 mM. In a further embodiment, the antioxidant is present in an amount of 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 or about 20 mM.

[0187] In addition to the anti-PD-1 antibody or antigen-binding fragment thereof, and rHuPH20 or variant or fragment thereof, non-reducing disaccharide, buffer, and antioxidant in the above amounts / concentrations, the formulations of the present invention may also contain a surfactant. Surfactants that may be useful in the formulations of the present invention include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (polysorbates, sold under the trade name Tween® (Uniquema Americas LLC, Wilmington, DE)) including polysorbate-20 (polyoxyethylene sorbitan monolaurate), polysorbate-40 (polyoxyethylene sorbitan monopalmitate), polysorbate-60 (polyoxyethylene sorbitan monostearate) and polysorbate-80 (polyoxyethylene sorbitan monooleate), and poloxamers such as poloxamer 188 or poloxamer 407.

[0188] The amount of surfactant contained in the formulation of the present invention is an amount sufficient to perform the desired function, i.e., the minimum amount necessary to stabilize the active pharmaceutical ingredient in the formulation (i.e., the anti-PD-1 antibody or its antigen-binding fragment, or rHuPH20 or its variant or fragment). Typically, the surfactant is present at a concentration of about 0.005% to about 0.2% w / v, or about 0.005% to about 0.1% w / v. In some embodiments of this aspect of the present invention, the surfactant is present in the formulation in an amount of about 0.01% to about 0.04%, about 0.01% to about 0.03%, about 0.01% to about 0.02%, about 0.015% to about 0.04%; about 0.015% to about 0.03%, about 0.015% to about 0.02%, about 0.02% to about 0.04%, about 0.02% to about 0.035%, or about 0.02% to about 0.03%. In certain embodiments, the surfactant is present in an amount of about 0.02%. In alternative embodiments, the surfactant is present in an amount of about 0.01%, about 0.015%, about 0.025%, about 0.03%, about 0.035%, or about 0.04%.

[0189] In an exemplary embodiment of the present invention, the surfactant is a nonionic surfactant selected from the group consisting of polysorbate 20 and polysorbate 80. In a preferred embodiment, the surfactant is polysorbate 80.

[0190] In certain embodiments, the formulation of the present invention contains about 0.01% to about 0.04% of PS80. In further embodiments, the formulation of the present invention contains PS80 in an amount of about 0.008%, about 0.01%, about 0.015%, about 0.02%, about 0.025%, about 0.03%, about 0.035%, about 0.04% or about 0.045%. In certain embodiments, the formulation of the present invention contains about 0.02% of PS80.

[0191] In certain embodiments, the formulations of the present invention contain from about 0.1% to about 0.4% poloxamer. In further embodiments, the formulations of the present invention contain from 0.1 to 0.2% of poloxamer 188 or 407. In further embodiments, the formulations of the present invention contain 0.2% of poloxamer 188. In further embodiments, the formulations of the present invention contain 0.1% of poloxamer 407.

[0192] Lyophilized pharmaceutical composition Lyophilized formulations of therapeutic proteins offer several advantages. Lyophilized formulations generally provide better chemical stability than solution formulations, thus extending the shelf life. Lyophilized formulations may also be reconstituted at different concentrations depending on clinical factors such as route of administration or dose. For example, a lyophilized formulation may be reconstituted at a high concentration (i.e., a small volume) if required for subcutaneous administration, or at a lower concentration if administered intravenously. High concentrations may also be required when high doses are needed for a particular subject, especially in subcutaneous administration where the injection volume must be minimized. One such lyophilized antibody formulation is disclosed in U.S. Patent No. 6,267,958, which is hereby incorporated by reference in its entirety. Another lyophilized formulation of a therapeutic protein is described in U.S. Patent No. 7,247,707, which is hereby incorporated by reference in its entirety.

[0193] Typically, lyophilized formulations are prepared with a view to reconstitution with a high concentration of drug product (DP), i.e., with a low volume of water. Dilution with water or an isotonic buffer can then be used immediately to dilute the DP to a lower concentration. Typically, excipients are included in the lyophilized formulations of the present invention at levels that will result in a substantially isotonic formulation when reconstituted at a high DP concentration, for example, for subcutaneous administration. Reconstitution with a greater amount of water to lower the DP concentration will necessarily reduce the isotonicity of the reconstituted solution, but such a reduction is of little significance for administrations other than subcutaneous, e.g., intravenous administration. If isotonicity is desired at a lower DP concentration, the lyophilized powder can be reconstituted with a standard small volume of water and then further diluted with an isotonic diluent such as 0.9% sodium chloride.

[0194] In one embodiment of the present invention, a formulation comprising a humanized anti-PD-1 antibody (or an antigen-binding fragment thereof) and rHuPH20 or a variant or fragment thereof is reconstituted and formulated as a lyophilized powder for use in subcutaneous administration. In certain embodiments, the lyophilized formulation is reconstituted with sterile water for injection prior to use. If desired, the reconstituted solution can be aseptically diluted with 0.9% sodium chloride Injection USP in a sterile IV container. In some embodiments, the target pH of the reconstituted formulation is 5.5 ± 0.5. In various embodiments, the lyophilized formulation of the present invention enables the anti-PD-1 antibody to be reconstituted at a high concentration, such as about 50, 60, 75, 100, 125, 130, 150, 165, 175, 185 or 200 mg / mL.

[0195] Lyophilized formulations are, by definition, essentially dry, and thus the concept of concentration is not useful for describing them. It is more useful to describe lyophilized formulations in terms of the weight of the components in a unit dose vial, but this is problematic as it varies with different doses or vial sizes. In the description of the lyophilized formulations of the present invention, it is useful to express the amount of a component as the ratio of the weight of the component to the weight of the drug substance (DS) in the same sample (e.g., vial). This ratio can be expressed as a percentage. Such a ratio reflects the inherent properties of the lyophilized formulations of the present invention, independent of vial size, administration, and reconstitution protocol.

[0196] In other embodiments, the formulation of the anti-human PD-1 antibody or antigen-binding fragment and rHuPH20 or a variant or fragment thereof is defined with respect to the pre-lyophilization solution used to make the lyophilized formulation, e.g., the pre-lyophilization solution. In one embodiment, the pre-lyophilization solution comprises the antibody or an antigen-binding fragment thereof at a concentration of about 25 - 200 mg / mL and rHuPH20 or a variant or fragment thereof at 150 - 8000 U / ml. Such a pre-lyophilization solution can have a pH of about 5.0 - 6.0 or about pH 5.5.

[0197] In yet other embodiments, a lyophilized formulation of an anti-human PD-1 antibody or antigen-binding fragment and rHuPH20 or its variant or fragment is defined with respect to a reconstitution solution generated from the lyophilized formulation. The present invention provides a liquid formulation reconstituted from a lyophilized formulation. The reconstitution solution contains the antibody or its antigen-binding fragment at a concentration of about 25, 30, 40, 50, 60, 75, 80, 90, 100, 120, 130, 150, 165, 167, 185 or 200 mg / mL, and rHuPH20 or its variant or fragment at 150, 300, 600, 900, 1000, 1500, 2000, 3000, 4000 or 5000 U / ml. Such a reconstitution solution can have a pH of about 5.5, or can be in the range of about pH 5.0 to about pH 6.0.

[0198] The lyophilized formulation of the present invention is formed by lyophilization (freeze-drying) of a pre-lyophilization solution. Freeze-drying is achieved by freezing the formulation and subsequently sublimating water at a temperature suitable for primary drying. Under these conditions, the temperature of the product is lower than the eutectic point or collapse temperature of the formulation. Typically, the storage temperature for primary drying is in the range of about -30 to 25 °C at an appropriate pressure typically in the range of about 50 to 250 mTorr (when the product remains frozen during primary drying). The size and type of the container (e.g., glass vial) holding the formulation, sample, and the volume of the liquid can determine the time required for drying, which can range from several hours to several days (e.g., 40 to 60 hours). The secondary drying stage can be carried out at about 0 to 40 °C, mainly depending on the type and size of the container and the type of protein used. The secondary drying time is determined by the desired residual moisture level in the product and typically takes at least about 5 hours. Typically, the water content of the lyophilized formulation is less than about 5%, preferably less than about 3%. The pressure can be the same as that used during the primary drying process. The freeze-drying conditions can be varied depending on the formulation and vial size.

[0199] In some cases, it may be desirable to lyophilize the protein formulation in a container and perform protein reconstitution therein to avoid the transfer step. The container in this case can be, for example, a 3, 5, 10, 20, 50 or 100 cc vial.

[0200] Reconstitution is generally performed at a temperature of about 25 °C to ensure complete hydration, but other temperatures may be used as needed. The time required for reconstitution depends, for example, on the type of diluent, excipients and amount of protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution.

[0201] Liquid pharmaceutical composition Liquid antibody formulations can be made by obtaining a buffer to exchange with the buffer in the liquid form of the drug substance (e.g., anti-human PD-1 antibody and / or rHuPH20 or its variants or fragments), and the drug substance in the final buffer is concentrated to the desired concentration. Excipients such as sucrose, methionine and polysorbate 80 are added to the drug substance and diluted to the final protein concentration using an appropriate buffer. The finally formulated drug substance is filtered, for example, using a 0.22 μm filter and filled into the final container (e.g., glass vial or syringe). Such liquid formulations are exemplified by a final liquid formulation containing 10 mM histidine pH 5.5, 7% sucrose, 0.02% polysorbate 80, 25 - 200 mg / mL of the anti-PD-1 antibody of the present invention, and 150 - 8000 U / ml of rHuPH20.

[0202] Protein expression Methods for producing antibodies include culturing antibody-secreting mammalian cells. Such cultured mammalian cells are typically generated by recombinant DNA techniques involving transient or stable transfection, for example, by transfecting a pooled plasmid construct (expression vector) from a cloning step into multiple host cells (e.g., mammalian, HEK 293 or CHO, bacterial, insect, yeast cells) for expression using cationic lipids, polyethyleneimine, Lipofectamine™ or ExpiFectamine™, or electroporation. Those skilled in the art are aware of numerous suitable means for transfecting to achieve the expression of recombinant antibodies. Alternatively, a production cell line of protein-secreting mammalian cells can be generated using methods for stable genomic integration of an expression cassette encoding the protein of interest.(See, for example, Zhang, Crispr-Cas Systems and Methods for Altering Expression Of Gene Products, WO 2014 / 093661 A2; Frendewey et al., Methods and Compositions for Targeted Modification of a Genome, U.S. Pat. No. 9,228,208 B2; Church et al., Multiplex Automated Genome Engineering, WO 2008 / 052101 A2, U.S. Pat. No. 8,153,432 B2; Bradley et al., Methods Cells and Organisms, U.S. 2015 / 0079680 A1; Begemann et al., Compositions and Methods for Modifying Genomes, WO 2017 / 141173 A2; Gill et al., Nucleic acid-guided nucleases, U.S. Pat. No. 9,982,279 B1; Minshull et al., Enhanced nucleic acid constructs for eukaryotic gene expression, U.S. Pat. No. 9,428,767 B2, U.S. Pat. No. 9,580,697 B2, U.S. Pat. No. 9,574,209 B2; Minshull et al., DNA Vectors, Transposons And Transposases For Eukaryotic Genome Modification, U.S. Pat. No. 10,041,077 B2). In other embodiments, expression cassettes that are efficiently integrated into eukaryotic transcriptional active hotspots and ensure long-term stable and consistent expression of a gene of interest (GOI) are described in International Publication No. WO 2020 / 068631. These expression cassettes can be transfected into various host CHO cell lines, including CHOK1SV (Lonza; Slough, UK), HD-BIOP1 (Horizon Discovery, UK), CHOZN® (Sigma-Aldrich, St. Louis, MO), and GS knockout CHO host cell lines. In one embodiment, the expression vector is (a) a first expression cassette comprising the following elements in order from upstream to downstream: a first insulator, EASE, a promoter, TPL, an insertion site for the gene of interest (GOI), an IRES, a polynucleotide encoding a eukaryotic selectable marker, a polyA signal, and a second insulator (b) two ITR sequences adjacent to the first expression cassette (c) a second expression cassette comprising a polynucleotide encoding a bacterial selectable marker, and (d) a bacterial plasmid origin of replication.

[0203] In one embodiment, the promoter is the SV40 promoter (Nature 273(5658):113-20 (1978), Proc. Natl. Acad. Sci. USA 81(1):23-27 (1984), GenBank: J02400.1). In another embodiment, the promoter is the hCMV immediate early enhancer / promoter (GenBank X17403.1). In another embodiment, the ITR is the piggyBac ITR. In one embodiment, the insulator is the chicken β-globin HS4 insulator.

[0204] In one embodiment, the eukaryotic selectable marker is neomycin phosphotransferase, histidinol dehydrogenase, hygromycin B phosphotransferase, xanthine-guanine phosphoribosyltransferase, dihydrofolate reductase, tryptophan synthase, puromycin N-acetyl-transferase, thymidine kinase, adenine phosphoribosyltransferase, glutamine synthetase, adenosine deaminase, or metallothionein-1. In one embodiment, the eukaryotic selectable marker is neomycin phosphotransferase. In another embodiment, the eukaryotic selectable marker is glutamine synthetase. In certain embodiments of the various expression vectors provided herein, the bacterial selectable marker is an ampicillin resistance gene, tetracycline resistance gene, hygromycin resistance gene, kanamycin resistance gene, blasticidin resistance gene, and the like. In one embodiment, the bacterial selectable marker is an ampicillin resistance gene.

[0205] Upstream cell culture process Surprisingly, the continuous perfusion upstream process provided pembrolizumab compositions having a low % Met105 oxidation, low % acidic species, and / or high % main species compared to pembrolizumab produced by a fed-batch process.

