Pharmaceutical compositions of programmed death receptor 1 (PD-1) antibodies and rHuPH20 or variants or fragments thereof

Abstract

The present invention provides a pharmaceutical composition of an anti-PD-1 antibody, or an antigen-binding fragment thereof, having an oxidation of less than or equal to about 3.0% of Met105 in the CDRH3 heavy chain region, and rHuPH20, or a variant or fragment thereof. The present invention also provides pharmaceutical compositions comprising a major species of anti-PD-1 antibodies and its acidic species, and rHuPH20 or a variant or fragment thereof wherein the amount of the acidic species is from 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

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 393,111, filed on July 28, 2022, the entire contents of which are incorporated herein by reference.

[0003] References to electronically submitted sequence listings

[0004] The contents of the electronic format of the Sequence Listing (25563-WO-SEQLIST.xml; size: 22,005 bytes; creation date: November 28, 2022) are incorporated herein by reference in its entirety. Technical Field

[0005] The present invention provides a pharmaceutical composition of 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 of an anti-PD-1 antibody or an antigen-binding fragment thereof and rHuPH20 or a variant or fragment thereof, wherein the anti-PD-1 antibody or an antigen-binding fragment thereof has less than or equal to about 3.0% oxidation of Met105 in the CDRH3 heavy chain region, and / or about 1.0%-12.0% acidic species. Background Art

[0006] Immune checkpoint therapy targeting the programmed death receptor 1 (PD-1) axis has produced breakthrough improvements in clinical responses in a variety of 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, 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) regulates T cell-mediated immune responses and may play a role in immune escape of human tumors (Pardoll DM. Nat Rev Cancer 2012, 12: 252-64). Binding of PD-1 to either of its ligands results in the delivery of inhibitory stimuli to T cells. Immunotherapies targeting the PD-1 axis include monoclonal antibodies against the PD-1 receptor (KEYTRUDA TM (pembrolizumab), Merck and Co., Inc., Kenilworth, NJ, and OPDIVO TM (nivolumab), Bristol-Myers Squibb, Princeton, NJ) and those that bind to PD-L1 ligands (MPDL3280A; TECENTRIQ TM (atezolizumab), Genentech, San Francisco, CA). Both treatments have demonstrated anti-tumor effects in a variety of cancer types.

[0007] Oxidation of methionine is one of the main degradation pathways in many protein drugs. Methionine residues in proteins are easily oxidized, resulting in the formation of methionine sulfoxide and, under extreme conditions, sulfone. Exposed methionine residues or methionine residues in antibody CDRs are likely to affect the biological activity of antibodies through oxidation. The main degradation pathways of pembrolizumab include 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 complexed with PD-1 shows that Met105 is part of the paratope and interacts with Lys131 in PD-1. Lee, JY et al. Nat. Commun. 7, 13354 (2016).

[0008] Therefore, it is desirable to obtain pembrolizumab compositions with low oxidation, particularly low oxidation at the Met105 position.

[0009] Deamidation of asparagine residues can lead to the formation of succinimide, which can then be converted to aspartic acid or isoaspartic acid. Deamidation in antibodies (especially in the CDR region) may affect the biological activity of the antibody. The crystal structure of the Fab fragment of pembrolizumab complexed with PD-1 shows that N52, N55 and N59 of CDRH2 are part of the paratope. PD-1 residues participate in water-mediated interactions with N52, N55 and N59. In addition, PD-1 residues also have direct interactions with N59. Lee, JY et al. Nat. Commun. 7, 13354 (2016). Therefore, it is also desirable to obtain a pembrolizumab composition with a low deamidation variant.

[0010] Hyaluronidase is an enzyme that degrades hyaluronic acid present in the extracellular matrix. Six types of hyaluronidase are known in the human body: Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1 and PH20 / SPAM1. PH20 / SPAM1 (hereinafter referred to as PH20) is expressed in the sperm plasma membrane and acrosome membrane.

[0011] Hyaluronidase hydrolyzes hyaluronic acid, thereby reducing the viscosity of hyaluronic acid in the extracellular matrix and increasing its permeability into tissue (skin). The subcutaneous region of the skin has a neutral pH of about 7.0 to 7.5. Among the various types of hyaluronidases, PH20 is widely used (Bookbinder et al., 2006). In embodiments using PH20, PH20 is typically co-administered with an antibody therapeutic injected subcutaneously (Bookbinder et al., 2006). rHuPH20 (also known as ) has been approved by the FDA for use as an adjuvant to increase the dispersion and absorption of other injected drugs. Summary of the invention

[0012] The present invention 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, wherein the anti-PD-1 antibody or an antigen-binding fragment thereof has less than or equal to about 3.0% oxidation of Met105 in the CDRH3 heavy chain region. The present invention also provides a pharmaceutical composition comprising an anti-human PD-1 antibody main species and an acidic species of the anti-human PD-1 antibody main species (wherein the amount of the acidic species is about 1.0%-12.0%) and rHuPH20 or a variant or fragment, wherein the anti-human PD-1 antibody main species comprises an 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. The present invention also provides a pharmaceutical composition, which comprises an anti-human PD-1 antibody main species and acidic species and basic species of the anti-human PD-1 antibody main species (wherein the amount of the main species is about 65%-95%) and rHuPH20 or a variant or fragment, wherein the anti-human PD-1 antibody main species comprises an 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. 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, wherein the pharmaceutical composition has one or more of the following: (i) less than or equal to about 3.0% oxidation of Met105 in the CDRH3 heavy chain region of the anti-human PD-1 antibody or its antigen-binding fragment; (ii) 1.0%-12.0% of the acidic species of the anti-human PD-1 antibody or its antigen-binding fragment; and (iii) acidic and basic species of the main species of the anti-human PD-1 antibody, wherein the amount of the main species is about 65%-85%. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment is produced or obtainable by a continuous perfusion process.

[0013] The present invention also provides a preparation of the above-mentioned pharmaceutical composition, which comprises:

[0014] a) about 5 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof; b) about 150-8000 U / ml of rHuPH20 or a variant or fragment thereof; c) a buffer; d) a non-reducing disaccharide; e) a non-ionic surfactant; and, optionally f) an antioxidant.

[0015] In one embodiment, the formulation comprises:

[0016] a) about 5 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0017] b) about 150-8000 U / ml of rHuPH20 or a variant or fragment thereof;

[0018] c) about 5 mM to about 20 mM buffer;

[0019] d) about 3% to about 10% weight / volume (w / v) of a non-reducing disaccharide selected from the group consisting of sucrose and trehalose;

[0020] e) from about 0.005% to about 0.10% nonionic surfactant; and, optionally

[0021] f) about 1 mM to about 30 mM antioxidant.

[0022] In a specific embodiment of the present 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.

[0023] In another aspect, the present invention provides a formulation comprising:

[0024] a) about 5 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody, wherein the antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the heavy chain variable region comprising three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8;

[0025] b) about 2000 U / ml of rHuPH20 or variant of SEQ ID NO: 17 or 18;

[0026] c) about 5 mM to about 20 mM histidine buffer; pH between about 5.0 to about 6.0;

[0027] d) about 3% to about 10% w / v non-reducing disaccharide;

[0028] f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0029] Surprisingly, under heat stress, certain embodiments of the above co-formulations have increased hyaluronidase activity of rHuPH20 or variants or fragments thereof compared to rHuPH20 or variants or fragments alone.The formulation can be a liquid formulation or a liquid formulation reconstituted from a lyophilized formulation.

[0030] Also provided herein is a method of treating cancer in a human patient in need thereof, the method comprising: administering to the patient an effective amount of a pharmaceutical composition of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Cell culture performance of continuous perfusion culture at 2 L and 50 L scale. Top left: viable cell density (VCD); top right: viability; bottom left: permeate titer; and bottom right: product screening.

[0032] Figure 2 : Total ion chromatograms generated from the reduced peptide digestion described in Example 3. Both the fed-batch produced pembrolizumab formulated drug product reference standard (top) and the continuously perfused produced pembrolizumab (bottom) were loaded in a similar manner, and the relative abundance shows that the peptides and retention times are the same for both samples. NL refers to normalized level; m / z refers to mass-to-charge ratio; FTMS refers to Fourier transform mass spectrometry, and ESI refers to electrospray ionization.

[0033] Figure 3 : Extracted ion chromatograms of the unmodified M105 peptide of the fed-batch produced pembrolizumab reference standard (top panel) and the continuous perfusion produced pembrolizumab (bottom panel). The retention time of the unmodified peptide containing M105 is about 37.8 min. NL refers to normalized level; m / z refers to mass-to-charge ratio; FTMS refers to Fourier transform mass spectrometry, ESI refers to electrospray ionization; RT refers to retention time.

[0034] Figure 4 : Extracted ion chromatograms of the modified M105 peptide of the fed-batch produced pembrolizumab reference standard (top panel) and the continuous perfusion produced pembrolizumab (bottom panel). The retention time of the modified peptide containing M105 is about 36.4 min. NL refers to normalized level; m / z refers to mass-to-charge ratio; FTMS refers to Fourier transform mass spectrometry, and ESI refers to electrospray ionization. RT refers to retention time.

[0035] Figure 5 : M105 oxidation of pembrolizumab in harvested cell culture fluid (HCCF) samples over time. The black circles are the results for each sample injection, and the line is a linear fit of the data and is described by y=0.038x+0.94, with an R-squared value of 0.9833.

[0036] Figure 6: Ion exchange chromatogram of pembrolizumab after protein A chromatography purification from HCCF in a continuous perfusion process. The retention time of each peak is annotated in the figure.

[0037] Figure 7 : Anion exchange chromatography of HCCF from continuous perfusion process and ion exchange chromatography of pembrolizumab after protein A chromatography purification. The retention time of each peak is marked in the figure.

[0038] Figure 8 : Overlay of the ion exchange chromatograms of pembrolizumab after the anion exchange step of the present invention and the pembrolizumab reference standard obtained from the fed-batch process. The diamonds depict the start and end of the integrated peak.

[0039] Fig. 9 : Total acidic species (acidic 1 + acidic variants + pre-main peak) % in the cell culture medium of the bioreactor (BRX), in the cell-free permeate (PERM), after the protein A chromatography step (PAP), and after anion exchange chromatography (AEXP) during the time course (in culture days).

[0040] Fig.10 : % of total major species (as measured by ion exchange) in the cell-free permeate (PERM), after the Protein A chromatography step (PAP), after anion exchange chromatography (AEXP) during the time course (in days in culture).

[0041] Fig.11 : Total basic species (basic 1 + basic 2 + basic variant A + basic variant B) % in the cell-free permeate (PERM), after the protein A chromatography step (PAP), after anion exchange chromatography (AEXP) during the time course (in days in culture).

[0042] Fig.12 : % of basic 1 species in the cell-free permeate (PERM), after the protein A chromatography step (PAP), after anion exchange chromatography (AEXP) during the time course (in days of culture).

[0043] Fig.13 : HIC-HP profile of pembrolizumab reference from fed-batch process. Pre-peak 3 contains antibody with Met105 oxidation in both heavy chains; Pre-peaks 1 and 2 contain antibody with Met105 oxidation in one heavy chain. Retention time (x-axis) and absorbance (y-axis) are shown.

[0044] Fig.14 : Linear fit calibration curve of the enzymatic activity assay as described in Example 6.

[0045] Fig.15.After heat stress, the enzymatic activity retention of rHuPH20 variant 1 in pembrolizumab produced by continuous perfusion process (CPP) co-formulation is enhanced.

[0046] Fig.16 .Enzymatic activity retention of rHuPH20 variant 1 in pembrolizumab CPP (165 mg / ml) co-formulations was enhanced after exposure to stainless steel. DETAILED DESCRIPTION

[0047] I. Definitions and Abbreviations

[0048] As used throughout the specification and appended claims, the following abbreviations apply:

[0049] API Active Pharmaceutical Ingredient

[0050] CDR Complementarity determining region in the variable region of an immunoglobulin

[0051] CHO Chinese Hamster Ovary

[0052] CI confidence interval

[0053] DS API

[0054] EC50 Concentration resulting in 50% efficacy or binding

[0055] ELISA enzyme-linked immunosorbent assay

[0056] FFPE formalin fixed, paraffin embedded

[0057] FR Framework Region

[0058] HC Heavy Chain

[0059] HNSCC Head and neck squamous cell carcinoma

[0060] HP-HIC High Performance Hydrophobic Interaction Chromatography

[0061] HP-IEX High Performance Ion Exchange Chromatography

[0062] HP-SEC High Performance Size Exclusion Chromatography

[0063] IC50 concentration causing 50% inhibition

[0064] IgG Immunoglobulin G

[0065] IHC Immunohistochemistry or Immunohistochemistry

[0066] mAb monoclonal antibody

[0067] NCBI National Center for Biotechnology Information

[0068] NSCLC Non-small cell lung cancer

[0069] PCR polymerase chain reaction

[0070] PD-1 Programmed death 1 (also known as programmed cell death 1 and programmed death receptor 1)

[0071] PD-L1 Programmed cell death 1 ligand 1

[0072] PD-L2 Programmed cell death 1 ligand 2

[0073] PS80 or PS-80 Polysorbate 80

[0074] SWFI Sterile Water for Injection

[0075] TNBC Triple-negative breast cancer

[0076] V H Immunoglobulin heavy chain variable region

[0077] VK immunoglobulin kappa light chain variable region

[0078] V L Immunoglobulin light chain variable region

[0079] v / v volume / volume

[0080] WFI Water for Injection

[0081] w / v weight / volume

[0082] In order to more easily understand the present invention, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by ordinary technicians in the field to which the present invention belongs.

[0083] As used throughout the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0084] Mentioning "or" means one or both possibilities, unless the context clearly indicates one of the indicated possibilities. In some cases, "and / or" is used to emphasize one or both possibilities.

[0085] As used herein, "acidic species" refers to an anti-PD-1 antibody species that is more acidic (e.g., as determined by cation exchange chromatography) than the main anti-PD-1 antibody species. Such acidic species are detected by various chromatographic purification methods for separating molecular variants according to charge, such as ion exchange, such as cation exchange chromatography (e.g., the method described in Example 5) or WCX-10 HPLC (weak cation exchange chromatography), optionally followed by mass spectrometry. Typically, acidic species have a lower isoelectric point (pI) than the main species and may have a more acidic character due to, for example, methionine oxidation, sialylation of asparagine residues, or deamidation variants of the antibody, or a combination thereof. Examples of acidic species include, but are not limited to, the present invention. Figure 6 or acidic variants, acidic 1, and pre-main peak identified in SEQ ID NO: 1 or 7. Any of the acidic species may also have one or more of the 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.

[0086] In one embodiment, the anti-PD-1 antibody acidic species is identified by one or more peaks eluting before the main peak as per a cation exchange method. In another embodiment, the anti-PD-1 antibody acidic species is identified by one or more peaks eluting before the main peak as per a weak cation exchange method. In ion exchange chromatography, "% acidic species" refers to the total area of ​​the acidic species peaks in the elution chromatogram divided by the total area of ​​all peaks.

[0087] As used herein, "acidic 1 species" refers to an acidic species in which one or more of deamidation, succinimide, aspartic acid or isoaspartic acid formation is present in one or more of N384, N389 and N390 of the heavy chain of the anti-PD-1 antibody major species. Such acidic 1 species are detected by various chromatographic purification methods for separating molecular variants according to charge, such as ion exchange, such as cation exchange chromatography (e.g., the method described in Example 5) or WCX-10 HPLC (weak cation exchange chromatography), followed by mass spectrometry analysis of the acidic species peak.

[0088] In one embodiment, the anti-PD-1 antibody acidic 1 species is Figure 6 or 7, and eluted according to the cation exchange method described in Example 5. In ion exchange chromatography, "% Acidic 1 species" refers to the total area of ​​the Acidic 1 peaks in the elution chromatogram divided by the total area of ​​all peaks.

[0089] As used herein, "acidic variant species" refers to the following acidic species of the main species of anti-PD-1 antibodies, which have one or more of deamidation, succinimide, aspartic acid or isoaspartic acid formation in one or more of N31, N52, N55, N59 and N61 of the heavy chain; or M105 oxidation in the heavy chain; or a combination thereof. Such acidic variants are detected by various chromatographic purification methods for separating molecular variants according to charge, such as ion exchange, such as cation exchange chromatography (e.g., the method described in Example 5) or WCX-10HPLC (weak cation exchange chromatography), followed by mass spectrometry analysis of the acidic species peak.

[0090] In one embodiment, the anti-PD-1 antibody acidic variant species are the following anti-PD-1 antibody species, which are as described by Figure 6 or 7, and eluted according to the cation exchange method described in Example 5. In ion exchange chromatography, "% acidic variant species" refers to the total area of ​​one or more acidic variant peaks in the eluted chromatogram divided by the total area of ​​all peaks.

[0091] As used herein, "basic species" refers to an anti-PD-1 antibody species that is more basic (e.g., as determined by cation exchange chromatography) than the main species of the anti-PD-1 antibody. Such basic species are detected by various chromatographic purification methods for separating molecular variants according to charge, such as ion exchange, such as cation exchange chromatography (e.g., the method described in Example 5) or WCX-10 HPLC (weak cation exchange chromatography), optionally followed by mass spectrometry. In general, basic species have a higher pI than the main species and may have stronger basic characteristics 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 α-amidation of a C-terminal leucine residue (SEQ ID NO: 14 or 15), truncation of the N-terminal amino acid residue in one or both heavy chains according to the amino acid sequence in any one of SEQ ID NO: 10-15, or a combination thereof). Examples of basic species include, but are not limited to, the invention. Figure 6 or basic variant A, basic variant B, basic 1, and basic 2 peaks identified in 7. Any of the basic species may also have one or more of the 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.

[0092] In one embodiment, the anti-PD-1 antibody basic species are identified by one or more peaks eluting after the main peak as per a cation exchange method. In another embodiment, the anti-PD-1 antibody basic species are identified by one or more peaks eluting after the main peak as per a weak cation exchange method. In ion exchange chromatography, "Basic Species %" refers to the total area of ​​one or more basic species peaks in the elution chromatogram divided by the total area of ​​all peaks.

[0093] As used herein, "main species" refers to anti-PD-1 antibody species that are identified as the majority of antibody species in a mixture with one or more acidic or basic species. Such main species are detected by various chromatographic purification methods for separating molecular variants according to charge, such as ion exchange, such as cation exchange chromatography (e.g., the method described in Example 5) or WCX-10HPLC (weak cation exchange chromatography), optionally followed by mass spectrometry. The mixture can be, for example, an antibody formulation from a mammalian cell and the result of its post-translational modification, upstream and downstream processing or storage. The main species can also have one or more of the 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.

[0094] In one embodiment, the major 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 major species is produced by Chinese hamster ovary cells, which contain 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.

[0095] In one embodiment, the major species is identified as the major peak according to the cation exchange method. In the ion exchange method, "% major species" refers to the total area of ​​the major peak divided by the total area of ​​all peaks in the elution chromatogram.

[0096] As used herein, "basic 1 species" refers to a basic species consisting of 2 heavy chains and 2 light chains, 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 each light chain consisting of the amino acid sequence of SEQ ID NO: 5; or a basic species consisting of two heavy chains and two light chains, 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, wherein the C-terminal leucine is α-amidated, and each light chain consisting of the amino acid sequence of SEQ ID NO: 5; or a combination thereof. Such basic 1 species are detected by various chromatographic purification methods for separating molecular variants according to charge, such as ion exchange, such as cation exchange chromatography (e.g., the method described in Example 5) or WCX-10 HPLC (weak cation exchange chromatography), followed by mass spectrometry analysis of the basic species peak.

[0097] In one embodiment, the anti-PD-1 antibody basic 1 species is Figure 6 or 7, and eluted according to the cation exchange method described in Example 5. In ion exchange chromatography, "Basic 1 species %" refers to the total area of ​​the Basic 1 peaks in the eluted chromatogram divided by the total area of ​​all peaks.

[0098] As used herein, "deamidated variants" refer to antibodies in which one or more asparagine residues have been deamidated. Deamidated variants can be in the form of succinimide, aspartic acid, or isoaspartic acid. That is, the neutral amide side chain has been converted to a residue with an overall acidic character.