[0206] The host cells used to produce antibodies in the present invention can be cultured in various cell culture media. Commercially available media such as CD-CHO liquid or CD-CHO AGT™ powder (Life Technologies), Ham’s F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing the host cells. Furthermore, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469, International Publication No. 90103430, International Publication No. 87 / 00195 or U.S. Patent Re. No. 30,985 can be used as the culture medium for the host cells. Any of these media can be supplemented with other components at appropriate concentrations, which would be known to those skilled in the art, if necessary.For example, hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, sodium bicarbonate, calcium, iron, potassium, zinc, copper sulfate, ferric citrate, manganese, magnesium, phosphate, etc.), nucleotides (such as adenosine, adenine, thymidine, cytidine, guanosine, uridine, purine, etc.), any of the 20 amino acids (such as tyrosine, cysteine, cystine, glutamic acid), vitamins or supplements (such as choline, inositol, thiamine, folic acid, biotin, calcium, nicotinamide, p-aminobenzoic acid, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, linoleic acid, selenite, glycine, putrescine, ethanolamine), selection agents that confer resistance or viability to selectable markers such as antibiotics (such as geneticin, neomycin, hygromycin B, puromycin, zeocin, gentamicin (trademark), etc.), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose, galactose or an equivalent energy source. Typically, the concentration of the cell culture medium for perfusion is increased to enable continuous growth of cells at a higher density. In one embodiment, a nonionic surfactant such as Kolliphor (registered trademark) P188 is added at 2 - 10 g / L to prevent cell death during the perfusion process and maintain high cell density and viability. See Xu et al., Bioprocess Biosyst Eng (2017) 40:1317 - 1326. In another embodiment, the copper concentration in the perfusion cell culture medium is 10 - 35 ppb. Depending on the cell density and copper uptake by the cells, the copper concentration in the perfusion bioreactor can range from 1 - 35 ppb. The culture medium is preferably serum-free. In some embodiments, the aqueous medium is liquid such that the host cells are cultured in a cell suspension within the liquid medium.

[0207] Culture conditions, such as temperature (for mammalian cells, typically about 37° ± 1°C), pH (not necessarily, but typically, cell culture media are maintained within the range of about pH 6.5 to 7.5), oxygenation, etc. will be apparent to those skilled in the art. In one embodiment, the dissolved oxygen level is about 15 to 100% and the pH is about 6.7 to 7.3. Clearly, the changes in temperature, pH, or other culture conditions over time, and the changes from place to place through the culture tank (i.e., bioreactor) are small, and there are operating ranges for these parameters. (See, for example, Oguchi et al., pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture, Cytotechnology. 52(3):199 - 207(2006); Al - Fageeh et al., The cold - shock response in cultured mammalian cells: Harnessing the response for the improvement of recombinant protein production, Biotechnol. Bioeng. 93:829 - 835(2006); Marchant, R.J. et al., Metabolic rates, growth phase, and mRNA levels influence cell - specific antibody production levels from in vitro cultured mammalian cells at sub - physiological temperatures, Mol. Biotechnol. 39:69 - 77(2008)).

[0208] When culturing transfected or transformed host cells, the antibody is directly secreted into the cell culture medium (by using an appropriate secretion - directed signal peptide) and recovered therefrom.

[0209] A perfusion bioreactor can be fluidly connected to an affinity chromatography step with a continuous flow coming from the bioreactor to the affinity chromatography system (either directly or indirectly via the operation of an intervening unit, such as a surge tank). Some embodiments of the present invention include a first single-use surge tank (SUSV1) adapted to receive a large amount of cell-free permeate removed from one or more perfusion bioreactors. These large amounts of permeate can be automatically and fluidly supplied from one or more perfusion bioreactors to SUSV1.

[0210] The anti-PD-1 antibody or antigen-binding fragment of the present invention can be produced or obtained by a process comprising the following.

[0211] a) A step of perfusing mammalian host cells in a cell culture medium in a perfusion bioreactor by applying a perfusion rate of at least about 0.25 to 6.0 volume per day per vessel (vvd), wherein the host cells comprise a polynucleotide encoding the light chain variable region of an antibody or antigen-binding fragment of an anti-human PD-1 antibody and a polynucleotide encoding the heavy chain variable region, or a polynucleotide encoding the light chain variable region and the heavy chain variable region, step b) A step of continuously recovering the antibody from the cell culture broth to obtain a recovered cell culture fluid c) Optionally, a step of transferring the recovered cell culture fluid (HCCF) to a surge tank during a residence time of about 0.5 to 8 hours, and d) A step of continuously purifying the recovered cell culture fluid in an affinity chromatography step to obtain a purified composition.

[0212] In one embodiment, the above method includes the following steps before step a).

[0213] (i) A step of inoculating a perfusion bioreactor with mammalian host cells at a cell density of about 0.2 to 0.6×10 6 cells / ml in the cell culture medium, step ii) Growing mammalian cells in a cell culture medium in a perfusion bioreactor to a cell density of about 1.0 - 6.0×10 6 cells / ml.

[0214] In one embodiment, in step i), the cell density is about 0.20 - 0.6×10 6 cells / ml on day 1. In one embodiment, in step i), the cell density is about 0.25 - 0.5×10 6 cells / ml on day 1. In another embodiment, in step i), the cell density is about 0.4 - 0.5×10 6 cells / ml on day 1. In another embodiment, in step a), perfusion is started on day 3. In another embodiment, in step a), perfusion is started at a cell density of about 2 - 10×10 6 cells / ml. In another embodiment, in step a), perfusion is started at a cell density of about 4 - 8×10 6 cells / ml. In another embodiment, step (ii) is growing mammalian host cells in a cell culture medium in a perfusion bioreactor to a cell density of about 2.0 - 6.0×10 6 cells / ml.

[0215] The perfusion rate may start at about 0.5 vvd and can be increased continuously, gradually or incrementally from about 0.5 vvd to 6 vvd. In one embodiment, in step a), the perfusion rate is about 0.5 vvd - 4 vvd. In one embodiment, in step a), the perfusion rate is about 0.5 vvd - 2 vvd. Preferably, the perfusion rate is kept constant after reaching the target. Alternatively, the perfusion rate can be adjusted throughout the continuous manufacturing process according to the continuously measured viable cell density. In one embodiment, the perfusion rate is about 0.5 vvd on day 3, about 1 vvd on day 4 and about 2 vvd on day 5. In a further embodiment, step d) is carried out after day 5. In a further embodiment, a specific cell density, for example about 80 - 100×10 6Cell / ml, or cell bleeding is performed to maintain a capacitance value (about 70 - 90 or 80 pF / cm). In one embodiment, the capacitance value is about 70 - 90 pF / cm during perfusion. In one embodiment, the titer of the permeating substance is 0.2 - 2 g / L. In one embodiment, the maximum cell density is about 80 - 100×10 6 cells / ml. In one embodiment, the maximum cell density is about 80 - 150×10 6 cells / ml. In one embodiment, the maximum cell density is about 100×10 6 cells / ml. In one embodiment, the mammalian host cell is a CHO cell. In one embodiment, the host cell contains a polynucleotide encoding a light chain and a polynucleotide encoding a heavy chain, or a polynucleotide encoding a light chain and a heavy chain, the light chain consists of the amino acid sequence shown in SEQ ID NO: 5, and the two heavy chains consist of the amino acid sequence shown in SEQ ID NO: 10. In another embodiment, the perfusion cell culture medium has a copper concentration of 10 - 35 ppb. In a further embodiment, the copper concentration in the perfusion bioreactor ranges from 1 - 3 ppb.

[0216] In another aspect, continuous recovery is performed by setting a constant permeation rate to obtain a cell-free permeate through a hollow fiber membrane connected to a perfusion bioreactor. In one embodiment, the perfusion rate is equal to the sum of the permeation rate and the cell bleeding rate. A typical perfusion system with a feed stream, a perfusion stream, and a cell bleeding stream is described in Goudar, C.T. & Chen, C. & Le, H. (2015) SBE special section: Biopharmaceuticals - Continuous processing in upstream operations. p111.

[0217] Downstream process The antibody or antigen-binding fragment composition from the upstream process can further undergo continuous or semi-continuous downstream purification, or downstream batch purification processes.

[0218] Affinity chromatography separates molecules based on highly specific interactions between the target molecule and the functional groups of the resin, such as those between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid. Some commonly used affinity chromatography resins include protein A or protein G resins for purifying antibodies, avidin-biotin resins for purifying biotin / avidin and their derivatives, glutathione resins for purifying GST-tagged recombinant proteins, heparin resins for separating plasma coagulation proteins, and IMAC resins for purifying proteins that specifically interact with metal ions. The operating conditions for each affinity chromatography depend on the mechanism of interaction and the factors that affect the interaction. Commercially available affinity chromatography resins include, but are not limited to, MabSelect Sure, UNOSphere SUPrATM, Affi-Gel® and Affi-Prep®. In one embodiment, the affinity chromatography step is protein A chromatography performed in binding and elution modes.

[0219] In one embodiment, the harvested cell culture fluid is purified by protein A affinity chromatography comprising the following steps.

[0220] a) Binding the HCCF to the stationary phase b) Eluting the antibody or antigen-binding fragment from the protein A stationary phase with an eluent c) Optionally, washing and disinfecting the stationary phase for repeated cycles.

[0221] In one embodiment, prior to step (a), equilibration of the stationary phase with an equilibration solution is performed. In one embodiment, there are one or more impurities in the flow-through of step a).

[0222] In another aspect of the method, after step a) but before step b), the method further comprises a step of washing the stationary phase with one or more washing liquids. In one embodiment, one or more impurities are removed from the washing step. In one embodiment, the washing liquid or eluent contains a salt, preferably a monovalent metal ion salt such as NaCl or KCl. In one embodiment, the washing liquid contains about 400 - 600 mM of NaCl or KCl. In another embodiment, the washing liquid contains about 500 mM of NaCl or KCl. In another embodiment, the washing liquid contains about 400 - 500 mM of NaCl or KCl. In a further embodiment, the first, second and third washing liquids contain about 5 - 20 mM of sodium phosphate, and the second washing liquid further contains about 400 - 600 mM of NaCl or KCl. In a further embodiment, the first, second and third washing liquids contain about 10 mM of sodium phosphate, and the second washing liquid further contains about 500 mM of NaCl or KCl.

[0223] In one embodiment, the pH of the washing liquid or eluent is about 6 - 7. In one embodiment, the pH of the washing liquid or eluent is about 6.5. In another embodiment, the eluent contains about 5 - 50 mM of sodium acetate. In another embodiment, the eluent contains about 5 - 30 mM of sodium acetate. In another embodiment, the eluent contains about 20 mM of sodium acetate.

[0224] In another aspect, the eluent contains about 5 - 50 mM of sodium acetate. In another embodiment, the eluent contains about 5 - 30 mM of sodium acetate. In another embodiment, the eluent contains about 20 mM of sodium acetate. In one embodiment, the eluent has a pH of about 3.5 - 3.6. In another embodiment, the eluent has a pH of about 3 - 4.

[0225] In one embodiment, the affinity chromatography is operated in a continuous multi-column chromatography system (of at least two, three or four columns) that allows for a continuous flow of HCCF to a chromatography skid.

[0226] In one embodiment, a perfusion bioreactor or perfusion system containing HCCF is fluidly connected, either indirectly or directly, to affinity chromatography. In one embodiment, the affinity chromatography is fluidly connected to the perfusion bioreactor.

[0227] In another embodiment, a perfusion bioreactor or system containing HCCF is fluidly connected to affinity chromatography via a surge tank. The surge tank is sized to control the residence time that the fluid control volume spends in the surge tank. In some embodiments of the present invention, the average residence time is from 0.5 to 30 hours. In some embodiments of the present invention, the average residence time is from 0.5 to 20 hours. In some embodiments of the present invention, the average residence time is from 0.5 to 8 hours. In some embodiments, the average residence time is 2 hours. In some embodiments, the average residence time is 1 hour. In some embodiments, the average residence time is 0.5 hour. The HCCF can be stored in the surge tank at 4 to 25°C. In other embodiments, the loading residence time (from exiting the perfusion bioreactor to loading onto the affinity chromatography) is within 30 hours. In other embodiments, the loading residence time is within 24 hours. In one embodiment, the flow rate of HCCF to the surge tank is equal to the supply rate from the surge tank to a continuous multi-column chromatography system. Alternatively, the HCCF can be stored in a container at -40°C to -80°C before being subjected to affinity chromatography. In one embodiment, the antibody is protected from light during storage in the surge tank or container.

[0228] In a further embodiment, the affinity chromatography may be fluidly connected, either directly or via a surge tank or a holding tank, and followed by one or more of the steps of virus inactivation, depth filtration, second chromatography, third polishing chromatography, virus filtration, ultrafiltration, diafiltration, single-pass tangential flow filtration, and in-line diafiltration.

[0229] In one aspect, affinity chromatography is fluidly connected, in a continuous flow, to a virus inactivation system and optionally, in a continuous flow, to a depth filtration, and to a second chromatography system, optionally a third polishing chromatography system, a virus filtration system, and an ultrafiltration / diafiltration system, all in a continuous flow to the upstream processing steps described above and in series with each other, with an optional intervening surge tank, see Figure 1C of WO 2020 / 168315. The final ultrafiltration step can be configured to maintain continuity through an in-line diafiltration step, which is a single-pass tangential flow filtration step and is fluidly connected or via an optional surge tank, through the in-line diafiltration product from the affinity chromatography step.

[0230] In another aspect, affinity chromatography is fluidly connected, in a continuous flow, to a virus inactivation system and optionally, in a continuous flow or batch mode, to a depth filtration system, a holding tank (HV1) for temporary storage of the virus inactivation product pool, a second chromatography system, an optional third polishing chromatography system, and an ultrafiltration / diafiltration system, in series with each other, optionally including an intervening surge tank or holding tank (i.e., a holding tank in the case of two or more batch steps or operations). See, for example, Figure 1D of WO 2020 / 168315. The final ultrafiltration step can be configured to maintain continuity through an in-line diafiltration step, which is a single-pass tangential flow filtration step and is directly connected or via an optional surge tank, through the in-line diafiltration product from the affinity chromatography step.