[0099] In one aspect of measuring the main species, acidic species or basic species, a Thermo Scientific ProPac WCX-10 column is used for the cation exchange method. In another embodiment, a Thermo Scientific ProPac WCX-10 column is used, the mobile phase (A) is 24mM MES pH 6.1, with 4% acetonitrile, and the mobile phase (B) is 20mM sodium phosphate, 95mM NaCl pH 8.0, with 4% acetonitrile, and the column temperature is 35°C. In one embodiment, a nonlinear gradient is used: 22%-22% B for 0min-0.6min; 22%-29% B for 0.6min-15.0min; 29%-70% B for 15.0min-30.0min; 70%-100% B for 30.0min-30.5min; and 100%-100% B from 30.5-33.0min. In a further embodiment, the cation exchange method is described in Example 5.

[0100] As used herein, "express" and "expression" refer to allowing or causing the information in a gene or coding sequence (e.g., RNA or DNA) to be expressed; for example, by activating cellular functions involved in the transcription and translation of the corresponding gene to produce a protein. A DNA sequence can be expressed in or by a cell to form an "expression product," such as RNA (e.g., mRNA) or a protein. The expression product itself can also be said to be "expressed" by the cell.

[0101] As used herein, "expression vector" or "expression construct" refers to a vector (e.g., a plasmid) by which a polynucleotide comprising regulatory sequences operably linked to a coding sequence can be introduced into a host cell, where the coding sequence is expressed using the host cell's transcription and translation machinery.

[0102] As used herein, "expression cassette" refers to a polynucleotide comprising elements sufficient to control gene expression, including but not limited to a promoter operably linked to a gene sequence or operably linked to a multiple cloning site for inserting the gene sequence, and a poly A signal. In some embodiments, the expression cassette further comprises one or more regulatory elements that can regulate the expression of the gene at the transcriptional, translational and / or chromatin levels.

[0103] As used herein, "promoter" or "promoter sequence" refers to a DNA fragment containing a regulatory region capable of recruiting RNA polymerase (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. Within the promoter sequence can be found a transcription start site (e.g., conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for RNA polymerase recruitment.

[0104] As used herein, an "enhancer" or "enhancer sequence" refers to a DNA regulatory region that enhances transcription from a promoter independent of its distance, position, or orientation from the promoter. In certain embodiments, the enhancer is immediately adjacent to the promoter. In some embodiments, the enhancer is distal to the promoter. In other embodiments, the promoter and enhancer are a combined sequence, referred to herein as a "combined enhancer / promoter."

[0105] As used herein, "internal ribosome entry site" or "IRES" refers to an RNA element or sequence that allows translation initiation in a cap-independent manner 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 allows translation initiation in a cap-independent manner by directly recruiting ribosomes. The IRES can be a wild-type IRES from any species or a variant or mutant thereof, whether naturally occurring or artificial.Examples of IRES 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 dependentupon IRES sequence and gene location. Biotechniques. 2006 Sep; 41(3): 283-4), the IRES element from the expression vector pInSRT-GFP (GenBank LC417349.1), the IRES element from the 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), an IRES element from the expression vector pIRESneo3 (Clontech / Takara Bio), an IRES element described in WO 2015 / 016786, WO 2015 / 021077, WO 2016 / 003368, WO 2016 / 074016 or WO 2013 / 092743, or a variant thereof.

[0106] As used herein, "regulatory element", "regulatory region" or "regulatory sequence" refers to a polynucleotide sequence that has the ability to regulate (e.g., start, activate, enhance, increase, decrease, inhibit, suppress or silence) gene expression. In certain embodiments, regulation is achieved by binding of cytokines to polynucleotide sequences. In other embodiments, regulation is achieved by interactions 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.

[0107] As used herein, "insulator" refers to a class of DNA elements or sequences that have the ability to isolate proximal DNA regions from surrounding chromosomal regions by preventing position effects. 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 that prevents the advancement of nearby condensed chromatin, which otherwise might silence expression. In other embodiments, an insulator can block an enhancer and act as a barrier.

[0108] As used herein, "expression enhancing sequence element" or "EASE" refers to a DNA element or sequence that can increase protein expression when the DNA element or sequence is placed upstream of a promoter that controls protein expression.

[0109] As used herein, "triplet leader sequence" or "TPL" refers to an RNA element or sequence in the 5' untranslated region of an adenoviral late-expressed mRNA that has the ability to initiate translation of the late-expressed mRNA in a cap-independent manner. As used herein, the term "triplet leader sequence" or "TPL" also encompasses a DNA sequence that can be transcribed into an RNA sequence in the 5' untranslated region of an adenoviral late-expressed mRNA that has the ability to initiate translation of the late-expressed mRNA in a cap-independent manner.

[0110] As used herein, "inverted terminal repeats" or "ITRs," in the context of transposon technology, refer to DNA elements or sequences at both ends of a transposon and its inverted version that signal that breakage and ligation should occur.

[0111] As used herein, "selectable marker" or "selection marker" refers to a protein that allows specific selection of cells expressing the protein by adding a corresponding selection agent to the culture medium. In certain embodiments, the selectable marker is a eukaryotic selectable marker that allows selection of eukaryotic cells expressing the marker protein. In some embodiments, the selectable marker is a bacterial selectable marker that allows selection of bacterial cells expressing the marker protein.

[0112] As used herein, "polynucleotide sequence," "nucleic acid sequence," or "nucleotide sequence" refers to a series of nucleotide bases (also called "nucleotides") in a nucleic acid (eg, DNA or RNA), and means any chain of two or more nucleotides.

[0113] As used herein, "host cell" refers to any cell of any organism used to produce a recombinant protein encoded by an expression vector or to propagate an expression vector introduced into a 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.

[0114] As used herein, the term "fed-batch culture" refers to a method of culturing cells in which additional nutrients are provided to the culture during the culture process. Fed-batch culture is typically stopped at some point and the cells and / or components in the culture medium are harvested. The product accumulates and remains in the bioreactor until the end of the run.

[0115] As used herein, "harvesting" an antibody or antigen-binding fragment involves separating the antibody or antigen-binding fragment from particulate matter that may include host cells, cell aggregates, and / or lysed cell fragments to form a cell-free fraction that is substantially free of host cells and cell debris, i.e., a cell-free "permeate". For example, such cells and cell debris are removed from a cell culture fluid by centrifugation, depth filtration, and / or microfiltration. For example, to prepare a cell-free permeate, a hollow fiber membrane or a series of filtration steps, such as depth filtration, may be used. "Continuous harvesting" refers to harvesting cell culture broth while performing antibody production in a bioreactor. During the process of removing culture medium via a perfusion system, protein products secreted in a bioreactor can be continuously harvested from the cell culture fluid by microfiltration, thereby isolating the target protein in the microfilter permeate leaving the perfusion system. The microfilter may be a tangential flow filtration (TFF) unit or an alternating tangential flow filtration (ATF) unit comprising a hollow fiber module. Commercially available TFF units include, but are not limited to TFF unit or 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 for the cell culture fluid containing the antibody or antigen binding fragment to maintain a consistent flow rate for the affinity chromatography step.

[0116] As used herein, "cell culture fluid" refers to the liquid medium containing host cells, cell debris, cell culture medium, antibodies or antigen-binding fragments during the cell growth and antibody production process.

[0117] As used herein, "cell culture harvest fluid" or "HCCF" refers to cell culture fluid obtained after harvesting the cell culture fluid, containing antibodies or antigen-binding fragments, which is substantially free of host cells and cell debris. In one embodiment, the HCCF is a cell-free permeate.

[0118] As used herein, "fluidically connected," "fluidically," or "fluidically connected to" or "fluidically receiving material from" refers to another step in a manufacturing process or from another system when material containing the protein of interest flows between steps or systems through a pipe, tube, or other closed conduit without manual loading or unloading.

[0119] As used herein, "continuous purification" in the context of HCCF refers to the uninterrupted flow of HCCF to at least one affinity stationary phase (at least for the loading step), and optionally to any wash or elution steps.

[0120] As used herein, "perfusion" or "perfusion" refers to a method of culturing cells in which additional fresh medium is continuously provided to the culture over a period of time (after the culture process begins), and the medium is simultaneously removed while the antibody or antigen-binding fragment is continuously harvested from the culture medium. Fresh medium typically provides the cells with nutritional supplements that are depleted during the culture process.

[0121] As used herein, "perfusion rate" refers to the rate at which fresh culture medium is provided and cell culture fluid is removed.

[0122] As used herein, "perfusion bioreactor" refers to a bioreactor for culturing cells in which equal volumes of culture medium can be added to and simultaneously removed from the reactor. In one embodiment, the cells are retained in the bioreactor. A perfusion bioreactor comprises a bioreactor and an operably attached perfusion system that provides a steady source of fresh nutrient medium and removes cellular waste products. The bioreactor and perfusion system of a perfusion bioreactor can be independent mechanical units that operate in coordination. Many commercially available examples include, but are not limited to, various Brand single-use bioreactors (SUB; GE Healthcare Life Sciences) and Brand perfusion flow path components and systems (Spectrum; Repligen), a skilled practitioner can appropriately combine the bioreactor and perfusion system into a perfusion bioreactor. Alternatively, the bioreactor and perfusion system can be assembled into a single mechanical unit, such as, but not limited to, the 3D Biotek brand perfusion bioreactor (Sigma-Aldrich).

[0123] As used herein, the term "surge vessel" refers to a well-mixed (provides well-mixed flow homogenization) storage reservoir, mixing vessel, feed tank, or collection vessel (or interchangeably, "collection tank") at the downstream end of a conduit, feeder, dam, pipe, or tube to absorb differential flow rates between two fluidly connected unit operations, such as the flow rate of permeate from a bioreactor and the flow rate of a first chromatography system under automatic control in a continuous or semi-continuous process embodiment of the present invention. The surge vessel absorbs changes or differences in flow rates by allowing the volume to fluctuate within the limits of a preset volume range between the fluidly connected unit operations.

[0124] As used herein, the term "residence time" refers to the average time a fluid solution spends in a container. For perfusion, this is the inverse of the exchange rate (e.g., 2VVD has an average residence time of 0.5 days). Sieving of solution components, retention of solution components by the membrane is neglected for residence time.

[0125] As used herein, "M105," "Met105," or "methionine 105" refers to the methionine in the heavy chain CDRH3 region in SEQ ID NO: 8 (RDYRFDMGFDY).

[0126] As used herein, "% oxidation of Met105" or "% oxidation of Met105" refers to a) the total amount of one or more anti-PD-1 antibody fragments of the invention with oxidized Met105 compared to the total amount of one or more anti-PD-1 antibody fragments of the invention with and without oxidized Met105 as determined by reduced peptide mapping, liquid chromatography and mass spectrometry; or b) the total amount of anti-PD-1 antibodies of the invention with oxidized Met105 compared to the total amount of anti-PD-1 antibodies of the invention with and without oxidized Met105 as determined in hydrophobic interaction chromatography (HIC).

[0127] "Binding" an antibody or antigen-binding fragment to a stationary phase means exposing the antibody or antigen-binding fragment to the stationary phase under appropriate conditions (pH and / or conductivity) such that the antibody or antigen-binding fragment becomes reversibly associated with the stationary phase through an interaction between the antibody or antigen-binding fragment and a ligand immobilized on the stationary phase.

[0128] As used herein, the term "equilibrium solution" refers to a solution used to equilibrate the stationary phase before the antibody or antigen-binding fragment is loaded onto the stationary phase. The equilibrating solution may comprise one or more salts and buffer substances. In one embodiment, the equilibrating solution has the same conditions as the loading solution comprising the antibody or antigen-binding fragment.

[0129] As used herein, the term "loading solution" refers to a solution used to load a composition comprising an antibody or antigen-binding fragment of interest and one or more impurities onto a stationary phase. The loading solution may optionally further comprise one or more of a buffer substance, and a salt.

[0130] As used herein, the term "wash solution" refers to a solution used to wash or re-equilibrate a stationary phase prior to eluting the antibody or antigen-binding fragment of interest. For washing, the conductivity and / or pH of the wash solution is such that impurities are removed from the stationary phase. For re-equilibration, the wash solution and the equilibration solution may be the same, but this is not required. The wash solution may contain one or more salts and buffer substances.

[0131] As used herein, "elution solution" refers to a solution used to elute the antibody or antigen-binding fragment of interest from a stationary phase. The elution solution may contain one or more salts or buffer substances. The presence of one or more of the salt, buffer substance, pH, or conductivity of the elution solution causes the antibody or antigen-binding fragment to elute from the stationary phase.

[0132] As used herein, the term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In a solution, electric current flows by ion transport. Therefore, as the number of ions present in an aqueous solution increases, the solution will have a higher conductivity. Conductivity is measured in mS / cm and can be measured using a conductivity meter sold by GE Healthcare. The conductivity of a solution can be measured by changing the concentration of ions in the solution. For example, the concentration of the buffer and / or the concentration of the salt (e.g., NaCl or KCl) in the solution can be changed to achieve the desired conductivity. Preferably, the salt concentration of the various buffers is modified to achieve the desired conductivity as described in the following examples.

[0133] As used herein, "purifying" an antibody or antigen-binding fragment of interest or a "purified composition" refers to increasing the purity of the antibody or antigen-binding fragment in a composition by removing (completely or partially) at least one impurity from the composition. Impurities may be host cell components, such as serum, proteins or nucleic acids, cell debris, growth medium, or antibody aggregates. The term is not intended to refer to the complete absence of such biomolecules or the absence of water, buffers or salts or components of a pharmaceutical composition comprising the antibody or antigen-binding fragment.

[0134] As used herein, a "continuous multi-column chromatography system" refers to a chromatography system containing at least two stationary phases having similar impurity separation functions, which allows at least one stationary phase to be loaded with a sample and at least one stationary phase to perform a non-loading step (one or more of equilibration, washing, elution, and regeneration).

[0135] As described herein, "stationary phase" refers to any surface on which one or more ligands can be fixed. The stationary phase can be a suspension, a discontinuous phase of discrete particles, a plate, a sensor, a chip, a capsule, a box, a resin, a bead, a monolith, a gel, a film or a membrane adsorbent, etc. The stationary phase can also be loaded into a purification column (e.g., filled with resin beads). Examples of materials for forming stationary phases 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 any of the above derivatives) and polysaccharides (e.g., cellulose, agarose and dextran). See Jansson, JC; Rydén, L. Protein Purification; Wiley: New York, 1998.

[0136] As used herein, "impurities" refer to substances other than the desired antibody or antigen-binding fragment. Impurities can be host cell proteins (HCPs), host cell DNA (HC-DNA), protein aggregates or fragments, and other undesirable protein modifications (i.e., oxidized species, acid variant species).

[0137] As used herein, "treating" or "treating" cancer refers to administering a composition of the invention to a subject having an immune disorder or a cancer disorder or diagnosed with cancer or a pathogen infection (e.g., viral, bacterial, fungal) to achieve at least one positive therapeutic effect, such as a decreased number of cancer cells, a decreased tumor size, a decreased rate of cancer cell infiltration into surrounding organs, or a decreased rate of tumor metastasis or tumor growth. "Treatment" may include one or more of the following: inducing / increasing anti-tumor immune responses, stimulating immune responses to pathogens, toxins and / or self-antigens, stimulating immune responses to viral infections, reducing the amount of one or more tumor markers, stopping or delaying the growth of tumors or blood cancers or diseases associated with the binding of PD-1 to its ligands PD-L1 and / or PD-L2 ("PD-1-associated diseases") such as cancer progression, stabilizing PD-1-associated diseases, inhibiting the growth or survival of tumor cells, eliminating or reducing the size of one or more cancerous lesions or tumors, reducing the level of one or more tumor markers, improving or eliminating clinical manifestations of PD-1-associated diseases, reducing the severity or duration of clinical symptoms of PD-1-associated diseases such as cancer, prolonging patient survival relative to the expected survival of similar untreated patients, and inducing complete or partial remission of cancerous conditions or other PD-1-associated diseases.

[0138] "Immune conditions" or "immune disorders" include, for example, pathological inflammation, inflammatory conditions, and autoimmune conditions or diseases. "Immune conditions" also refer to infections, persistent infections, and proliferative conditions, such as cancers, tumors, and angiogenesis, including infections, tumors, and cancers that resist eradication by the immune system. "Cancerous conditions" include, for example, cancers, cancer cells, tumors, angiogenesis, and precancerous conditions such as dysplasia.

[0139] The positive treatment effect of cancer can be measured in a variety of ways (see WA Weber, J. Nucl. Med. 50: 1S-10S (2009)). For example, with respect to tumor growth inhibition, according to the NCI standard, T / C≦42% is the minimum level of anti-tumor activity. T / C<10% is considered a high level of anti-tumor activity, where T / C (%) = median tumor volume of treatment / median tumor volume of control × 100. In some embodiments, the treatment achieved by administering the composition of the present invention is any one of progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS). PFS, also known as "time to tumor progression", refers to the length of time that cancer does not grow during and after treatment, and includes the amount of time that the patient experiences complete remission or partial remission, and the amount of time that the patient experiences stable disease. DFS refers to the length of time that the patient remains disease-free during and after treatment. OS refers to the extension of life expectancy compared to the original or untreated individual or patient. While embodiments of the compositions, methods of treatment, and uses of the invention may not be effective in achieving a positive therapeutic effect in every patient, it should achieve a positive therapeutic effect in a statistically significant number of subjects, as determined by any statistical test known in the art, such as Student's t-test, chi2 test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0140] As used herein, the term "patient" (also referred to herein as "subject" or "individual") refers to a mammal (e.g., rat, mouse, dog, cat, rabbit), most preferably a human, that can be treated with the composition or the composition of the invention. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a pediatric patient. Those "in need of treatment" include those patients who may benefit from treatment with the composition or the composition of the invention, such as patients with cancer or immune diseases.

[0141] As used herein, the term "antibody" refers to any form of antibody that exhibits a desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized antibodies, fully human antibodies and chimeric antibodies.

[0142] Typically, the basic antibody structural unit comprises a tetramer. Each tetramer includes two pairs of identical polypeptide chains, each pair having a "light" chain (about 25 kDa) and a "heavy" chain (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The variable region of each light chain / heavy chain pair forms an antibody binding site. Therefore, typically, a complete antibody has two binding sites. The carboxyl terminal portion of the heavy chain can define a constant region primarily responsible for effector function. Typically, human light chains are divided into kappa and lambda light chains. In addition, human heavy chains are typically classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D" region of more than about 10 amino acids. See generally Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989).

[0143] Typically, the variable domains of the heavy and light chains each include three hypervariable regions, also referred to as complementarity determining regions (CDRs), which are located within relatively conservative framework regions (FRs). The CDRs are typically aligned by the framework regions to enable binding to specific epitopes. Typically, from N-terminus to C-terminus, the light and heavy chain variable domains each include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Generally, the amino acid assignments for each domain are based on the definitions in 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.

[0144] As used herein, "pharmaceutically effective amount" or "effective amount" refers to an amount of a therapeutic composition or composition introduced into a patient sufficient to treat a disease or condition. Those skilled in the art recognize that this level may vary depending on the patient's characteristics such as age, weight, etc.

[0145] When modifying the amount of a substance or composition (e.g., mM or M), the percentage of a component of a composition (v / v or w / v), the pH of a solution / composition, or the value of a parameter of a step in a characterization method, etc., the term "about" refers to possible changes in the value due to, for example, typical measurements, handling, and sampling procedures involved in the preparation, characterization, and / or use of the substance or composition; due to instrumental errors in these procedures; due to differences in the manufacture, source, or purity of ingredients used to make or use the composition or perform the procedure; etc. In certain embodiments, "about" may mean a change of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% of the stated value.

[0146] As used herein, the terms "cancer," "cancerous," or "malignant" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma, and sarcoma. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, lymphocytic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer.