[0231] In some embodiments of the present invention, a virus inactivation step follows the affinity chromatography step. For virus inactivation, the collection of the affinity chromatography eluate is pooled into one of two virus inactivation pool tanks. After separate amounts of eluate are collected in the pool tank, the pool volume undergoes an automatic pH adjustment from the elution pH to the virus inactivation pH (pH = 3.4 - 3.7) by adding an acidic solution, followed by adjustment to a neutral pH by adding a base. In some embodiments, the neutralization pH is 4.0 - 7.5. In some embodiments, the neutralization pH is 4 - 6. In some embodiments, the neutralization pH is 7.2. During this automatic adjustment cycle, fresh affinity chromatography eluate is collected into the second virus inactivation pool tank. By switching between the two virus inactivation pool tanks, it becomes possible to continuously operate the unit. In some embodiments of the present invention, the virus inactivation product (VIP) is transferred to a surge tank that continuously supplies the operation of other downstream units.

[0232] In some embodiments of the present invention, the operation downstream of the virus inactivation system / neutralization system may be supplied to a second chromatography step or filtered by depth filtration to obtain a filtered virus inactivation product pool (FVIP), including continuous processing by transferring the VIP to a surge tank. The semi - continuous flow in the operation of these units is maintained by circulating either the depth filtration consumables or the second chromatography column at regular intervals. In these examples, the flow is temporarily stopped during the flushing and regeneration stages between consumable switch - outs and during the unload process of the chromatography stage.

[0233] In some embodiments of the present invention, the operation downstream of the virus inactivation system / neutralization system involves continuous processing of a virus inactivation product pool (optionally filtered by depth filtration to obtain a filtered virus inactivation product pool (FVIP)). In such embodiments, the virus inactivation product pool is collected in a collection tank, and in subsequent batch processes or operations, a purified product pool or virus-free filtrate may optionally be collected in another collection tank between processes. In such individual operations, batch, or batch-mode processing, a (one or more) collection tank or interchangeably a "(one or more) collection tank" (which in certain embodiments may also be regarded as a "(one or more) feed tank" for subsequent processes) from one process lacks automatic control of the surge layer, and the collection tank (or feed tank) may physically resemble a surge tank, but such a collection tank (or interchangeably, "collection tank") or feed tank is called a "holding tank" or interchangeably "HV" (e.g., HV1, HV2, HV3, HV4, or HV5). The "holding tank" may be a single-use holding tank (SUHV) distinct from a single-use collection tank for virus (SUCV, e.g., SUCV1 or SUCV2) in a continuous or semi-continuous format set of manufacturing process steps or operations. See FIG. 1D of WO 2020 / 168315.

[0234] After the affinity chromatography step, a second and / or third chromatography step can be continued to remove, for example, protein aggregates, host cell proteins or DNA. IEX chromatography separates molecules based on their net charge. The separation occurs as a result of competition between the charged molecule of interest and the counterion of the oppositely charged ligand groups on the IEX chromatography resin. The strength of binding of a molecule to the IEX resin depends on the net charge of the molecule, which is affected by operating conditions such as pH and ionic strength. IEX resins include AEX resins and CEX resins. AEX resins can contain substituents such as diethylaminoethyl (DEAE), trimethylaminoethyl (TMAE), quaternary aminoethyl (QAE) and quaternary amine (O) groups. CEX resins can contain substituents such as carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S) groups. Cellulose IEX resins, such as DE23, DE32, DE52, CM-23, CM-32 and CM-52, are available from Whatman Ltd., Maidstone, Kent, UK. Sephadex-based and cross-linked IEX resins are also known. For example, DEAE-, QAE-, CM-, and SP-Sephadex, as well as DEAE-, Q-, CM-, and S-Sepharose, and Sepharose are all available from GE Healthcare, Piscataway, NJ. Furthermore, ethylene glycol-methacrylate copolymers derived from both DEAE and CM, such as TOYOPEARL™ DEAE-650S or M and TOYOPEARL™ CM-650S or M, are available from Toso Haas Co., Philadelphia, PA. POROSTM HS, POROSTM HQ, POROSTM XS are available from Thermo Fisher Scientific, Waltham, MA. In one embodiment, the second chromatography step is AEX chromatography performed in flow-through mode at a pH of about 6.5 to 8.0. In one embodiment, the pH is about 6.5 to 7.5.

[0235] After purification by the chromatography step, the antibody or antigen-binding fragment can be processed by a series of filtration steps including nanofiltration for virus removal and ultrafiltration for concentration and buffer exchange. Nanofiltration can be operated batchwise by use of a holding tank or continuously by circulating the nanofiltration membrane at appropriate frequencies. The ultrafiltration step may be carried out conventionally in batch mode by feeding the unit operation from a holding tank containing the virus-filtered nanofiltration product. Alternatively, ultrafiltration can be carried out continuously by using single-pass ultrafiltration followed by inline diafiltration either directly connected or via the use of an intermediate surge tank.

[0236] Method of Use The present invention also relates to a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of any of the pharmaceutical compositions of the present invention, i.e., any of the compositions described herein. In some embodiments of this method, the pharmaceutical composition is administered to the subject by subcutaneous administration.

[0237] In any of the methods of the present invention, the cancer can be selected from the group consisting of melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, Merkel cell carcinoma, cutaneous squamous cell carcinoma, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, endometrial cancer, cervical cancer, thyroid cancer, salivary gland cancer, prostate cancer (e.g., hormone-resistant adenocarcinoma of the prostate), pancreatic cancer, colon cancer, liver cancer, thyroid cancer, glioblastoma, glioma, and other neoplastic malignancies.

[0238] In one embodiment, the cancer is melanoma, non-small cell lung cancer, It is head and neck cancer, urothelial cancer, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma, endometrial cancer, cutaneous squamous cell carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, prostate cancer, glioblastoma, or a cancer with a high amount of tumor gene mutations or MSI-H cancer.

[0239] In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the lung cancer is squamous non-small cell lung cancer. In some embodiments, the lung cancer is non-squamous non-small cell lung cancer. In one embodiment, the NSCLC is metastatic.

[0240] In alternative embodiments, the lung cancer is small cell lung cancer (SCLC). In one embodiment, the SCLC is metastatic.

[0241] In some embodiments, the lymphoma is Hodgkin lymphoma.

[0242] In other embodiments, the lymphoma is non-Hodgkin lymphoma. In certain embodiments, the lymphoma is mediastinal large B-cell lymphoma. In some embodiments, the lymphoma is diffuse large B-cell lymphoma (DLBCL).

[0243] In some embodiments, the breast cancer is triple-negative breast cancer.

[0244] In some embodiments, the breast cancer is ER+ / HER2- breast cancer.

[0245] In some embodiments, the bladder cancer is urothelial cancer.

[0246] In some embodiments, the head and neck cancer is nasopharyngeal cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is salivary gland cancer. In other embodiments, the cancer is squamous cell carcinoma of the head and neck.

[0247] In some embodiments, the cancer is metastatic colorectal cancer having a high level of microsatellite instability (MSI-H).

[0248] In some embodiments, the cancer is a solid tumor having a high level of microsatellite instability (MSI-H).

[0249] In some embodiments, the cancer is a solid tumor having a high amount of mutations. In one embodiment, the tumor mutational burden (TMB) is 10 or more mutations / megabase as determined by an FDA-approved assay. In one embodiment, the tumor is metastatic or unresectable.

[0250] In some embodiments, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, recurrent or refractory classical Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal cancer, gastric cancer, DLBCL, and hepatocellular carcinoma.

[0251] In other embodiments of the above treatment methods, the cancer is a hematological malignancy. In certain embodiments, the hematological malignancy is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), DLBCL, EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), or small lymphocytic lymphoma (SLL).

[0252] Malignancies that exhibit improved disease-free survival and overall survival in relation to the presence of tumor-infiltrating lymphocytes in a biopsy or surgical specimen, such as melanoma, colorectal cancer, liver cancer, kidney cancer, gastric / esophageal cancer, breast cancer, pancreatic cancer, and ovarian cancer, are encompassed by the methods and treatments described herein. Such cancer subtypes are known to be susceptible to immune control by T lymphocytes. Further included are refractory or recurrent malignancies whose growth can be inhibited using the antibodies described herein.

[0253] In some embodiments, the pharmaceutical composition of the present invention is administered to a subject having cancer, including ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, and melanoma, characterized by increased expression of PD-L1 and / or PD-L2 in the tested tissue sample. Further cancers that can benefit from treatment with an anti-PD-1 antibody, such as the humanized anti-PD-1 antibody pembrolizumab, include those associated with persistent infection by viruses such as human immunodeficiency virus, hepatitis virus classes A, B, and C, Epstein-Barr virus, and human papillomavirus, which are known to be causally related to, for example, Kaposi's sarcoma, liver cancer, nasopharyngeal cancer, lymphoma, cervical cancer, vulvar cancer, anal cancer, penile cancer, and oral cancer.

[0254] In one embodiment, the present invention includes a method of treating cancer in a human patient in need thereof, comprising administering to the patient an effective amount of any pharmaceutical composition of the present invention.

[0255] In one embodiment, the present invention includes a method of treating unresectable or metastatic melanoma in a human patient in need thereof, comprising administering to the patient an effective amount of any pharmaceutical composition of the present invention.

[0256] In one embodiment, the present invention includes a method of treating metastatic non-small cell lung cancer (NSCLC) in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the patient has a tumor with high PD-L1 expression [(Tumor Proportion Score (TPS) ≥ 50%)]. In other embodiments, the patient has a tumor with PD-L1 expression (TPS ≥ 1%). In still other embodiments, the patient has been previously treated with platinum-containing chemotherapy or has not been treated. In certain embodiments, the patient had disease progression during or after administration of platinum-containing chemotherapy. In one embodiment, the NSCLC is metastatic or stage III.

[0257] In certain embodiments, the PD-L1 TPS is determined by an FDA-approved test.

[0258] In certain embodiments, the patient's tumor does not have genomic aberrations of EGFR or ALK.

[0259] In certain embodiments, the patient's tumor has genomic aberrations of EGFR or ALK and the patient has received treatment for the EGFR or ALK aberration before or after administration of the composition of the invention and the disease has progressed.

[0260] In certain embodiments, the patient has a tumor with a PD-L1 expression CPS ≥ 1%.

[0261] In one embodiment, the invention includes a method of treating non-squamous non-small cell lung cancer (NSCLC) in a patient in need thereof, the method comprising administering to the patient an effective amount of the pharmaceutical composition of the invention, pemetrexed, and platinum chemotherapy. In one embodiment, the invention is a method of treating non-squamous non-small cell lung cancer (NSCLC) in a human patient in need thereof, the method comprising: (1) administering to the patient an effective amount of the pharmaceutical composition of the invention; and (2) administering to the patient an effective amount of pemetrexed and carboplatin. In certain embodiments, the patient has not been previously treated with an anti-cancer therapeutic agent prior to initiating combination therapy regimen with the composition of the invention, pemetrexed, and carboplatin. In certain embodiments, the patient has metastatic non-squamous non-small cell lung cancer.

[0262] In certain embodiments, pemetrexed is administered to the patient in an amount of 500 mg / m 2 ². In a sub-embodiment, pemetrexed is administered to the patient by intravenous infusion every 21 days. In certain embodiments, the infusion time is about 10 minutes.

[0263] In an embodiment of the invention where the patient is treated with the pharmaceutical composition of the invention in combination with pemetrexed, the invention further comprises administering to the patient about 400 μg to about 1000 μg of folic acid once daily, starting about 7 days before administering pemetrexed to the patient and continuing until about 21 days after the last dose of pemetrexed is administered to the patient. In certain embodiments, the folic acid is administered orally. In some embodiments, the invention comprises about 1 mg of vitamin B 12 and further comprises administering to the patient about 1 week before the first administration of pemetrexed and every about 3 cycles of pemetrexed administration (i.e., about every 9 weeks). In certain embodiments, vitamin B 12 is administered intramuscularly. In certain embodiments, the invention further comprises administering to the patient about 4 mg of dexamethasone twice daily on the day before, on the day of, and on the day after pemetrexed administration. In certain specific embodiments, dexamethasone is administered orally.

[0264] In one embodiment, the invention is a method of treating squamous non-small cell lung cancer (NSCLC) in a human patient in need thereof, comprising: (1) administering to the patient an effective amount of the pharmaceutical composition of the invention; and (2) administering to the patient an effective amount of paclitaxel or protein-bound paclitaxel and carboplatin. In certain embodiments, the patient has not been previously treated with an anticancer therapeutic agent prior to starting the combination treatment regimen. In certain embodiments, the patient has metastatic squamous non-small cell lung cancer.

[0265] In one embodiment, the invention comprises a method of treating recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) in a human patient in need thereof, comprising administering to the patient an effective amount of any pharmaceutical composition of the invention. In certain embodiments, the patient has been previously treated with platinum-containing chemotherapy. In certain embodiments, the patient had disease progression during or after platinum-containing chemotherapy. In certain embodiments, the patient's tumor expresses PD-L1 [Combined Positive Score (CPS) ≥1]. In one embodiment, the treatment is performed in combination with platinum and FU.

[0266] In one embodiment, the present invention includes a method of treating refractory classical Hodgkin lymphoma (cHL) in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the patient has relapsed after two or more lines of therapy for cHL. In certain embodiments, the patient is an adult patient. In alternative embodiments, the patient is a pediatric patient.

[0267] In one embodiment, the present invention includes a method of treating locally advanced or metastatic urothelial carcinoma in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the patient is ineligible for cisplatin-containing chemotherapy. In certain embodiments, the patient has disease progression during or after platinum-containing chemotherapy, or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy. In certain embodiments, the patient's tumor expresses PD-L1 [Combined Positive Score (CPS) ≥1]. In one embodiment, the patient has high-risk muscle-invasive bladder cancer that is non-responsive to Bacillus Calmette-Guerin (BCG).