[0147] As used herein, the terms "PD-1 binding fragment", "antigen binding fragment thereof", and "binding fragment thereof" include fragments or derivatives of antibodies that still substantially retain their biological activity of binding to antigen (human PD-1) and inhibiting its activity (e.g., blocking the binding of PD-1 to PDL1 and PDL2). Therefore, the term "antibody fragment" or PD-1 binding fragment refers to a portion of a full-length antibody, typically 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 the PD-1 inhibitory activity. In some embodiments, the binding fragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (or more) of its PD-1 inhibitory activity, although any binding fragment with sufficient affinity to exert the desired biological effect is useful. In some embodiments, an antigen-binding fragment binds to its antigen with an affinity that is at least two times greater than that of an unrelated antigen, preferably at least ten times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater. In one embodiment, the antibody has an affinity of greater than about 10 9The affinity of liters / mole is determined by, for example, Scatchard analysis. Munsen et al. (1980) Analyt. Biochem. 107: 220-239. It is also intended that the PD-1 binding fragment may include variants with conservative amino acid substitutions that do not substantially alter their biological activity.

[0148] As used herein, "humanized antibody" refers to an antibody form comprising sequences from non-human (e.g., mouse) antibodies and human antibodies. Such antibodies contain the minimum sequence derived from non-human immunoglobulin. Generally, humanized antibodies will include substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin sequences. Humanized antibodies also optionally include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. The humanized form of rodent antibodies typically includes the same CDR sequence as the parent rodent antibody, although certain amino acid substitutions may be included for increasing affinity, increasing the stability of the humanized antibodies, or other reasons.

[0149] The antibodies of the present invention also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, for example, U.S. Pat. No. 5,624,821; WO2003 / 086310; WO2005 / 120571; WO2006 / 0057702; Presta (2006) Adv. Drug Delivery Rev. 58: 640-656. Such modifications can be used to enhance or inhibit various responses of the immune system, which may have a beneficial effect on diagnosis and treatment. Changes in the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and the addition of multiple Fcs. Changes in Fc can also change the half-life of the antibody in the therapeutic antibody. The longer the half-life, the lower the frequency of administration, which brings about an increase in convenience and a reduction in the use of substances. See Presta (2005) J. Allergy Clin. Immunol. 116: 731 at 734-35.

[0150] As used herein, "conservatively modified variants" or "conservative substitutions" refer to amino acid substitutions known to those skilled in the art and that can generally be made without altering the biological activity of the resulting molecule, even in essential regions of the polypeptide. Such exemplary substitutions are preferably made according to those listed in Table 1, as follows:

[0151] Table 1. Exemplary conservative amino acid substitutions

[0152]

[0153]

[0154] In addition, those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide will not significantly alter biological activity. See, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th edition).

[0155] The phrase "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of" as used throughout the specification and claims means including any recited elements or groups of elements, and optionally including other elements having properties similar or different from the recited elements, which do not substantially change the basic or novel properties of the specified dosage regimen, method, or composition. As a non-limiting example, a binding compound consisting essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, which do not substantially affect the properties of the binding compound.

[0156] Throughout the specification and claims, the word "comprising" or variations such as "comprise", "comprises" or "comprised of" are used in an inclusive sense, which specifies the presence of the recited features but does not exclude the presence or addition of other features, the presence or addition of which may substantially enhance the operation or utility of any embodiment of the invention, unless the context requires otherwise due to the express language or necessary implication.

[0157] As used herein, "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules constituting the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically include a variety of different antibodies with different amino acid sequences in their variable domains (particularly their CDRs, which are typically specific for different epitopes). The modifier "monoclonal" represents the characteristics of an antibody obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of antibodies by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in 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.

[0158] When applied to a subject diagnosed with or suspected of having cancer, "tumor" refers to a malignant or potentially malignant tumor or tissue mass of any size, including primary tumors and secondary tumors. Solid tumors are abnormal growths or tissue masses that usually do not contain cysts or fluid areas. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, epithelial cancers, and lymphomas. Leukemias (cancers of the blood) do not usually form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

[0159] The term "tumor size" refers to the overall size of a tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by a variety of methods known in the art, such as by measuring the size of one or more tumors after removal from a subject, such as using calipers, or in vivo using imaging techniques (e.g., bone scans, ultrasound, CT or MRI scans).

[0160] The “tumor proportion score (TPS)” refers to the percentage of tumor cells expressing PD-L1 on the cell membrane at any intensity (weak, moderate, or strong). Linear partial or complete cell membrane staining was interpreted as PD-L1 positivity.

[0161] The "mononuclear inflammatory density score (MIDS)" refers to the ratio of the number of mononuclear inflammatory cells (MICs) expressing PD-L1 (large and small lymphocytes, monocytes, and macrophages within tumor nests and adjacent supporting stroma) that infiltrate or are adjacent to the tumor to the total number of tumor cells. MIDS is recorded on a scale of 0 to 4, where 0 = absent; 1 = present, but less than 1 MIC per 100 tumor cells (<1%); 2 = at least 1 MIC per 100 tumor cells, but less than 1 MIC per 10 tumor cells (1%-9%); 3 = at least one MIC per 10 tumor cells, but fewer MICs than tumor cells (10%-99%); 4 = at least as many MICs as tumor cells (≥100%).

[0162] The “combined positive score (CPS)” is the ratio of the number of PD-L1-positive tumor cells and PD-L1-positive mononuclear inflammatory cells (MICs) within the tumor nests and adjacent supporting stroma (numerator) to the total number of tumor cells (denominator; i.e., the number of PD-L1-positive and PD-L1-negative tumor cells). PD-L1 expression was considered positive at any intensity, i.e., weak (1+), moderate (2+), or strong (3+).

[0163] "PD-L1 expression positivity" refers to a tumor proportion score, monocyte inflammation density score, or combined positive score of at least 1%; AIS ≥ 5; or increased PD-L1 expression (protein and / or mRNA) levels in malignant cells and / or tumor-infiltrating immune cells compared with appropriate controls.

[0164] "Microsatellite instability (MSI)" refers to a form of genomic instability associated with defective DNA mismatch repair in tumors. See Boland et al., Cancer Research 58, 5258-5257, 1998. In one embodiment, five microsatellite markers recommended by the National Cancer Institute (NCI) can be used for MSI analysis: BAT25 (GenBank Accession No. 9834508), BAT26 (GenBank Accession No. 9834505), D5S346 (GenBank Accession No. 181171), D2S123 (GenBank Accession No. 187953), D17S250 (GenBank Accession No. 177030). Other markers, such as BAT40, BAT34C4, TGF-β-RII and ACTC, can be used. Commercially available kits for MSI analysis include, for example, Promega MSI multiplex PCR assay, CDx (F1CDx) is a next-generation sequencing-based in vitro diagnostic device that uses DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples.

[0165] "High frequency of microsatellite instability" or "microsatellite high instability (MSI-H)" means if two or more of the above five NCI markers show instability or ≥30%-40% of the total markers show instability (ie, there are insertion / deletion mutations).

[0166] As used herein, "non-MSI-H cancer" refers to microsatellite stable (MSS) and MSI-low (MSI-L) cancers.

[0167] "Microsatellite stable (MSS)" means if none of the five NCI markers above show instability (ie, have insertion / deletion mutations).

[0168] "Proficient mismatch repair (pMMR) cancer" refers to tumor samples that normally express MMR proteins (MLH1, PMS2, MSH2, and MSH6) based on IHC. Commercially available kits for MMR analysis include the Ventana MMR IHC assay.

[0169] “Mismatch repair-deficient (dMMR) cancer” refers to tumor samples with low expression of one or more MMR proteins (MLH1, PMS2, MSH2, and MSH6) based on IHC.

[0170] As used herein, "variable region" or "V region" refers to a fragment of an IgG chain that is variable in sequence between different antibodies. It extends to Kabat residue 109 in the light chain and to residue 113 in the heavy chain.

[0171] "Pembrolizumab" (formerly known as MK-3475, SCH 900475, and lambloid), also referred to herein as "pembro", is a humanized IgG4 mAb, the structure of which is described in WHO Drug Information, Vol. 27, No. 2, pp. 161-162 (2013), and which comprises the heavy and light chain amino acid sequences and CDRs described in Table 2. Pembrolizumab has been approved by the U.S. FDA as KEYTRUDA TM As described in the prescribing information of (Merck & Co., Inc., Whitehouse Station, NJ USA; first approved in the United States in 2014).

[0172] As used herein, "pembrolizumab variant" means a monoclonal antibody comprising heavy and light chain sequences that are substantially identical to those in pembrolizumab, except that there are three, two, or one conservative amino acid substitutions at positions located outside the light chain CDRs and six, five, four, three, two, or one conservative amino acid substitutions at positions located outside the heavy chain CDRs, e.g., the variant positions are located in the FR region or the constant region, and optionally have a deletion of the C-terminal lysine residue of the heavy chain. In other words, pembrolizumab and pembrolizumab variants comprise the same CDR sequences, but differ from each other by having conservative amino acid substitutions at no more than three or six other positions in their full-length light and heavy chain sequences, respectively. Pembrolizumab variants are substantially identical to pembrolizumab in terms of the following properties: binding affinity to PD-1 and the ability to block binding of PD-L1 and PD-L2 to PD-1, respectively.

[0173] The term "buffer" encompasses those agents that maintain the solution pH of the compositions of the invention within an acceptable range.

[0174] The terms "pharmaceutical composition," "formulation," or "pharmaceutical formulation" refer to a preparation containing a pharmaceutically acceptable excipient in a form that renders the active ingredient effective, and which contains no additional components that are toxic to a subject to which the composition would be administered.

[0175] The terms "lyophilization", "lyophilized" and "freeze-dried" refer to a process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. Excipients may be included in the pre-lyophilization formulation to enhance the stability of the lyophilized product during storage.

[0176] "Pharmaceutically acceptable" refers to excipients (carriers, additives) and compositions that can reasonably be administered to a subject to provide an effective dose of the active ingredient employed, and are "generally regarded as safe", e.g., they are physiologically tolerable and do not generally produce allergic or similar adverse reactions when administered to humans, such as stomach upset, etc. In another embodiment, the term refers to molecular entities and compositions that are approved by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0177] A "reconstituted" formulation is one 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, e.g., parenteral or intravenous administration, and may optionally be suitable for subcutaneous administration.

[0178] "Reconstitution time" is the time required to rehydrate a lyophilized formulation with a solution into a clear, particle-free solution.