[0268] In one embodiment, the present invention includes a method of treating unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair-deficient solid tumors in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the patient has had disease progression after prior anti-cancer treatment.

[0269] In one embodiment, the present invention includes a method of treating unresectable or metastatic, microsatellite instability-high (MSI-H) or mismatch repair-deficient solid tumors or colorectal cancer in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the patient had disease progression after prior treatment with fluoropyrimidine, oxaliplatin, and irinotecan.

[0270] In one embodiment, the present invention includes a method for treating recurrent locally advanced or metastatic gastric cancer in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention.

[0271] In one embodiment, the present invention includes a method for treating recurrent locally advanced or metastatic gastroesophageal junction adenocarcinoma in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the patient's tumor expresses PD-L1 [Combined Positive Score (CPS) ≥ 1]. In certain embodiments, the patient has disease progression during or after two or more previous lines of therapy including fluoropyrimidine and platinum-containing chemotherapy. In certain embodiments, the patient has disease progression during or after two or more previous lines of therapy including HER2 / neu targeted therapy.

[0272] In one embodiment, the present invention includes a method for treating recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the patient's tumor expresses PD-L1 [Combined Positive Score (CPS) ≥ 1].

[0273] In one embodiment, the present invention includes a method for treating recurrent locally advanced or metastatic cervical cancer in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the patient's tumor expresses PD-L1 [Combined Positive Score (CPS) ≥ 1].

[0274] In one embodiment, the present invention includes a method for treating hepatocellular carcinoma in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention. In one embodiment, the present invention includes a method for treating recurrent locally advanced or metastatic Merkel cell carcinoma in a human patient, comprising administering to the patient the composition of the present invention. In one embodiment, the present invention includes a method for treating recurrent or metastatic cutaneous squamous cell carcinoma in a human patient, comprising administering to the patient the composition of the present invention.

[0275] In one embodiment, the present invention includes a method for treating advanced renal cell carcinoma in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention and axitinib. In one embodiment, the present invention includes a method for treating advanced endometrial carcinoma in a human patient in need thereof, comprising administering to the patient the composition of the present invention and lenvatinib. In one embodiment, the endometrial carcinoma is not MSI-H or dMMR.

[0276] In one embodiment, the present invention includes a method for treating cancer in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention, wherein the patient has cancer selected from the group consisting of melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, and salivary gland cancer.

[0277] In one embodiment, the present invention includes a method for treating small cell lung cancer in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention.

[0278] In one embodiment, the present invention includes a method for treating non-Hodgkin lymphoma in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the non-Hodgkin lymphoma is mediastinal large B-cell lymphoma. In certain embodiments, the non-Hodgkin lymphoma is diffuse large B-cell lymphoma.

[0279] In one embodiment, the present invention includes a method for treating breast cancer in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention. In certain embodiments, the breast cancer is triple-negative breast cancer and may be combined with chemotherapy. In certain embodiments, the breast cancer is ER+ / HER2- breast cancer. In certain embodiments, the patient's tumor expresses PD-L1 [Combined Positive Score (CPS) ≧1].

[0280] In one embodiment, the present invention includes a method for treating nasopharyngeal cancer in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention.

[0281] In one embodiment, the present invention includes a method for treating thyroid cancer in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention.

[0282] In one embodiment, the present invention includes a method for treating salivary gland cancer in a human patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition of the present invention.

[0283] As described above, in some embodiments of the methods of the present invention, the method further comprises administering an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-LAG3 antibody or antigen-binding fragment thereof, an anti-GITR antibody or antigen-binding fragment thereof, an anti-TIGIT antibody or antigen-binding fragment thereof, an anti-CD27 antibody or antigen-binding fragment thereof. In one embodiment, the additional therapeutic agent is a Newcastle disease virus vector expressing IL-12. In a further embodiment, the additional therapeutic agent is dinaciclib. In still further embodiments, the additional therapeutic agent is a STING agonist. In one embodiment, the additional therapeutic agent is coxsackievirus CVA21.

[0284] Suitable routes of administration of the additional therapeutic agent may include, for example, intramuscular, subcutaneous, and parenteral delivery including intrathecal, direct intraventricular, intravenous, and intraperitoneal. The drug can be administered by various conventional methods such as intraperitoneal, parenteral, arterial or intravenous injection.

[0285] The selection of the dosage of an additional therapeutic agent depends on several factors including the serum or tissue turnover rate of the entity, the level of the symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissues or organs in the individual being treated. The dosage of the additional therapeutic agent must be an amount that results in an acceptable level of side effects. Thus, the dosage and frequency of administration of each additional therapeutic agent (e.g., a biological or a chemical therapeutic agent) will depend in part on the specific therapeutic agent, the severity of the cancer being treated, and the characteristics of the patient. Guidelines are available for selecting appropriate dosages of antibodies, cytokines and small molecules. See, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602; Physicians’ Desk Reference 2003 (Physicians’ Desk Reference, 57th Ed); Medical Economics Company; ISBN:1563634457; 57th edition (November 2002).Determination of an appropriate dosage regimen may be made by a clinician using, for example, parameters or factors that are known or suspected to affect treatment, or are predicted to affect treatment, in the art, e.g., a patient's medical history (e.g., prior therapies), the type and stage of cancer being treated, and biomarkers of response to one or more of the therapeutic agents in combination therapy.

[0286] References to a variety of literature are available to facilitate selection of a pharmaceutically acceptable carrier or excipient for an additional therapeutic agent. See, for example, Remington’s Pharmaceutical Sciences and U.S.Pharmacopeia:National Formulary, Mack Publishing Company, Easton, PA (1984); Hardman, et al. (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0287] The pharmaceutical antibody composition can be administered by continuous infusion or by doses at intervals such as once a day, 1 to 7 times a week, once a week, two weeks, three weeks, monthly, every other month, etc. A preferred dosage protocol is one that includes the maximum dose or dosing frequency that avoids significant undesirable side effects. The total weekly dose is generally at least 0.05 μg / kg, 0.2 μg / kg, 0.5 μg / kg, 1 μg / kg, 10 μg / kg, 100 μg / kg, 0.2 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg body weight or more. See, for example, Yang et al. (2003) New Engl. J. Med. 349:427-434; Herold et al. (2002) New Engl. J. Med. 346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji et al. (20003) Cancer Immunol. Immunother. 52:133-144. The desired dose of a small molecule therapeutic, such as a peptidomimetic, natural product or organic chemical substance, is approximately the same as that of an antibody or polypeptide on a molar / kg basis.

[0288] In certain embodiments, administration includes administering to a subject escalating doses of the pharmaceutical composition or compositions of the invention at 1.0, 3.0, and 10 mg / kg over the course of treatment. The composition may be a reconstituted liquid composition or a liquid composition that has not been previously lyophilized. The time course may vary and can continue as long as the desired effect is obtained. In certain embodiments, the dose escalation will continue up to a dose of about 10 mg / kg. In certain embodiments, the subject has a histological or cytological diagnosis of melanoma or other forms of solid tumors and, in certain instances, the subject may have a non-measurable disease. In certain embodiments, the subject is being treated with other chemotherapeutic agents, while in other embodiments, the subject is treatment-naive.

[0289] In further additional embodiments, the dosing regimen includes administering a dose of 1, 3, or 10 mg / kg of any of the pharmaceutical compositions or compositions described herein over the course of the treatment. In such dosing regimens, the interval between administrations will be about 14 days (±2 days). In certain embodiments, the interval between administrations will be about 21 days (±2 days).

[0290] In certain embodiments, the dosing regimen will include administering doses from about 0.005 mg / kg to about 10 mg / kg using intra-patient dose escalation. In certain embodiments, doses of 5 mg / kg or 10 mg / kg will be administered at intervals of every three weeks or every two weeks. In further additional embodiments, a dose of 3 mg / kg will be administered to melanoma patients or patients with other solid tumors at three-week intervals. In these embodiments, the patient is assumed to have an inoperable disease. However, the patient may have had surgery in the past.

[0291] In certain embodiments, the subject will be administered a 30-minute IV infusion of any of the pharmaceutical compositions or compositions described herein. In certain embodiments for escalating doses, the interval between administrations will be about 28 days (±1 day) between the first and second doses. In certain embodiments, the interval between the second and third administrations is about 14 days (±2 days). In certain embodiments, the interval between administrations, for administrations after the second administration, will be about 14 days (±2 days). In certain embodiments, the interval between administrations, for administrations after the second administration, will be about three weeks. In certain embodiments, the interval between administrations, for administrations after the second administration, will be about six weeks.

[0292] In certain embodiments, the use of cell surface markers and / or cytokine markers as described in WO 2012 / 018538 or WO 2008 / 156712 will be used in bioassays for monitoring, diagnosis, patient selection, and / or treatment regimens involving blockade of the PD-1 pathway. Subcutaneous administration can be performed by injection using a syringe or by using other injection devices (e.g., Inject-ease® device), injector pen, or needleless devices (e.g., MediJector and BioJector®).

[0293] Embodiments of the invention also include one or more of the pharmaceutical compositions or formulations described herein for use in (a) therapy (e.g., in a human body), (b) a medicament, (c) inducing or enhancing an anti-tumor immune response, (d) decreasing the number of one or more tumor markers in a patient, (e) stopping or delaying the growth of a tumor or blood cancer, (f) stopping or delaying the progression of a PD-1 related disease, (g) stopping or delaying the progression of cancer, (h) stabilizing a PD-1 related disease, (i) inhibiting the growth or survival of tumor cells, (j) eliminating or reducing the size of one or more cancerous lesions or tumors, (k) reducing the progression, onset or severity of a PD-1 related disease, (l) reducing the severity or duration of clinical symptoms of a PD-1 related disease such as cancer, (m) extending the survival of a patient compared to the survival expected for untreated similar patients, (n) inducing complete or partial remission of a cancerous condition or other PD-1 related disease, or (o) for use in the treatment of cancer, (i) for use, (ii) for use as a medicament or composition, or (iii) for use in the preparation of a medicament.

[0294] General methods Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 1989 2nd Edition, 2001 3rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods are also described in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which includes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), complex carbohydrate and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0295] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and protein glycosylation are described (e.g., see Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques are available for characterizing ligand / receptor interactions (e.g., see Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0296] Monoclonal antibodies, polyclonal antibodies, and humanized antibodies can be prepared (e.g., see Shepherd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; see U.S. Patent No. 6,329,511).

[0297] Although different embodiments of the present invention have been described herein with reference to the accompanying drawings, it should be understood that the present invention is not limited to those exact embodiments, and various changes and modifications can be made by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0298] [Examples] [Example 1] Continuous perfusion process In the following procedures and examples, a glutamine synthetase knockout CHO host cell (expressing pembrolizumab) containing a polynucleotide encoding a light chain having the amino acid sequence shown in SEQ ID NO: 5 and a polynucleotide encoding a heavy chain having the amino acid sequence shown in SEQ ID NO: 10 was used.

[0299] 3 L bioreactor operation A glass bioreactor (3 L, Sartorius Stedim, Gottingen, Germany) equipped with a marine impeller (70 mm in diameter) and a perforated sparger (14 holes of 0.5 mm in diameter) was used. The bioreactor was inoculated at a target cell density of 0.5×10 6 cells / mL. Dissolved oxygen (DO) was controlled to 30 - 60% of air saturation using pure O2 and a stirring speed that was gradually increased in the range of 260 - 450 rpm, and the O2 sparging rate was controlled to <0.30 vvm to avoid excessive foam formation. EX-CELL® antifoam agent (Sigma-Aldrich, St. Louis, MO) was added as needed for foam control. The overlay air was controlled at 0.1 L / min. The temperature was maintained at 36.5 °C throughout the culture period. During the culture period, the pH of the bioreactor was controlled at 7.0 ± 0.3.

[0300] A commercially available basal medium was used for the first bioreactor batch growth, and then a perfusion medium was used for medium exchange. The copper concentration of the perfusion medium was 10 - 35 ppb. The bioreactor started with a working volume of 1.6 L and was maintained at the same level during perfusion. On day 3, when the cell density reached 2 - 4×10 6 cells / mL, medium exchange was started and the perfusion rate was increased according to a predetermined schedule starting from 0.5 vvd. On day 4, a perfusion rate of 1 vvd was applied, and the maximum perfusion rate of 2 vvd was reached from day 5 until the end of production. A TFF system equipped with a magnetic levitation pump was used for cell retention and medium exchange (KrosFlo® KML System, Repligen, Waltham, MA). The TFF system continuously circulates the cell culture fluid through a hollow fiber module while collecting the permeate at a predetermined rate. The specifications of the hollow fiber module are as follows: module length: 32 cm; effective filtration surface area: 0.09 m 2; Fiber lumen ID: 1.4 mm; pore size: 0.2 μm (Pall, Port Washington, NY). A TFF cross-flow rate of 1.0 L / min was used in all experiments. When the cell density reached 100×10 6 cells / mL or the capacitance target of 80 pF / cm, the cell bleed pump was activated to maintain the cell density or biovolume (capacitance, pF / cm cell bleed was automatically controlled by a capacitance probe (Incyte DN12, Hamilton Bonaduz AG, Switzerland). The perfusion culture period was 28 - 32 days. The copper concentration in the bioreactor ranged from 1 - 35 ppb. Continuous harvest was started 5 days after a perfusion rate of 2 vvd to the bioreactor was achieved.

[0301] 50 L Bioreactor Operation An Xcellerx XDR 50 single-use bioreactor (GE healthcare, Marlborough, MA) was used as the production bioreactor for this study. The bioreactor was inoculated with a target cell density of 0.5×10 6 cells / mL with a dissolved oxygen (DO) controlled to 30 - 60% air saturation using a pure O2 supply. The agitation of the bioreactor was controlled at 86 - 93 rpm to ensure proper mixing and mass transfer. EX-CELL® antifoam (Sigma-Aldrich, St. Louis, MO) was added as needed for foam control. The overlay air was controlled at 0.1 - 0.5 L / min. The temperature was maintained at 36.5 °C throughout the culture period. During the culture period, the pH of the bioreactor was controlled at 7.0 ± 0.3.

[0302] A commercially available basal medium was used for the first bioreactor batch growth, followed by a perfusion medium for medium exchange. The copper concentration of the perfusion medium was 32.5 ppb. The bioreactor started with a working volume of 50 L and was maintained at the same level during perfusion. On day 3, when the cell density reached 2 - 4×10 6Once the cell density reached [X] cells / mL, the medium exchange was initiated, and the perfusion rate was increased according to a predetermined schedule starting from 0.5 vvd on day 3, reaching 1 vvd on day 4, and 2 vvd (average residence time of 0.5 days) on day 5. A TFF system equipped with a magnetic levitation pump was used for cell retention and medium exchange (KrosFlo® KML System, Repligen, Waltham, MA). The TFF system continuously circulates the cell culture fluid through a hollow fiber module while collecting the permeate at a predetermined rate. Hollow fibers with a pore size of 0.2 µm (Pall, Port Washington, NY) were used as the cell retention device for this study. A TFF cross-flow rate of 10.5 L / min was used in this study. When the cell density reached [X] cells / mL or the capacitance target of 80 - 85 pF / cm, the cell bleed pump was activated to maintain the cell density or biovolume. (Capacitance, pF / cm cell bleed was automatically controlled by a capacitance probe (Incyte DN12, Hamilton Bonaduz AG, Switzerland)). The perfusion culture period was 28 days. Continuous harvest began 5 days after the perfusion rate of 2 vvd to the bioreactor was achieved. 6 When the cell density reached [X] cells / mL or the capacitance target of 80 - 85 pF / cm, the cell bleed pump was activated to maintain the cell density or biovolume. (Capacitance, pF / cm cell bleed was automatically controlled by a capacitance probe (Incyte DN12, Hamilton Bonaduz AG, Switzerland)). The perfusion culture period was 28 days. Continuous harvest began 5 days after the perfusion rate of 2 vvd to the bioreactor was achieved.

[0303] Note: The [X] in the translation represents the placeholder in the original text which is not fully provided. You may need to fill in the correct value according to the complete original text.Samples were collected daily from the bioreactor and the permeate line. Viable cell density (VCD) and viability were measured using the trypan blue exclusion method with a Cedex Hi-Res cell counter (Roche Diagnostics GmbH, Mannheim, Germany). Cell diameter was measured and reported using the same cell counter. Offline pH, pO2, and pCO2 were measured using an ABL80 blood gas analyzer (Radiometer, Denmark). Glucose, lactate, ammonium, and lactate dehydrogenase (LDH) were measured using an RX Daytona+ or Imola analyzer (Randox Laboratories, Ltd., Crumlin, UK). Bioreactor supernatant and permeate antibody titers were analyzed using an Agilent 1100 high performance liquid chromatography (HPLC) equipped with a Protein A column (Agilent Technologies, Santa Clara, CA).

[0304] [Example 2] Downstream purification Protein A affinity chromatography Protein A affinity chromatography functions as a primary capture step using MabSelect SuRe resin from GE Healthcare (trademark) for the majority of the purification. The product binds to the resin, while impurities such as media components and host cell proteins do not bind and remain in the column flow-through fraction. The Protein A step is operated continuously using a continuous multi-column chromatography skid, enabling a continuous flow of HCCF from the bioreactor to be fed to the skid (BioSMB PD, Sartorius Stedim Biotech GmbH Goettingen, Germany). This is achieved by utilizing four equivalent columns filled with the same Protein A resin on the skid. At any given point in the Protein A process, while three columns are dedicated to loading, the fourth is undergoing a non-loading process (equilibration, washing, elution, regeneration).

[0305] The HCCF from the bioreactor is connected to a continuous multi-column chromatography skid via a 3 L single-use surge tank (Cercell, Herlev, Denmark). The HCCF flow rate to the single-use surge tank matches the feed rate from the surge tank to the continuous multi-column chromatography system, maintaining a constant volume in the tank. The operating volume range in the surge tank provided an average residence time of 15 - 45 minutes of the fluid in the tank with a target value of 30 minutes. As shown in Figure 5, due to oxidative instability, the residence time of HCCF loading onto the Protein A column is optimal at less than 24 hours.

[0306] Operationally, the same steps as in single-column batch mode chromatography, including steps for column equilibration, loading, washing, and regeneration, are performed in multi-column Protein A chromatography. After equilibrating the column with 10 mM sodium phosphate, pH 6.5, the loading step was initiated. The protein was loaded to a capacity of 50 g / L of Protein A resin. After the loading step of the Protein A process, three washing steps were performed with 10 mM sodium phosphate, pH 6.5, and 10 mM sodium phosphate and 0.5 M sodium chloride to elute loosely bound impurities and increase the purity of the antibody in the product stream. The product was eluted at 20 mM sodium acetate pH 3.6 via a low pH shift and monitored by on-line spectrophotometry at an absorbance of 280 nm. The processes in the surge tank and the Protein A process were carried out at ambient temperature (20 ± 5 °C).

[0307] After Protein A chromatography, the protein was processed through additional purification and filtration steps to achieve an ultrafiltration product in a continuous process connected via an intermediate surge tank that included virus inactivation, depth filtration, anion exchange chromatography, virus filtration, single-pass tangential flow filtration, and in-line diafiltration.

[0308] Virus inactivation / Depth filtration The Protein A affinity pool was adjusted to low pH (target pH 3.6) with 1 M acetic acid to inactivate any potential viruses. This step was carried out at ambient temperature (20 ± 5 °C). After virus inactivation, the pool was adjusted to the target pH 5.5 with 1 M Tris and filtered through a charged depth filter (A1HC) and a 0.22 μm filter for clarification (Millipore Sigma, Burlington MA). The filtered and neutralized virus-inactivated product (FNVIP) can be stored at 2 - 8 °C prior to further processing in the subsequent anion exchange chromatography step.

[0309] Anion exchange Anion exchange chromatography (AEX) using POROS HQ 50 from Applied Biosystems is the first polishing step in this process. The pembrolizumab antibody flows through the column, while potential residual impurities such as host cell proteins (HCP), DNA, aggregates, and viruses bind to the resin. The column was equilibrated and washed with 25 mM sodium phosphate pH 7.2. Prior to the start of the process, the pH of the post-virus inactivation pool was adjusted to the target pH 7.2 using 1 M Tris. The UV absorbance of the column effluent was monitored online at a wavelength of 280 nm and used to collect the AEX pool. The pH of the pool was adjusted to pH 5.5 using 1 M acetic acid to continue processing in the post-process surge tank.

[0310] Virus filtration The AEX product was filtered through a 0.1 μm prefilter or equivalent in line with a Planova 20N (mean pore size 19 nm) virus removal filter (Asahi Kasei Bioprocess, Glenview, IL). The prefilter and filter connections were autoclaved and aligned in line with the nanofilter in a biosafety cabinet. The filter was then continuously flushed in a closed mode with 10 mM histidine, 10 mM methionine, pH 5.4.

[0311] Ultrafiltration concentration and diafiltration The virus filtration product was first concentrated 8-fold by a single-pass tangential flow filtration (SPTFF) module (Pall Corporation, Westboro, MA, Port Washington, NY) using a 30 kDa molecular weight cut-off. The product from the SPTFF step was then diafiltered at 10 mM histidine, 10 mM methionine, pH 5.4, resulting in buffer exchange of the concentrated protein stream. Buffer exchange was performed using a six-stage in-line diafiltration (ILDF) module (Pall Corporation, Westboro, MA, Port Washington, NY) containing a 30 kDa MW cut-off membrane. The diafiltration product, also referred to as the ultrafiltration product, was further filtered through a sterile filter and collected in a storage vessel. Once a sufficient mass of the ultrafiltration product had accumulated, the ultrafiltration products were pooled and further concentrated to a final concentration of 190 - 200 g / L by a standard batch ultrafiltration process using an Ultracel 30 kDa molecular weight cut-off membrane (Millipore, Burlington, MA). Stock excipients were added to achieve a final drug substance formulation of 165 mg / mL in 10 mM histidine, 10 mM methionine, 7% (w / v) sucrose and 0.02% (w / v) PS-80 buffer pH 5.5.

[0312] [Example 3] Method for determining the M105 oxidation level Reductive peptide mapping, liquid chromatography and mass spectrometry Sample preparation In the project or the sample of the drug substance (DS) was diluted with water to 5 mg / mL. A total of 20 μL of the diluted sample (containing 100 μg) was denatured and reduced in a final solution (100 μL) containing 6 M guanidine-HCl, 50 μM Tris-HCl, 50 μM EDTA and 200 μM DTT. The mixed sample was incubated in a thermomixer at 37 °C for 30 minutes while shaking at 300 rpm. After mixing and spin-down, each sample was alkylated with 5 μL of iodoacetamide (IAM) (1 M) protected from light at 25 °C for 30 minutes. A total of 5 μL of DTT (200 μM) was added to block unreacted iodoacetamide (IAM). 500 μL of lysyl endopeptidase (Lys-C) (Wako, 125-05061) enzyme (1:10 (wt:wt)) was added to the protein sample and mixed well by slowly pipetting up and down 3 times. The digest was incubated in a thermomixer at 37 °C for 60 minutes. The digest was quenched with 15 μL of 20% TFA. The digested sample was analyzed by LC-MS within 24 hours after sample digestion. Separately, the digested sample was stored at -80 °C for future analysis.

[0313] LC-MS method and data analysis Using Waters Acquity liquid chromatography, 20 μL of the sample was injected into a column (UPLC HSS T3 100 Å, 1.8 μm, 2.1 mm × 150 mm, P / N: 186003540). The autosampler was set at 5 °C. Mobile phase A was 0.02% TFA in water, mobile phase B was 0.02% TFA in acetonitrile, and the gradient was 0.1% B from 0 to 5 minutes, 0.1% - 10% B from 5 to 7 minutes, and then a linear increase to 35% B over the next 38 minutes. MS1 data was collected using a Q Exactive Orbitrap MS (Thermo). Chromeleon and Xcalibur were used for data analysis. Using the extracted ion chromatograms (EIC) of M105 unmodified and modified peptides (two charge states (+3 and +4) and three isotope ions each for each peptide), the M105 oxidation % was determined using the formula [peak area of the extracted ion chromatogram (EIC) of the modified peptide] / [peak area of the EIC of the modified peptide + peak area of the EIC of the unmodified peptide] × 100. See Figures 2 - 4. The %CV (coefficient of variation, calculated as (standard deviation / mean) × 100) of M105 oxidation was less than 20%.

Table 4

[0314] The formulation of the fed-batch manufactured pembrolizumab pharmaceutical reference standard is 25 mg / mL of pembrolizumab in 10 mM histidine buffer, pH 5.5, containing 7% (w / v) sucrose and 0.02% (w / v) polysorbate 80. The formulation of the pembrolizumab formulated drug substance manufactured by continuous perfusion is 165 mg / mL of pembrolizumab, 10 mM L-histidine, 7% (w / v) sucrose, 0.02% (w / v) PS80, and 10 mM L-methionine, pH 5.5.

[0315] The oxidation %M105 of the fed-batch manufactured pembrolizumab formulated drug reference standard was approximately 4.9%, while the pembrolizumab samples manufactured by continuous perfusion contained 0.5% - 3.0% of oxidized M105. Some representative samples from Protein A purification, ultrafiltration product (non-formulated DS), and formulated DS are shown in Table 3.

[0316] Hydrophobic interaction chromatography (HP-HIC) For the measurement of Met105 oxidation %, high-performance hydrophobic interaction chromatography (HP-HIC) was used to separate and quantify the oxidized product from the non-oxidized molecules. Pre-peak 3 contains antibodies with Met105 oxidation in both heavy chains, and Pre-peaks 1 and 2 contain antibodies with Met105 oxidation in either of the heavy chains (Figure 13). The percentages of the pre-peaks, as well as the main and post-peak percentages, were determined. The HP-HIC method was carried out by diluting the sample to 5.0 mg / mL in purified water. The sample was then injected (10 μL) into an HPLC equipped with a Tosoh Phenyl-5PW column at 30 °C and a UV detector at 280 nm. For HIC analysis, a mobile phase containing the following components (Mobile phase A: 5 mM sodium phosphate in 2% acetonitrile, pH 7.0; Mobile phase B: 400 mM ammonium sulfate, 5 mM sodium phosphate in 2% acetonitrile, pH 6.9;) was used. The gradient was 0% mobile phase A from 0 to 2 minutes, 0% - 100% mobile phase A from 2 to 52 minutes, after which the column was washed and equilibrated. The pembrolizumab reference manufactured by a fed-batch process, which had undergone batch Protein A, virus inactivation, depth filtration, and AEX purification, was tested for Met105 oxidation %. Representative values of Met105 oxidation % of the unfiltered samples after virus inactivation and AEX purification of the pembrolizumab reference were approximately 5.5%.

[0317] [Example 4] Measurement of the change in M105 oxidation of pembrolizumab over time in HCCF The oxidation rate was determined by repeatedly measuring the oxidation amount of the HCCF sample according to Example 1 using ProA-HIC 2D-LC described below Example 3.

[0318] Samples were taken from the bioreactor, filtered to remove cells, and HCCF was obtained. Under sterile conditions, the HCCF sample was aliquoted into 200 μL into 48 wells of a 96-well plate. The plate was covered with an aluminum foil lid to maintain sterility and shield the sample from light. With the internal lighting turned off, the sample was immediately placed in an Agilent 1290 autosampler set at 25 °C. Each well of the sample was sequentially analyzed over time by two-dimensional liquid chromatography separation.

[0319] The chromatograms of each obtained sample were integrated to determine the percentage of oxidation, and a summary of the data is shown in Figure 5. The linear regression of the data shows a percentage of oxidation starting at 0.94% and increasing to a maximum of 1.95% at approximately 0.038% per hour over the next 26 hours.