[0179] A "stable" formulation is one in which the protein substantially retains its physical stability and / or chemical stability and / or biological activity after storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in, for example, the following literature: Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured for a selected time period at a selected temperature. For example, in one embodiment, a stable formulation is one in which no significant changes are observed at refrigerated temperatures (2° C.-8° C.) for at least 12 months. In another embodiment, a stable formulation is one in which no significant changes are observed at refrigerated temperatures (2° C.-8° C.) for at least 18 months. In another embodiment, a stable formulation is one in which no significant changes are observed at room temperature (23° C.-27° C.) for at least 3 months. In another embodiment, a stable formulation is a formulation in which no significant changes are observed at room temperature (23°C-27°C) for at least 6 months. In another embodiment, a stable formulation is a formulation in which no significant changes are observed at room temperature (23°C-27°C) for at least 12 months. In another embodiment, a stable formulation is a formulation in which no significant changes are observed at room temperature (23°C-27°C) for at least 18 months. The stability criteria for antibody formulations are as follows. Typically, no more than 10%, preferably no more than 5%, of the antibody monomers are degraded according to SEC-HPLC measurements. Typically, the formulation is colorless, or transparent to slightly milky white by visual analysis. Typically, the concentration, pH, and osmotic pressure of the formulation do not vary by more than + / -10%. The potency is typically within the range of 60%-140% of the control or reference, preferably 80%-120%. Typically, no more than 10%, preferably 5%, of antibody clipping is observed, i.e., the percentage of low molecular weight substances as determined by, for example, HP-SEC. Typically, no more than 10%, preferably no more than 5% aggregation of the antibodies is observed, ie the percentage of high molecular weight species as determined by, for example, HP-SEC.

[0180] An antibody "maintains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation when visually inspected for color and / or clarity or when measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering. Changes in protein conformation can be assessed by fluorescence spectroscopy to determine protein tertiary structure and FTIR spectroscopy to determine protein secondary structure.

[0181] An antibody “retains its chemical stability” in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or shearing (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping coupled with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate assay, etc.), and isomerization (assessed by methods such as isoaspartate content assay, peptide mapping, etc.).

[0182] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared. The biological activity of the antibody can be determined, for example, by antigen binding assays. The formulations of the invention include antibodies and fragments thereof that are biologically active when reconstituted or in liquid form.

[0183] The term "isotonic" means that the intended formulation has substantially the same osmotic pressure as human blood. Isotonic formulations typically have an osmotic pressure of about 270mOsm-328mOsm. Mild hypotonic pressure is 250mOsm-269mOsm, and mild hypertonic pressure is 328mOsm-350mOsm. Osmotic pressure can be measured, for example, using a vapor pressure or ice-type osmometer.

[0184] A "non-reducing disaccharide" is a disaccharide that cannot act as a reducing agent because it does not contain or cannot be converted to contain a free aldehyde group or a free ketone group. Examples of non-reducing disaccharides include, but are not limited to, disaccharides such as sucrose and trehalose.

[0185] "PH 20" refers to the wild-type PH20 hyaluronidase of SEQ ID NO:16.

[0186] "Unit" or "U" refers to one unit of hyaluronidase activity: the amount of rHuPH20 or variant or fragment thereof that causes a change in optical density at 600nm under conditions suitable for hyaluronic acid and enzyme reaction, and is calculated according to a calibration curve using an activity standard. Examples of assays are described in Example 4 of US2022 / 0089738 and Example 6 below. Hyaluronic acid (HA) binds to albumin, and the albumin-HA complex produces turbidity. When HA is hydrolyzed by hyaluronidase, the turbidity of the albumin-HA complex decreases. Therefore, the assay measures turbidity to determine the hyaluronidase activity of rHuPH20 or variant or fragment thereof. Hyaluronidase activity is based on the following reaction:

[0187] Hyaluronic acid --> disaccharides and monosaccharides + smaller hyaluronic acid fragments. One skilled in the art understands that the hyaluronidase activity expressed in units / mg hyaluronidase may vary depending on the purity of the hyaluronidase, the manufacturing process, etc.

[0188] Anti-PD-1 antibodies and antigen-binding fragments thereof

[0189] In some embodiments, the anti-human PD-1 antibody or antigen-binding fragment thereof used in the composition of the present invention comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises three light chain CDRs of CDRL1, CDRL2 and CDRL3, and the heavy chain variable region comprises three heavy chain CDRs of CDRH1, CDRH2 and CDRH3.

[0190] In one embodiment of the 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.

[0191] 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 and a heavy chain variable region, the light chain variable region comprises SEQ ID NO: 4 or consists thereof and the heavy chain variable region comprises SEQ ID NO: 9 or consists thereof.

[0192] In another embodiment, the composition of the invention comprises an antibody or antigen-binding fragment having a V LDomain or V H One of the domains has a V having at least 95%, 90%, 85%, 80%, 75% sequence homology L Domain and / or V H In another embodiment, the antibody or antigen-binding fragment of the composition of the present invention comprises a V domain having up to 1, 2, 3, 4 or 5 or more amino acid substitutions. L and V H domain and exhibited specific binding to PD-1.

[0193] 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 can be used, including IgG1, IgG2, IgG3, and IgG4. Different constant domains can be attached to the V domains provided herein. L and V H Region. For example, if the specific intended use of the antibody (or fragment) of the invention requires a change in effector function, a heavy chain constant domain other than IgG1 may be used. Although IgG1 antibodies have a long half-life and effector functions, such as complement activation and antibody-dependent cellular toxicity, such activities may not be required for all uses of the antibody. In this case, for example, an IgG4 constant domain may be used.

[0194] In an embodiment of the present invention, the anti-PD-1 antibody comprises a light chain and a heavy chain, the light chain comprising or consisting of an amino acid residue sequence as shown in SEQ ID NO: 5, and the heavy chain comprising or consisting of an amino acid residue sequence as shown in SEQ ID NO: 10. In another embodiment, the anti-human PD-1 antibody consists of two light chains and two heavy chains, wherein the two light chains consist of the amino acid sequence shown in SEQ ID NO: 5, wherein the two heavy chains consist 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 pembrolizumab variant.

[0195] Typically, the amino acid sequence variants of the anti-PD-1 antibodies and antigen-binding fragments of the invention will have similarities to the reference antibody or antigen-binding fragment (e.g., heavy chain, light chain, V H 、V Lor humanized sequence) with an amino acid sequence having at least 75% amino acid sequence identity, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, 98% or 99%. Identity or homology with respect to a sequence is defined herein as 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, if necessary, to obtain the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions of the antibody sequence should not be interpreted as affecting sequence identity or homology.

[0196] Sequence identity refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. Sequence identity can be determined using the BLAST algorithm, wherein the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence. The following references relate to the BLAST algorithm that is often used for sequence analysis: BLAST algorithm: Altschul, SF, et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W., et al., (1993) Nature Genet. 3: 266-272; Madden, TL, et al., (1996) Meth. Enzymol. 266: 131-141; Altschul, SF, et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J., et al., (1997) Genome Res.7:649-656; Wootton, JC, et al., (1993) Comput. Chem. 17:149-163; Hancock, JM, et al., (1994) Comput. Appl. Biosci. 10:67-70; Alignment scoring system: Dayhoff, MO, et al., "A model of evolutionary change in proteins." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. MO Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, RM, et al., "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure. Structure, (1978) vol.5, suppl.3." MO Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, (1991) J. Mol. Biol. 219:555-565; States, DJ, et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, SF, et al., (1993) J. Mol. Evol.36:290-300; Alignment statistics: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, SF "Evaluating the statistical significance of multiple distinct local alignments." in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York.

[0197] Likewise, any type of light chain can be used in the compositions and methods herein. Specifically, kappa, lambda, or variants thereof can be used in the compositions and methods herein.

[0198] Table 2. Exemplary PD-1 antibody sequences

[0199]

[0200]

[0201]

[0202] rHuPH20 and its fragments

[0203] rHuPH20, also known as Consisting of the amino acid sequence in SEQ ID NO: 17, which is amino acid residues 36-482 of wild-type human PH20 in SEQ ID NO: 16 (amino acid residues 1-36 are a signal peptide sequence). In one embodiment, rHuPH20 or a variant or fragment is amino acid residues 36-464, 36-465, 36-466, 36-467, 36-468, 36-469, 36-470, 36-471, 36-472, 36-473, 36-474, 36-475, 36-476, 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO: 16; Amino acid residues 37-464, 37-465, 37-466, 37-467, 37-468, 37-469, 37-470, 37-471, 37-472, 37-473, 37-474, 37-475, 37-476, 37-477, 37-478, 37-479, 37-480, 37-481, 37-482 or 37-483 of NO:16; Amino acid residues 38-464, 38-465, 38-466, 38-467, 38-468, 38-469, 38-470, 38-471, 38-472, 38-473, 38-474, 38-475, 38-476, 38-477, 38-478, 38-479, 38-480, 38-481, 38-482 or 38-483 of NO:16; 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; 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 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 of SEQ ID NO: 16; or In a preferred embodiment, the rHuPH20 variant consists of 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 that disclosed in U.S. Patent No. 7,767,429, which is incorporated herein by reference in its entirety. ;

[0204] Table 3: Hyaluronidases and exemplary variants

[0205]

[0206]

[0207] Compositions comprising anti-PD-1 antibodies or antigen-binding fragments and rHuPH20 or variants or fragments

[0208] The present invention provides a composition comprising an anti-human PD-1 antibody or an antigen-binding fragment thereof having less than about 3.0% oxidation of methionine 105, and rHuPH20 or a variant or fragment thereof, wherein the anti-human PD-1 antibody or an antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the heavy chain variable region comprises three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8. In one embodiment, the anti-human PD-1 antibody or an antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4 and a heavy chain variable region comprising the amino acid sequence set forth 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 set forth in SEQ ID NO:5 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:10. In a further embodiment, the anti-human PD-1 antibody consists of two light chains and two heavy chains, wherein in at least a portion of the anti-human PD-1 antibody, the two light chains consist of the amino acid sequence set forth in SEQ ID NO:5, and the two heavy chains consist of the amino acid sequence set forth in any one of SEQ ID NOs:10-15 or a combination thereof. In a further embodiment, the anti-human PD-1 antibody consists of two light chains and two heavy chains, wherein in at least a portion of the anti-human PD-1 antibody, the two light chains consist of the amino acid sequence set forth in SEQ ID NO:5, and the two heavy chains consist of the amino acid sequence set forth in SEQ ID NO:11.