[0320] [Example 5] Ion exchange (IEX) method for measuring acidic species of anti-PD-1 antibody In the IEX method, a Waters Alliance LC system (Milford, MA, USA) was used, and ProPac WCX-10 from Thermo Scientific (p / n: 054993, particle size 10 μm, diameter 4 mm, length 250 mm) was selected with a loading of 80 μg of sample. Mobile phase (A) 24 mM MES pH 6.1 containing 4% acetonitrile, and mobile phase (B) 20 mM sodium phosphate, 95 mM NaCl pH 8.0 containing 4% acetonitrile were used as a non-linear S-shaped, pH gradient, and the separation was 0.5 mL min -1The column temperature was 35°C and monitored over 34 min at a flow rate of 22% B. The gradient used was 22% to 22% B from 0.6 min to 15.0 min, 22% to 29% B from 0.6 min to 15.0 min, 29% to 70% B from 15.0 min to 30.0 min, 70% to 100% B from 30.0 min to 30.5 min, and 100% to 100% B from 30.5 min to 33.0 min. The mobile phase (C) was 10 mM CHES pH 8.0, 40 mM Tris, 15 mM EDTA, 200 mM NaCl, and 4% acetonitrile, with a flow rate of 0.5 mL min from 33.1 to 34.0 min. -1 Strip the column with 1.0 mL min -1 The column was re-equilibrated at 22% B for 34.5–44.5 min at a flow rate of 0.5 mL min−1. -1 The elution was monitored at 280 nm for peak detection. Assay variation was determined to be within 1%.

[0321] The identified chemical composition of each peak of the pembrolizumab drug substance reference sample (manufactured by a fed-batch process) was determined by collecting samples of each peak and performing peptide mapping and reverse-phase liquid chromatography, followed by analysis by mass spectrometry and MS / MS using the same methods as in Example 3. The main peak was determined to primarily contain antibodies having the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:11 for both the light and heavy chains, respectively. Oxidation (e.g., methionine 105) and deamidation of asparagine residues of the aforementioned antibodies (e.g., N31, N52, N55, N59, or N61 in the heavy chain of SEQ ID NO:11) were detected in the acidic variant peak. Deamidation of asparagine residues of the aforementioned antibodies (e.g., N384, N389, or N390 in the heavy chain of SEQ ID NO:11) was detected in the acidic 1 peak. In the basic 1 peak, antibodies were detected that contained one heavy chain consisting of the amino acid sequence of SEQ ID NO: 11, one heavy chain consisting of the amino acid sequence of SEQ ID NO: 12, and two light chains consisting of the amino acid sequence of SEQ ID NO: 5, or one heavy chain consisting of the amino acid sequence of SEQ ID NO: 11, one heavy chain consisting of the amino acid sequence of SEQ ID NO: 14 in which the C-terminal leucine is alpha-amidated, and two light chains consisting of the amino acid sequence of SEQ ID NO: 5.

[0322] Ion exchange chromatograms and percentages of acidic species, main peaks, and basic species from the samples after protein A chromatography (PAP) and anion exchange chromatography (AEXP) according to the procedures of Examples 1-2 are shown in FIGS. 6-7 and Table 4. The total of acidic species (acidic variants, acid 1, and pre-main) for the PAP sample and the AEXP sample are 7.86% and 8.76%, respectively. The total of basic species (basic 1, basic variant A, basic 2, basic variant B) for the PAP sample and the AEXP sample are 16.95% and 18.19%, respectively. Ion exchange chromatograms and percentages of acidic species, main peaks, and basic species from the pembrolizumab reference obtained by the flow addition method are shown in FIGS. 8 and Table 5. The total of acidic species (acidic variants, acid 1, and pre-main) for the reference sample is 16.56%. The total of basic species (basic 1, basic variant A, basic 2, basic variant B) for the reference sample is 23.76%. The total of basic variants (basic variant A and basic variant B) for the reference sample is 6.23%. In summary, the pembrolizumab sample prepared by the continuous perfusion process of the present invention had a high proportion of the main species because the proportion of acidic species in the mixture was low. In other batches of pembrolizumab samples prepared by a continuous perfusion process substantially similar to Examples 1-2, the main peak was about 74-80%. [Table 5]

[0323] [Table 6]

[0324] FIGS. 9-12 also provide the time courses of total acidic species %, main species %, total basic species %, and basic 1 species % of the cell-free permeate (PERM) prepared according to the procedures of Examples 1-2 after the protein A chromatography step (PAP) and after the anion exchange chromatography (AEXP) as a function of the number of culture days.

[0325] [Example 6] Hyaluronidase Activity Assay Enzyme activity was determined by turbidity assay using a Molecular Devices SpectraMax M5e microplate reader. Calibration standards and test samples were prepared by diluting them to the working concentrations outlined in Table 6 with a cooled enzyme diluent (20 mM sodium phosphate, 77 mM sodium chloride, 0.01% bovine serum albumin (BSA), pH 7.0, 25 °C). [Table 7]

[0326] Fifty microliters of the dilution standards and samples were transferred in triplicate to a clear-bottom 96-well plate (Plate 1). As a blank control, 50 μL of the enzyme diluent solution was added to a dedicated well on the plate. The plate was sealed and incubated at 37 °C for 10 minutes. After incubation, 50 μL of a hyaluronic acid solution (0.06% hyaluronic acid, 300 mM phosphate, pH 5.35, 37 °C) at 37 °C was added to each well containing the solution using a multichannel pipette. The plate was sealed and then incubated at 37 °C for exactly 45 minutes while shaking at 600 rpm. Before removing Plate 1 from the incubation, a second plate (Plate 2) was prepared with 200 μL of an acidic albumin solution (24 mM sodium acetate, 79 mM acetic acid, 0.1% BSA, pH 3.75, 25 °C) in each well to create the same well layout as Plate 1. After the exact 45-minute incubation of Plate 1, 40 μL of the solution was removed from each well using a multichannel pipette and added to the corresponding well of Plate 2 (containing the acidic albumin solution). Plate 2 was incubated at 25 °C for 20 minutes in the plate chamber of the microplate reader. The microplate reader was set to read the absorbance at 600 nm 5 seconds after shaking. After the 20-minute incubation, the absorbance at 600 nm was read for each well.

[0327] Calibration curve generation The absorbance values at three points from the calibration standard were averaged and subtracted by the average absorbance of the blank. The absolute value of the obtained corrected absorbance was plotted against the volume measurement activity value of the calibration standard (e.g., 15, 12, 10, 7.5, 5, 2 units / mL). The plot was fitted to a first-order polynomial (Figure 14), and the obtained equation was used to determine the enzyme activity of the test sample.

[0328] Calculation of the enzyme activity of the test sample The absorbance values at three points were averaged and subtracted by the average absorbance of the blank. The absolute value of the obtained corrected absorbance was input into the fitting equation from the calibration curve to determine the calculated activity of the test sample. The corrected absorbance values outside the calibration curve were discarded. The value was corrected for dilution by multiplying the assay volume measurement activity by the dilution factor used to prepare the solution. For example, if the estimated enzyme activity of the formulation or standard stock solution was 1500 units / mL and it was diluted to 12, 10, and 7.5 units / mL for analysis, the dilution factors were 125, 150, and 200, respectively. The finally calculated enzyme activity values were averaged and reported in units / mL.

[0329] [Example 7] Evaluation of the stability of rHuPH20 variant 1 containing pembrolizumab CPP after heat stress Co-formulated samples containing pembrolizumab and rHuPH20 variant 1 produced by a continuous perfusion process (CPP) were prepared at the concentrations shown in Table 7. All formulations contained 7% w / v sucrose, 0.02% w / v PS-80, 10 mM methionine in 10 mM histidine buffer at pH 5.5 and were filled into 2R vials with a fill volume of 1.6 mL. [Table 8]

[0330] After incubating each sample at 35 °C for 1 week, the enzyme activity was measured. Figure 15 shows the activity data of the heat stress samples normalized by the activity values observed for the control (initial) samples. Surprisingly, the data show that in pembrolizumab CPP at concentrations of 5 - 165 mg / mL, the enzyme activity and stability of rHuPH20 variant 1 after heat stress in the presence of pembrolizumab CPP are enhanced compared to rHuPH20 variant 1 alone. The retention of enzyme activity against heat stress shows a dependence on the pembrolizumab CPP concentration. The data indicate that at concentrations above 50 mg / mL pembrolizumab CPP, the enhancement of rHuPH20 variant 1 enzyme activity was unexpectedly higher than the enzyme activity observed at lower concentrations of pembrolizumab CPP. Furthermore, at concentrations of pembrolizumab CPP above 100 mg / mL, no effect on rHuPH20 variant 1 enzyme activity was observed after heat stress compared to the control samples.

[0331] [Example 8] Evaluation of the stability of recombinant human hyaluronidase PH20 variant 1 containing pembrolizumab CPP after stainless steel exposure Test formulations of rHuPH20 variant 1 co-formulated with pembrolizumab CPP were prepared in polyethylene terephthalate copolyester glycol-modified (PETG) bottles (125 mL) having the composition outlined in Table 8. Each formulation was exposed to an SS solid cylinder at 25 °C (protected from light) for 72 hours. All formulations were filtered using a 0.22 μm PES filter. Control samples were prepared similarly in the absence of stainless steel exposure. All samples were stepwise fractionated for stability (protected from light) at 25 °C for 4 weeks to evaluate rHuPH20 variant 1 activity. [Table 9]

[0332] Figure 16 shows enzyme activity data normalized to the activity value of the non-exposed sample for samples exposed to stainless steel. Interestingly, the enzyme activity of rHuPH20 variant 1 co-formulated with pembrolizumab CPP showed enhanced activity and stability after storage at 25°C for 4 weeks after exposure to stainless steel, compared to rHuPH20 variant 1 alone. After storage at 25°C for 4 weeks, samples of the enzyme alone exposed to stainless steel showed a decrease in activity compared to the control sample. The enzyme activity of the samples co-formulated with pembrolizumab CPP was not affected after storage, indicating enhanced enzyme stability and activity in the presence of pembrolizumab CPP.

[0333] [Example 9] Evaluation of the effect of sucrose (a heat stabilizer) in a co-formulation of pembrolizumab reference and rHuPH20 variant 1 Co-formulations of pembrolizumab reference and rHuPH20 variant 1 (165 mg / mL pembrolizumab, 2000 units / mL rHuPH20 variant 1 in 0.2 mg / mL of PS-80, 10 mM methionine in 10 mM histidine buffer at pH 5.5) were prepared with or without 7% sucrose. Both formulations were filled into 2R glass vials with a fill volume of 1.6 mL. The samples were heat stressed at 35°C for up to 2 weeks. [Table 10]

[0334] Stressed samples were analyzed by microflow imaging (MFI) to determine the effect of the heat stabilizer on the subvisible particle population. Table 10 shows the concentration (number of particles / mL) of particles greater than 2 μm. The data indicate that the co-formulation prepared without the heat stabilizer had an increase in the number of particles after incubation at 35°C for 2 weeks compared to the co-formulation prepared with the heat stabilizer. [Table 11]

[0335] All references cited in this specification are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent were specifically and individually indicated to be incorporated by reference, even if such citation is not directly adjacent to a dedicated statement of incorporation by reference, provided that each is clearly identified in accordance with 37 C.F.R.§1.57(b)(2). Inclusion of a dedicated statement of incorporation by reference, if any, in this specification does not in any way weaken this general description of incorporation by reference. Citation of a reference in this specification is not intended as an admission that the reference is appropriate prior art, nor does it constitute any admission as to the content or date of these publications or documents. To the extent that a cited reference provides a definition of a term that is inconsistent with the definition provided in this specification, the definition provided in this specification shall be used to construe the claimed invention.

Claims

1. A pharmaceutical composition comprising an anti-human PD-1 antibody comprising a light chain variable region containing three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a heavy chain variable region containing three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8, wherein the oxidation is about 0.1% to 3.0% of methionine 105, rHuPH20 or a variant or fragment thereof, amino acid residues 36 to 464, 36 to 465, 36 to 466, 36 to 467, 36 to 468, 36 to 469, 36 to 470, 36 to 471, 36 to 472, 36 to 473, 36 to 474, 36 to 475, 36 to 476, 36 to 477, 3 , 38 to 464, 38 to 465, 38 to 466, 38 to 467, 38 to 468, 38 to 469, 38 to 470, 38 to 471, 38 to 472, 38 to 473, 38 to 474, 38 to 475, 38 to 476, 38 to 477, 38 to 478, 38 to 479, 38 to 480, 38 to 481, 38 to 482, 38 to 483, 39 to 464, 39 to 465, 39 to 466, 39 to 467, 39 to 468, 39 to 469, 39 to 470, 39 to 471, 39 to 472, 39 to 473, 39 to 474, 39 to 475, 39 to 476, 39 to 477, 39 to 478, 39 to 479, 39 to 480, 39 to 481, 39 to 482, 39 to 483, 40 to 464, 40 to 465, 40 to 466, 40 to 467, 40 to 468, 40 to 469, 40 to 470, 40 to 471, 40 to 47 , 41 to 464, 41 to 465, 41 to 466, 41 to 467, 41 to 468, 41 to 469, 41 to 470, 41 to 471, 41 to 472, 41 to 473, 41 to 474, 41 to 475, 41 to 476,A pharmaceutical composition comprising rHuPH20 or a variant or fragment thereof that is 41-477, 41-478, 41-479, 41-480, 41-481, 41-482, 41-483, 42-464, 42-465, 42-466, 42-467, 42-468, 42-469, 42-470, 42-471, 42-472, 42-473, 42-474, 42-475, 42-476, 42-477, 42-478, 42-479, 42-480, 42-481, 42-482, or 42-483, and a pharmaceutically acceptable carrier.,

2. The pharmaceutical composition according to claim 1, wherein the anti-human PD-1 antibody comprises a light chain variable region containing the amino acid sequence shown in SEQ ID NO: 4 and a heavy chain variable region containing the amino acid sequence shown in SEQ ID NO:

9.

3. The pharmaceutical composition according to claim 1, wherein the anti-human PD-1 antibody consists of two light chains and two heavy chains, the two light chains consist of the amino acid sequence shown in SEQ ID NO: 5, and the two heavy chains consist of the amino acid sequence shown in any one of SEQ ID NOs: 10 to 15, or a combination thereof.

4. The pharmaceutical composition according to claim 1, wherein the anti-human PD-1 antibody consists of two light chains and two heavy chains, in a part of the anti-PD-1 antibody, the two light chains consist of the amino acid sequence shown in SEQ ID NO: 5, and the two heavy chains consist of the amino acid sequence shown in SEQ ID NO:

11.

5. The pharmaceutical composition according to claim 1, wherein the antibody is a pembrolizumab variant.

6. The pharmaceutical composition according to claim 4, wherein the oxidation of methionine 105 is 0.1 to 3.0%.

7. The pharmaceutical composition according to any one of claims 1 to 5, wherein the oxidation of methionine 105 is 0.2 to 3.0%.

8. The pharmaceutical composition according to any one of claims 1 to 5, wherein the oxidation of methionine 105 is 0.5 to 3.0%.

9. A main species comprising an antibody consisting of two heavy chains and two light chains, wherein each light chain comprises a variable light region comprising three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2 and CDRL3 of SEQ ID NO: 3, and each heavy chain comprises a variable heavy region comprising three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7 and CDRH3 of SEQ ID NO: 8, and an acidic species of the main species, and the amount of the acidic species is 1.0 to 12.0%. An anti-human PD-1 antibody, rHuPH20 or a variant or fragment thereof, which is amino acid residues 36 to 464, 36 to 465, 36 to 466, 36 to 467, 36 to 468, 36 to 469, 36 to 470, 36 to 471, 36 to 472, 36 to 473, 36 to 474, 36 to 475, 36 to 476, 36 to 477, 36 to 478, 36 to 479, 36 to 480, 36 to 481, 36 to 482, 36 to 483, 37 to 464, 37 to 465, 37 to 466, 37 to 467, 37 to 468, 37 to 469, 37 to 470, 37 to 471, 37 to 472, 37 to 473, 37 to 474, 37 to 475, 37 to 476, 37 to 477, 37 to 478, 37 to 479, 37 to 480, 37 to 481, 37 to 482, 37 to 483, 38 to 464, 38 to 465, 38 to 466, 38 to 467, 38 to 468, 38 to 469, 38 to 470, 38 to 471, 38 to 472, 38 to 473, 38 to 474, 38 to 475, 38 to 476, 38 to 477, 38 to 478, 38 to 479, 38 to 480, 38 to 481, 38 to 482, 38 to 483, 39 to 464, 39 to 465, 39 to 466, 39 to 467, 39 to 468, 39 to 469, 39 to 470, 39 to 471, 39 to 472, 39 to 473, 39 to 474, 39 to 475, 39 to 476, 39 to 477, 39 to 478, 39 to 479, 39 to 480, 39 to 481, 39 to 482, 39 to 483, 40 to 464, 40 to 465, 40 to 466, 40 to 467, 40 to 468, 40 to 469, 40 to 470, 40 to 471, 40 to 472, 40 to 473, 40 to 474, 40 to 475, 40 to 476, 40 to 477, 40 to 478, 40 to 479, 40 to 480, 40 to 481, 40 to 482, 40 to 483, 41 to 464, 41 to 465, 41 to 466, 41 to 467, 41 to 468, 41 to 469,A pharmaceutical composition comprising rHuPH20 or a variant or fragment thereof, which is 41 to 470, 41 to 471, 41 to 472, 41 to 473, 41 to 474, 41 to 475, 41 to 476, 41 to 477, 41 to 478, 41 to 479, 41 to 480, 41 to 481, 41 to 482, 41 to 483, 42 to 464, 42 to 465, 42 to 466, 42 to 467, 42 to 468, 42 to 469, 42 to 470, 42 to 471, 42 to 472, 42 to 473, 42 to 474, 42 to 475, 42 to 476, 42 to 477, 42 to 478, 42 to 479, 42 to 480, 42 to 481, 42 to 482, or 42 to 483, and a pharmaceutically acceptable carrier.,

10. A main species comprising an antibody consisting of two heavy chains and two light chains, wherein each light chain comprises a light chain variable region comprising three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2 and CDRL3 of SEQ ID NO: 3, and each heavy chain comprises a heavy chain variable region comprising three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7 and CDRH3 of SEQ ID NO: 8, and an acidic species and a basic species of the main species, and the amount of the main species is 65 to 95%, an anti-human PD-1 antibody, rHuPH20 or a variant or fragment thereof, amino acid residues 36 to 464, 36 to 465, 36 to 466, 36 to 467, 36 to 468, 36 to 469, 36 to 470, 36 to 471, 36 to 472, 36 to 473, 36 to 474, 36 to 475, 36 to 476, 36 to 477, 36 to 478, 36 to 479, 36 to 480, 36 to 481, 36 to 482, 36 to 483, 37 to 464, 37 to 465, 37 to 466, 37 to 467, 37 to 468, 37 to 469, 37 to 470, 37 to 471, 37 to 472, 37 to 473, 37 to 474, 37 to 475, 37 to 476, 37 to 477, 37 to 478, 37 to 479, 37 to 480, 37 to 481, 37 to 482, 37 to 483, 38 to 464, 38 to 465, 38 to 466, 38 to 467, 38 to 468, 38 to 469, 38 to 470, 38 to 471, 38 to 472, 38 to 473, 38 to 474, 38 to 475, 38 to 476, 38 to 477, 38 to 478, 38 to 479, 38 to 480, 38 to 481, 38 to 482, 38 to 483, 39 to 464, 39 to 465, 39 to 466, 39 to 467, 39 to 468, 39 to 469, 39 to 470, 39 to 471, 39 to 472, 39 to 473, 39 to 474, 39 to 475, 39 to 476, 39 to 477, 39 to 478, 39 to 479, 39 to 480, 39 to 481, 39 to 482, 39 to 483, 40 to 464, 40 to 465, 40 to 466, 40 to 467, 40 to 468, 40 to 469, 40 to 470, 40 to 471, 40 to 472, 40 to 473, 40 to 474, 40 to 475, 40 to 476, 40 to 477, 40 to 478, 40 to 479, 40 to 480, 40 to 481, 40 to 482, 40 to 483, 41 to 464, 41 to 465, 41 to 466, 41 to 467, 41 to 468,A pharmaceutical composition comprising rHuPH20 or a variant or fragment thereof which is 41 to 469, 41 to 470, 41 to 471, 41 to 472, 41 to 473, 41 to 474, 41 to 475, 41 to 476, 41 to 477, 41 to 478, 41 to 479, 41 to 480, 41 to 481, 41 to 482, 41 to 483, 42 to 464, 42 to 465, 42 to 466, 42 to 467, 42 to 468, 42 to 469, 42 to 470, 42 to 471, 42 to 472, 42 to 473, 42 to 474, 42 to 475, 42 to 476, 42 to 477, 42 to 478, 42 to 479, 42 to 480, 42 to 481, 42 to 482, or 42 to 483, and a pharmaceutically acceptable carrier.,

11. The pharmaceutical composition according to claim 9 or 10, wherein each heavy chain consists of the amino acid sequence of SEQ ID NO: 11 and each light chain consists of the amino acid sequence of SEQ ID NO:

5.

12. A main species prepared from Chinese hamster ovary cells, comprising a polynucleotide encoding a light chain and a polynucleotide encoding a heavy chain, or a polynucleotide encoding a light chain and a heavy chain, wherein the heavy chain consists of the amino acid sequence of SEQ ID NO: 10, 13 or 15, and the light chain consists of the amino acid sequence of SEQ ID NO: 5, and an acidic species of the main species, and an anti-human PD-1 antibody, rHuPH20 or a variant or fragment thereof, wherein the amount of the acidic species is 1.0 to 12.0%, and the amino acid residues 36 to 464, 36 to 465, 36 to 466, 36 to 467, 36 to 468, 36 to 469, 36 to 470, 36 to 471, 36 to 472, 36 to 473, 36 to 474, 36 to 475, 36 to 476, 36 to 477, 36 to 478, 36 to 479, 36 to 480, 36 to 481, 36 to 482, 36 to 483, 37 to 464, 37 to 465, 37 to 466, 37 to 467, 37 to 468, 37 to 469, 37 to 470, 37 to 471, 37 to 472, 37 to 473, 37 to 474, 37 to 475, 37 to 476, 37 to 477, 37 to 478, 37 to 479, 37 to 480, 37 to 481, 37 to 482, 37 to 483, 38 to 464, 38 to 465, 38 to 466, 38 to 467, 38 to 468, 38 to 469, 38 to 470, 38 to 471, 38 to 472, 38 to 473, 38 to 474, 38 to 475, 38 to 476, 38 to 477, 38 to 478, 38 to 479, 38 to 480, 38 to 481, 38 to 482, 38 to 483, 39 to 464, 39 to 465, 39 to 466, 39 to 467, 39 to 468, 39 to 469, 39 to 470, 39 to 471, 39 to 472, 39 to 473, 39 to 474, 39 to 475, 39 to 476, 39 to 477, 39 to 478, 39 to 479, 39 to 480, 39 to 481, 39 to 482, 39 to 483, 40 to 464, 40 to 465, 40 to 466, 40 to 467, 40 to 468, 40 to 469, 40 to 470, 40 to 471, 40 to 472, 40 to 473, 40 to 474, 40 to 475, 40 to 476, 40 to 477, 40 to 478, 40 to 479, 40 to 480, 40 to 481, 40 to 482, 40 to 483, 41 to 464, 41 to 465, 41 to 466, 41 to 467, 41 to 468, 41 to 469, 41 to 470, 41 to 471,A pharmaceutical composition comprising rHuPH20 or a variant or fragment thereof which is 41 to 472, 41 to 473, 41 to 474, 41 to 475, 41 to 476, 41 to 477, 41 to 478, 41 to 479, 41 to 480, 41 to 481, 41 to 482, 41 to 483, 42 to 464, 42 to 465, 42 to 466, 42 to 467, 42 to 468, 42 to 469, 42 to 470, 42 to 471, 42 to 472, 42 to 473, 42 to 474, 42 to 475, 42 to 476, 42 to 477, 42 to 478, 42 to 479, 42 to 480, 42 to 481, 42 to 482, or 42 to 483, and a pharmaceutically acceptable carrier.,

13. The pharmaceutical composition according to any one of claims 9 to 12, wherein the oxidation of methionine 105 is about 0.2 to 3.0%.

14. The pharmaceutical composition according to any one of claims 9 to 12, wherein the oxidation of methionine 105 is about 0.5 to 3.0%.

15. The pharmaceutical composition according to any one of claims 9 to 14, wherein the amount of the acidic species is about 6 to 10%.

16. The pharmaceutical composition according to any one of claims 9 to 15, which contains one acidic species and the amount of the one acidic species is about 1 to 4%.

17. The pharmaceutical composition according to any one of claims 9 to 16, which contains an acidic variant species and the amount of the acidic variant species is about 1 to 5%.

18. The pharmaceutical composition according to any one of claims 9 to 17, wherein the amount of the main species is about 65 to 85%.

19. The pharmaceutical composition according to any one of claims 9 to 17, wherein the amount of the main species is about 70 to 80%.

20. The pharmaceutical composition according to any one of claims 9 to 19, wherein the amount of the basic species is about 12 to 27%.

21. The pharmaceutical composition according to any one of claims 9 to 20, wherein the main species, acidic species, acidic 1 species, acidic variant species or basic species are identified by cation exchange chromatography, optionally followed by mass spectrometry.

22. The pharmaceutical composition according to claim 21, wherein a cation exchange column selected from the group consisting of ProPac WCX-10, Sepax Proteomix WCX-NP1.7, Thermo MabPac SCX-10G and Thermo MabPac SCX50G is used.

23. The pharmaceutical composition according to claim 21, wherein a weak cation exchange column using a carboxylate functional group is used.

24. The main species, acidic species, acidic 1 species, acidic variant or basic species are determined by applying a gradient of 22% to 22% B at 0 to 0.6 minutes, 22% to 29% B at 0.6 to 15.0 minutes, 29% to 70% B at 15.0 to 30.0 minutes, 70% to 100% B at 30.0 to 30.5 minutes, 100% to 100% B at 30.5 to 33.0 minutes using ProPac WCX-10 with mobile phase (A) 24 mM MES containing 4% acetonitrile, pH 6.1, and mobile phase (B) 95 mM NaCl containing 20 mM sodium phosphate, 4% acetonitrile, pH 8.0, column temperature 35°C, and the chromatogram is created using detection at 280 nm. The pharmaceutical composition according to claim 21.

25. The pharmaceutical composition according to any one of claims 1 to 8 and 13 to 24, wherein the methionine 105 oxidation % is measured by reduced peptide mapping, liquid chromatography and mass spectrometry.

26. The pharmaceutical composition according to any one of claims 1 to 8 and 13 to 24, wherein the oxidation percentage of the methionine 105 is measured by hydrophobic interaction chromatography (HIC) using a Tosoh Phenyl-5PW column and a mobile phase containing the following components (mobile phase A: 5 mM sodium phosphate in 2% acetonitrile, pH 7.0; mobile phase B: 400 mM ammonium sulfate, 5 mM sodium phosphate in 2% acetonitrile, pH 6.9), with a gradient where mobile phase A is 0% from 0 to 2 minutes, mobile phase A is 0 to 100% from 2 to 52 minutes, and a detection wavelength of 280 nm.

27. The pharmaceutical composition according to any one of claims 1 to 26, comprising about 200 to 800 mg of the anti-human PD-1 antibody.