[0209] In one embodiment, the oxidation of methionine 105 is about 0.1-3.0%. In one embodiment, the oxidation of methionine 105 is 0.1-3.0%. In one embodiment, the oxidation of methionine 105 is about 0.2%-3.0%. In one embodiment, the oxidation of methionine 105 is 0.2%-3.0%. In another embodiment, the oxidation of methionine 105 is about 0.5-3.0%. In another embodiment, the oxidation of methionine 105 is 0.5-3.0%. In another embodiment, the oxidation of methionine 105 is about 0.5-2.5%. In another embodiment, the oxidation of methionine 105 is 0.5-2.5%. In another embodiment, the oxidation of methionine 105 is about 0.5-2.0%. In another embodiment, the oxidation of methionine 105 is 0.5-2.0%. In another embodiment, the oxidation of methionine 105 is about 0.5-1.5%. In another embodiment, the oxidation of methionine 105 is 0.5-1.5%.

[0210] In one embodiment, methionine oxidation % is measured by reduced peptide mapping followed by liquid chromatography and mass spectrometry. In one embodiment, reduced peptide mapping comprises the following steps: denaturing and reducing 100 μg of anti-PD-1 antibody in 100 μL 6M guanidine hydrochloride, 50 μM Tris-HCl, 50 μM EDTA, and 200 μM DTT; incubating the mixed sample in a thermomixer at 37°C with 300 rpm shaking for 30 min; alkylating with 5 μL 1M iodoacetamide protected from light at 25°C for 30 min; blocking unreacted iodoacetamide with 5 μL 200 μM DTT; adding lysyl endopeptidase (Lys-C) (Wako, 125-05061) enzyme (1:10 (wt:wt)) in 500 μL; incubating the digest in a thermomixer at 37°C for 60 min; quenching the digest with 15 μL 20% TFA.

[0211] In one embodiment, liquid chromatography and mass spectrometry include the following steps: inject 20 μL of the digested sample at 5°C onto a Waters Acquity liquid chromatography column (UPLC HSS T3 1.8 μm, 2.1 mm×150 mm, P / N: 186003540) with mobile phase A being 0.02% TFA in water and mobile phase B being 0.02% TFA in acetonitrile with a gradient of 0.1% B from 0 to 5 min, 0.1%-10% B from 5 to 7 min, followed by a linear increase to 35% B over the next 38 min, and MS1 data were collected using a QExactive Orbitrap MS (Thermo Fisher Scientific).

[0212] In another embodiment, 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 comprising a gradient of 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 of 0% mobile phase A from 0 to 2 minutes, 0%-100% mobile phase A from 2 to 52 minutes. Met 105 oxidation % is determined by the percentage of the pre-main peak (containing Met105 oxidation) relative to the sum of the pre-main peak (peak before the main peak), the main peak (most species), and the post-peak (peak after the main peak).

[0213] In a further aspect, the present invention provides a composition comprising an anti-human PD-1 antibody major species (which comprises an antibody consisting of two heavy chains and two light chains, each light chain comprising a light chain variable region, the light chain variable region comprising three light chain CDRs, the three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and each heavy chain comprising a heavy chain variable region, the heavy chain variable region comprising three heavy chain CDRs, the three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8) and acidic and basic species of the anti-human PD-1 antibody major species (wherein the amount of the major species is 65%-95%); and rHuPH20 or a variant or fragment. In one embodiment, the anti-human PD-1 antibody major species 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.

[0214] In a further aspect, the present invention provides a composition comprising an anti-human PD-1 antibody main species (which comprises an antibody consisting of two heavy chains and two light chains, each light chain comprising a light chain variable region, the light chain variable region comprising three light chain CDRs, the three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and each heavy chain comprising a heavy chain variable region, the heavy chain variable region comprising three heavy chain CDRs, the three heavy chain CDRs comprising 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 anti-human PD-1 antibody main species (wherein the amount of the acidic species is 1.0%-12.0%); and rHuPH20 or a variant or fragment thereof. In one embodiment, the anti-human PD-1 antibody main species 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.

[0215] In a further aspect, the present invention provides a composition comprising an anti-PD-1 antibody major species (which comprises an 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) and an acidic species of the anti-PD-1 antibody major species (wherein the amount of the acidic species is about 1.0-12.0%); and rHuPH20 or a variant or fragment. In another aspect, the present invention provides a composition comprising an anti-PD-1 antibody major species (which comprises an 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) and an acidic species and a basic species of the anti-PD-1 antibody major species (wherein the amount of the major species is about 65-95%); and rHuPH20 or a variant or fragment. In a further aspect, the present invention provides a composition comprising an anti-PD-1 antibody major species produced by Chinese hamster ovary cells (the Chinese hamster ovary cells contain a polynucleotide encoding a light chain and a polynucleotide encoding a heavy chain, or polynucleotides 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 anti-PD-1 antibody major species (wherein the amount of the acidic species is 1.0%-12.0%); and rHuPH20 or a variant or fragment. In one embodiment, the amount of the major species is about 65%-95%. In one embodiment, the amount of the major species is about 65%-85%. In one embodiment, the amount of the major species is 65%-80%. In another embodiment, the amount of the major species is about 70%-85%. In a further embodiment, the amount of the major species is about 70%-75%. In a further embodiment, the amount of the major species is at least about 65%.

[0216] 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 1 species is about 1%-4%. In another embodiment, the amount of acidic 1 species is about 2%-4%. In another embodiment, the amount of acidic 1 species 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 another embodiment, the amount of acidic variant species is about 2%-4%. In another embodiment, the composition further comprises about 12%-27% of alkaline species. In another embodiment, the composition further comprises about 15%-20% of alkaline species. In another embodiment, the amount of alkaline 1 species is about 4%-12%. In another embodiment, the amount of alkaline 1 species is about 8%-12%.

[0217] 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 1 species is 1%-4%. In another embodiment, the amount of acidic 1 species is 2%-4%. In another embodiment, the amount of acidic 1 species 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 another embodiment, the composition further comprises 12%-27% of alkaline species. In another embodiment, the composition further comprises 15%-20% of alkaline species. In a further embodiment, the amount of alkaline 1 species is 4%-12%. In a further embodiment, the amount of alkaline 1 species is 8%-12%.

[0218] In one aspect of measuring 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 MAbPacSCX-10G or Thermo MAbPac SCX50G. Those skilled in the art will appreciate that any industry equivalent post using the aforementioned post. In another embodiment, the weak cation exchange column with carboxylic acid functional group is used. In a further embodiment, the weak cation exchange column with carboxylic acid functional group has a particle diameter of 10um, a diameter of 4mm, and a length of 250mm. In one embodiment, the Thermo Scientific ProPac WCX-10 post is used for cation exchange method. In another embodiment, a Thermo Scientific ProPac WCX-10 column is used at 35°C with mobile phase (A) 24 mM MES pH 6.1, with 4% acetonitrile, and mobile phase (B) 20 mM sodium phosphate, 95 mM NaCl pH 8.0, with 4% acetonitrile, and a chromatogram is generated using detection at 280 nm. In one embodiment, the non-linear gradient is with: 22%-22% B for 0 min-0.6 min; 22%-29% B for 0.6 min-15.0 min; 29%-70% B for 15.0 min-30.0 min; 70%-100% B for 30.0 min-30.5 min; and 100%-100% B from 30.5-33.0 min. In a further embodiment, the cation exchange method is described in Example 5. In another aspect of measuring the major species, acidic species, or basic species, anion exchange chromatography is used.

[0219] In one embodiment, the antibody is produced by mammalian cells.

[0220] In another embodiment, the antibody is produced by CHO cells.

[0221] In another aspect of the above-mentioned embodiment, the composition comprises an antibody of about 5mg / ml-200mg / ml. In one embodiment, the composition comprises an antibody of about 25mg / ml-165mg / ml. In one embodiment, the composition comprises an antibody of about 25mg / ml. In one embodiment, the composition comprises an antibody of about 120mg / ml. In one embodiment, the composition comprises an antibody of about 130mg / ml. In one embodiment, the composition comprises an antibody of about 165mg / ml. In another embodiment, the composition comprises an antibody of 200mg-800mg. In another embodiment, the composition comprises an antibody of 200mg. In another embodiment, the composition comprises an antibody of 400mg.

[0222] In another aspect, the present invention provides a pharmaceutical composition or formulation as disclosed therein, comprising about 165 mg / mL of an anti-human PD-1 antibody, 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. In one embodiment, the pharmaceutical composition comprises about 130 mg / mL of an anti-human PD-1 antibody, 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.

[0223] In some embodiments of the formulations of the present invention, rHuPH20 or variants or fragments thereof are present at a concentration of about 1000 U / ml. In another embodiment, the concentration of rHuPH20 or variants or fragments thereof is about 1500 U / ml. In another embodiment, the concentration of rHuPH20 or variants or fragments thereof is about 2000 U / ml. In another embodiment, the concentration of rHuPH20 or variants or fragments thereof is about 3000 U / ml. In another embodiment, the concentration of rHuPH20 or variants or fragments thereof is about 4000 U / ml. In another embodiment, the concentration of rHuPH20 or variants or fragments thereof is about 5000 U / ml. In another embodiment, the concentration of rHuPH20 or variants or fragments thereof is about 6000 U / ml. In a further embodiment, the concentration of rHuPH20 or variants or fragments thereof is about 1000 U / ml-6000 U / ml. In a further embodiment, the concentration of rHuPH20 or variant or fragment thereof is about 2000 U / ml-5000 U / ml.

[0224] In some embodiments of the formulations of the invention, rHuPH20 or variants or fragments thereof are present at a concentration of about 150 U / ml. In another embodiment, the concentration of rHuPH20 or variants or fragments thereof is about 300 U / ml. In another embodiment, the concentration of rHuPH20 or variants or fragments thereof is about 600 U / ml. In another embodiment, the concentration of rHuPH20 or variants or fragments thereof is about 750 U / ml. In further embodiments, the concentration of rHuPH20 or variants or fragments thereof is about 150 U / ml-5000 U / ml.