28. a) about 5 mg / mL to about 175 mg / mL of the anti-human PD-1 antibody according to any one of claims 1 to 26, b) about 150 U / ml to 8000 U / ml of the rHuPH20 or its variant or fragment, c) a buffer of about 5 mM to about 20 mM at a pH of about 5.0 to 6.0, d) a non-reducing disaccharide of about 3% to about 10% weight / volume (w / v) selected from the group consisting of sucrose and trehalose, e) optionally, a non-ionic surfactant of about 0.005% to about 0.10% (w / v), and f) optionally, an antioxidant of about 1 mM to about 30 mM The pharmaceutical composition according to any one of claims 1 to 26, comprising.

29. The pharmaceutical composition according to claim 28, having a pH of about 5.2 to about 5.

8.

30. The pharmaceutical composition according to any one of claims 28 to 29, wherein the buffer is a histidine buffer.

31. The pharmaceutical composition according to any one of claims 28 to 30, wherein the buffer is a histidine buffer at a pH of about 5.5 and is present at a concentration of about 8 mM to about 12 mM.

32. The pharmaceutical composition according to any one of claims 28 to 31, wherein sucrose or trehalose is about 6% to about 8% weight / volume (w / v).

33. The pharmaceutical composition according to any one of claims 28 to 31, wherein the non-reducing disaccharide is sucrose present at about 7% w / v.

34. The pharmaceutical composition according to any one of claims 28 to 33, wherein the non-ionic surfactant is polysorbate 80, 60, 40 or 20.

35. The pharmaceutical composition according to claim 34, wherein the non-ionic surfactant is present at about 0.005 to 0.02% w / v.

36. The pharmaceutical composition according to claim 35, wherein the non-ionic surfactant is present at about 0.02% w / v.

37. The pharmaceutical composition according to any one of claims 28 to 36, wherein the antioxidant is L-methionine or a pharmaceutically acceptable salt thereof.

38. The pharmaceutical composition according to any one of claims 28 to 36, wherein the antioxidant is L-methionine or a pharmaceutically acceptable salt thereof and is present at a concentration of about 5 mM to about 20 mM.

39. The pharmaceutical composition according to any one of claims 28 to 38, wherein the concentration of the anti-human PD-1 antibody is about 5 mg / mL to about 165 mg / mL.

40. The pharmaceutical composition according to any one of claims 28 to 39, wherein the concentration of the anti-human PD-1 antibody is about 50 mg / mL to about 175 mg / mL.

41. The pharmaceutical composition according to any one of claims 28 to 40, wherein the concentration of the rHuPH20 or its variant or fragment is 2000 U / ml.

42. a) The anti-human PD-1 antibody of about 5 mg / mL to about 165 mg / mL b) rHuPH20 or its variant or fragment of about 2000 U / ml c) A histidine buffer of about 5 mM to about 20 mM with a pH of about 5.0 to about 6.0 d) Sucrose of about 6% to about 8% w / v, and f) Optionally, L-methionine of about 5 mM to about 20 mM, or a pharmaceutically acceptable salt thereof The pharmaceutical composition according to claim 28, comprising.

43. a) The anti-human PD-1 antibody of about 5 mg / mL to about 165 mg / mL b) rHuPH20 or its variant or fragment of about 2000 U / ml c) A histidine buffer of about 5 mM to about 20 mM with a pH of about 5.0 to about 6.0 d) Sucrose of about 6% to about 8% w / v e) Polysorbate 80 or 20 of about 0.01% to about 0.04% w / v, and f) Optionally, L-methionine of about 5 mM to about 20 mM, or a pharmaceutically acceptable salt thereof The pharmaceutical composition according to claim 28, comprising.

44. a) The anti-human PD-1 antibody of about 5 mg / mL to about 165 mg / mL b) rHuPH20 or its variant or fragment of about 2000 U / ml c) A histidine buffer of about 10 mM with a pH of about 5.5 d) about 7% w / v sucrose, and f) optionally, about 10 mM L-methionine, or a pharmaceutically acceptable salt thereof The pharmaceutical composition according to claim 28, comprising

45. a) about 5 mg / mL to about 165 mg / mL of said anti-human PD-1 antibody b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof c) about 10 mM histidine buffer at a pH of about 5.5 d) about 7% w / v sucrose e) about 0.02% w / v polysorbate 80, and f) optionally, about 10 mM L-methionine, or a pharmaceutically acceptable salt thereof The pharmaceutical composition according to claim 28, comprising

46. The pharmaceutical composition according to any one of claims 28 to 45, wherein the concentration of said anti-human PD-1 antibody is about 50 mg / mL to about 165 mg / mL.

47. The pharmaceutical composition according to any one of claims 28 to 45, wherein the concentration of said anti-human PD-1 antibody is about 100 mg / mL to about 165 mg / mL.

48. The pharmaceutical composition according to any one of claims 28 to 45, wherein the concentration of said anti-human PD-1 antibody is about 130 mg / mL to about 165 mg / mL.

49. The pharmaceutical composition according to any one of claims 28 to 45, wherein the concentration of said anti-human PD-1 antibody is about 130 mg / mL.

50. The pharmaceutical composition according to any one of claims 28 to 45, wherein the concentration of said anti-human PD-1 antibody is about 165 mg / mL.

51. The pharmaceutical composition according to any one of claims 1 to 50, wherein the pharmaceutical composition is for subcutaneous administration.

52. The pharmaceutical composition according to any one of claims 1 to 51, wherein said rHuPH20 or a variant or fragment thereof consists of amino acid residues 36 to 477, 36 to 478, 36 to 479, 36 to 480, 36 to 481, 36 to 482 or 36 to 483 of SEQ ID NO:

16.

53. The pharmaceutical composition according to any one of claims 1 to 51, comprising rHuPH20 of SEQ ID NO:

18.

54. The pharmaceutical composition according to any one of claims 1 to 51, comprising an rHuPH20 variant of SEQ ID NO:

17.

55. Said anti-human PD-1 antibody is a) perfusing Chinese hamster ovary cells in a cell culture medium in a perfusion bioreactor by applying a perfusion rate of at least about 0.5 to 6.0 vessel volumes per day (vvd) and maintaining a capacitance value of 70 to 90 pF / cm, wherein the host cell comprises a polynucleotide encoding the light chain variable region and a polynucleotide encoding the heavy chain variable region, or a polynucleotide encoding the light chain variable region and the heavy chain variable region, the step of perfusing; b) continuously recovering the antibody from the cell culture broth to obtain a recovered cell culture fluid; c) continuously purifying the recovered cell culture fluid in an affinity chromatography step to obtain a purified composition A pharmaceutical composition according to any one of claims 1 to 54, produced or obtainable by a method comprising.

56. In step a), the perfusion is started when the cell density reaches about 2 to 10×10 ６ cells / ml. The pharmaceutical composition according to claim 55.

57. The pharmaceutical composition according to any one of claims 55 to 56, wherein in step a), the perfusion rate is from about 0.5 vvd to 2 vvd.

58. The pharmaceutical composition according to any one of claims 55 to 57, wherein in step a), the perfusion rate is about 0.5 vvd on day 3, about 1 vvd on day 4 and about 2 vvd on day 5, and the purified composition is obtained from step c) after day 5.

59. The pharmaceutical composition according to any one of claims 55 to 58, wherein the copper concentration in the perfusion bioreactor is in the range of 1 to 35 ppb.

60. The pharmaceutical composition according to any one of claims 55 to 59, wherein the continuous recovery is performed by setting a constant permeation rate to obtain a cell-free permeate through a hollow fiber membrane connected to the perfusion bioreactor.

61. The pharmaceutical composition according to any one of claims 55 to 60, wherein the affinity chromatography step is protein A affinity chromatography operated in a continuous multi-column chromatography system and the loading residence time is within about 30 hours.

62. The pharmaceutical composition according to any one of claims 55 to 61, wherein the host cell comprises a polynucleotide encoding the light chain and a polynucleotide encoding the heavy chain, or a polynucleotide encoding the light chain and the heavy chain, the light chain consisting of the amino acid sequence shown in SEQ ID NO: 5, and the heavy chain consisting of the amino acid sequence shown in SEQ ID NO:

10.

63. a) An anti-human PD-1 antibody at about 5 mg / mL to about 165 mg / mL, comprising a light chain variable region containing three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a heavy chain variable region containing three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8; b) About 2000 U / ml of rHuPH20 or variant of SEQ ID NO: 17 or 18; c) A histidine buffer at about 5 mM to about 20 mM with a pH of about 5.0 to about 6.0; d) Sucrose at about 6% to about 8% w / v; f) Optionally, L-methionine at about 5 mM to about 20 mM, or a pharmaceutically acceptable salt thereof A pharmaceutical composition comprising the above.

64. a) The anti-human PD-1 antibody at about 5 mg / mL to about 165 mg / mL; b) The rHuPH20 or variant at about 2000 U / ml; c) A histidine buffer at about 5 mM to about 20 mM with a pH of about 5.0 to about 6.0; d) Sucrose at about 6% to about 8% w / v; e) Polysorbate 80 or 20 at about 0.01% to about 0.04% w / v, and f) Optionally, L-methionine at about 5 mM to about 20 mM, or a pharmaceutically acceptable salt thereof The pharmaceutical composition according to claim 63, comprising the above.

65. a) The anti-human PD-1 antibody at about 5 mg / mL to about 165 mg / mL; b) The rHuPH20 or variant at about 2000 U / ml; c) A histidine buffer at about 10 mM with a pH of about 5.5; d) Sucrose at about 7% w / v, and f) Optionally, L-methionine at about 10 mM, or a pharmaceutically acceptable salt thereof The pharmaceutical composition according to claim 63, comprising the above.

66. a) The anti-human PD-1 antibody at about 5 mg / mL to about 165 mg / mL; b) The rHuPH20 or variant at about 2000 U / ml; c) A histidine buffer at about 10 mM with a pH of about 5.5; d) Sucrose at about 7% w / v; e) Polysorbate 80 at about 0.02% w / v, and f) Optionally, L-methionine at about 10 mM, or a pharmaceutically acceptable salt thereof The pharmaceutical composition according to claim 63, comprising the above.

67. The pharmaceutical composition according to any one of claims 63 to 66, wherein the concentration of the anti-human PD-1 antibody is about 50 mg / mL to about 165 mg / mL.

68. a) An anti-human PD-1 antibody of about 50 mg / mL to about 165 mg / mL, comprising a light chain variable region comprising three light chain CDRs including CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and a heavy chain variable region comprising three heavy chain CDRs including CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO:

8. b) About 2000 U / ml of rHuPH20 or variant of SEQ ID NO: 17 or 18. c) A buffer with a pH of about 5.0 to about 6.

0. d) About 3% to about 10% w / v of a non-reducing disaccharide. e) About 0.005% to about 0.4% w / v of a non-ionic surfactant, and f) Optionally, about 5 mM to about 20 mM of L-methionine, or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising the above.

69. a), b), c) A histidine or acetate buffer with a pH of about 5.0 to about 6.

0. d) About 6% to about 8% w / v of sucrose or trehalose. e) Optionally, about 0.02% to about 0.2% w / v of a non-ionic surfactant, and f) Optionally, about 5 mM to about 20 mM of L-methionine, or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 68, comprising the above.

70. a), b), c) About 1 mM to about 30 mM of a histidine or acetate buffer with a pH of about 5.0 to about 6.

0. d) About 6% to about 8% w / v of sucrose or trehalose. e) Optionally, about 0.01% to about 0.05% w / v of polysorbate 80, and f) Optionally, about 5 mM to about 20 mM of L-methionine, or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 68, comprising the above.

71. a), b), c) About 1 mM to about 30 mM of a histidine or acetate buffer with a pH of about 5.0 to about 6.

0. d) About 6% to about 8% w / v of sucrose or trehalose. e) About 0.1% to about 0.4% w / v of a poloxamer. f) Optionally, about 5 mM to about 20 mM of L-methionine, or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 68, comprising the above.

72. a), b), c) About 1 mM to about 30 mM of a histidine or acetate buffer with a pH of about 5.0 to about 6.

0. d) About 6% to about 8% w / v of sucrose or trehalose. e) Optionally, 0.1% to about 0.2% w / v of poloxamer 188 or 407. f) Optionally, about 5 mM to about 20 mM of L-methionine, or a pharmaceutically acceptable salt thereof The pharmaceutical composition according to claim 68, comprising **Claim 73** The pharmaceutical composition according to any one of claims 63 to 72, wherein the concentration of the anti-human PD-1 antibody is about 75 mg / mL to about 165 mg / mL. **Claim 74** The pharmaceutical composition according to any one of claims 63 to 72, wherein the concentration of the anti-human PD-1 antibody is about 100 mg / mL to about 165 mg / mL. **Claim 75** The pharmaceutical composition according to any one of claims 63 to 72, wherein the concentration of the anti-human PD-1 antibody is about 130 mg / mL to about 165 mg / mL. **Claim 76** The pharmaceutical composition according to any one of claims 63 to 72, wherein the concentration of the anti-human PD-1 antibody is about 130 mg / mL. **Claim 77** The pharmaceutical composition according to any one of claims 63 to 72, wherein the concentration of the anti-human PD-1 antibody is about 165 mg / mL. **Claim 78** The pharmaceutical composition according to any one of claims 63 to 77, wherein the anti-human PD-1 antibody comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 4 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:

9. **Claim 79** The pharmaceutical composition according to any one of claims 63 to 77, wherein the anti-human PD-1 antibody consists of two light chains and two heavy chains, the two light chains consisting of the amino acid sequence shown in SEQ ID NO: 5, and the two heavy chains consisting of the amino acid sequence shown in any one of SEQ ID NOs: 10 to 15, or a combination thereof. **Claim 80** The pharmaceutical composition according to any one of claims 1 to 79, which is a liquid. **Claim 81** The pharmaceutical composition according to any one of claims 1 to 79, which is a reconstitution solution from a lyophilized formulation. **Claim 82** The pharmaceutical composition according to any one of claims 1 to 81, which is contained in a glass vial or an injection device. **Claim 83** A method for treating cancer in a human patient in need of cancer treatment, the method comprising administering to the patient an effective amount of the pharmaceutical composition according to any one of claims 1 to 82.

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