[0225] Specific aspects and embodiments of pharmaceutical compositions

[0226] In one embodiment, the pharmaceutical composition comprises:

[0227] a) about 5 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0228] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0229] c) about 5 mM to about 20 mM histidine buffer; pH between about 5.0 to about 6.0;

[0230] d) about 6% to about 8% w / v sucrose;

[0231] e) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0232] In another embodiment, the pharmaceutical composition comprises:

[0233] a) about 5 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0234] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0235] c) about 5 mM to about 20 mM histidine buffer, pH between about 5.0 and about 6.0;

[0236] d) about 6% to about 8% w / v sucrose;

[0237] e) about 0.01% to about 0.04% w / v polysorbate 80 or 20; and

[0238] f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0239] In another embodiment, the pharmaceutical composition comprises:

[0240] a) about 5 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0241] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0242] c) about 10 mM histidine buffer, pH about 5.5;

[0243] d) about 7% w / v sucrose;

[0244] e) optionally, about 10 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0245] In a further embodiment, the pharmaceutical composition comprises:

[0246] a) about 5 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0247] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0248] c) about 10 mM histidine buffer, pH about 5.5;

[0249] d) about 7% w / v sucrose;

[0250] e) about 0.02% w / v polysorbate 80; and

[0251] f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0252] In one embodiment, the present invention provides a pharmaceutical composition comprising:

[0253] a) about 50 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0254] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0255] c) about 5 mM to about 20 mM histidine buffer;

[0256] d) about 6% to about 8% w / v sucrose;

[0257] e) from about 0.01% to about 0.04% w / v polysorbate 80; and

[0258] f) about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0259] In one embodiment, the present invention provides a pharmaceutical composition comprising:

[0260] a) about 50 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0261] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0262] c) about 5 mM to about 20 mM histidine buffer; and

[0263] d) about 6% to about 8% w / v sucrose.

[0264] In one embodiment, the present invention provides a pharmaceutical composition comprising:

[0265] a) about 50 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0266] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0267] c) about 5 mM to about 20 mM histidine buffer;

[0268] d) about 6% to about 8% w / v sucrose; and

[0269] e) from about 0.01% to about 0.04% w / v polysorbate 80.

[0270] In one embodiment, the present invention provides a pharmaceutical composition comprising:

[0271] a) about 50 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0272] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0273] c) about 5 mM to about 20 mM histidine buffer;

[0274] d) about 6% to about 8% w / v sucrose; and

[0275] e) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof. In one embodiment, the present invention provides a pharmaceutical composition comprising:

[0276] a) about 50 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0277] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0278] c) about 10 mM histidine buffer;

[0279] d) about 10 mM L-methionine or a pharmaceutically acceptable salt thereof;

[0280] e) about 7% w / v sucrose; and

[0281] f) about 0.02% w / v polysorbate 80.

[0282] In one embodiment, the present invention provides a pharmaceutical composition comprising:

[0283] a) about 50 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0284] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0285] c) about 10 mM histidine buffer; and

[0286] d) about 7% w / v sucrose.

[0287] In one embodiment, the present invention provides a pharmaceutical composition comprising:

[0288] a) about 50 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0289] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0290] c) about 10 mM histidine buffer;

[0291] d) about 7% w / v sucrose; and

[0292] e) about 0.02% w / v polysorbate 80.

[0293] In one embodiment, the present invention provides a pharmaceutical composition comprising:

[0294] a) about 50 mg / mL to about 175 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0295] b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof;

[0296] c) about 10 mM histidine buffer;

[0297] d) about 7% w / v sucrose; and

[0298] e) about 10 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0299] In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 5-165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 50-165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 75-165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 75-175 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 100-175 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 100-165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 130-165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 150-165 mg / ml. In the aforementioned embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 130 mg / ml. In the aforementioned embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 165 mg / ml.

[0300] In any of the above specific aspects and embodiments, one of the anti-PD-1 antibodies or antigen-binding fragments thereof described in the section "Compositions comprising an anti-PD-1 antibody or antigen-binding fragment and rHuPH20 or variant or fragment" is used.

[0301] In any of the specific aspects and embodiments below, one of the anti-PD-1 antibodies or antigen-binding fragments thereof described in the "Anti-PD-1 Antibodies or Antigen-binding Fragments" section is used.

[0302] In another aspect, the present invention provides a pharmaceutical composition comprising:

[0303] a) about 5 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the heavy chain variable region comprising three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8;

[0304] b) about 2000 U / ml of rHuPH20 or variant of SEQ ID NO: 17 or 18;

[0305] c) about 5 mM to about 20 mM histidine buffer; pH between about 5.0 to about 6.0;

[0306] d) about 3% to about 10% w / v non-reducing disaccharide;

[0307] e) optionally, from about 0.005% to about 0.4% w / v of a nonionic surfactant;

[0308] f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0309] In one embodiment, the non-reducing disaccharide is selected from the group consisting of 6% to 8% w / v sucrose and trehalose.

[0310] In yet another aspect, the present invention provides a pharmaceutical composition comprising:

[0311] a) about 5 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the heavy chain variable region comprising three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8;

[0312] b) about 2000 U / ml of rHuPH20 or variant of SEQ ID NO: 17 or 18;

[0313] c) about 5 mM to about 20 mM histidine buffer; pH between about 5.0 to about 6.0;

[0314] d) about 6% to about 8% w / v sucrose;

[0315] f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0316] In another aspect, the present invention provides a pharmaceutical composition comprising:

[0317] a) about 5 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0318] b) about 2000 U / ml of rHuPH20 or variants;

[0319] c) about 5 mM to about 20 mM histidine buffer, pH between about 5.0 and about 6.0;

[0320] d) about 6% to about 8% w / v sucrose;

[0321] e) about 0.01% to about 0.04% w / v polysorbate 80 or 20; and

[0322] f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0323] In another aspect, the present invention provides a pharmaceutical composition comprising:

[0324] a) about 5 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0325] b) about 2000 U / ml of rHuPH20 or variants;

[0326] c) about 10 mM histidine buffer, pH about 5.5;

[0327] d) about 7% w / v sucrose; and

[0328] f) optionally, about 10 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0329] In another aspect, the present invention provides a pharmaceutical composition comprising:

[0330] a) about 5 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof;

[0331] b) about 2000 U / ml of rHuPH20 or variants;

[0332] c) about 10 mM histidine buffer, pH about 5.5;

[0333] d) about 7% w / v sucrose;

[0334] e) about 0.02% w / v polysorbate 80; and

[0335] f) optionally, about 10 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0336] In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 50-165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 100-165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 130-165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 150-165 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.

[0337] In yet another aspect, the present invention provides a pharmaceutical composition comprising:

[0338] a) about 50 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the heavy chain variable region comprising three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8;

[0339] b) about 2000 U / ml of rHuPH20 or variant of SEQ ID NO: 17 or 18;

[0340] c) a buffer having a pH between about 5.0 and about 6.0;

[0341] d) about 3% to about 10% w / v non-reducing disaccharide;

[0342] e) from about 0.005% to about 0.4% w / v of a nonionic surfactant;

[0343] f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0344] In yet another aspect, the present invention provides a pharmaceutical composition comprising:

[0345] a) about 50 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the heavy chain variable region comprising three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8;

[0346] b) about 2000 U / ml of rHuPH20 or variant of SEQ ID NO: 17 or 18;

[0347] c) a histidine or acetate buffer at a pH between about 5.0 and about 6.0;

[0348] d) about 6% to about 8% w / v sucrose or trehalose;

[0349] e) optionally, from about 0.02% to about 0.2% w / v of a nonionic surfactant;

[0350] f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0351] In yet another aspect, the present invention provides a pharmaceutical composition comprising:

[0352] a) about 50 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the heavy chain variable region comprising three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8;

[0353] b) about 2000 U / ml of rHuPH20 or variant of SEQ ID NO: 17 or 18;

[0354] c) about 1 mM to about 30 mM histidine or acetate buffer, pH between about 5.0 and about 6.0;

[0355] d) about 6% to about 8% w / v sucrose or trehalose;

[0356] e) optionally, from about 0.01% to about 0.05% w / v polysorbate 80;

[0357] f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0358] In yet another aspect, the present invention provides a pharmaceutical composition comprising:

[0359] a) about 50 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody or an antigen-binding fragment thereof, wherein the antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprising three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the heavy chain variable region comprising three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8;

[0360] b) about 2000 U / ml of rHuPH20 or variant of SEQ ID NO: 17 or 18;

[0361] c) about 1 mM to about 30 mM histidine or acetate buffer, pH between about 5.0 and about 6.0;

[0362] d) about 6% to about 8% w / v sucrose or trehalose;

[0363] e) optionally, from about 0.1% to about 0.4% w / v of a poloxamer, preferably from 0.1% to about 0.2% w / v of poloxamer 188 or 407;

[0364] f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

[0365] In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 100-165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 130-165 mg / ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibody or antigen-binding fragment is about 150-165 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.

[0366] In any of the above specific aspects and embodiments, rHuPH20, or one of its variants or fragments, such as described in the section entitled "rHuPH20, or a variant or fragment thereof" is used.

[0367] In some embodiments of the invention, any formulation described herein is in an aqueous solution. In alternative embodiments, the invention provides a lyophilized formulation prepared by lyophilizing an aqueous formulation to provide a reconstituted formulation of the invention, as discussed more fully below.

[0368] Formulation excipients

[0369] In an embodiment of the invention, the non-reducing disaccharide is sucrose. In a further embodiment, the non-reducing disaccharide is trehalose.

[0370] In some embodiments, the non-reducing disaccharide is about 6% to about 8% w / v sucrose. In some embodiments, the non-reducing disaccharide is about 6% to about 8% w / v trehalose.

[0371] In further embodiments, sucrose, trehalose is present in an amount 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.

[0372] In addition to the anti-PD-1 antibody or antigen-binding fragment thereof and rHuPH20 or variant or fragment thereof, and the above-mentioned amount / concentration of non-reducing disaccharides, the formulation of the present invention may also include a buffer. In some embodiments, the buffer is present in an amount of about 5mM to about 20mM. In further embodiments, the buffer has a pH in the range of about 5.0 to about 6.0. In 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.

[0373] In specific 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 specific embodiments of the invention, the buffer is histidine or acetate at 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, it is preferred that the buffer is not acetate because the acetate buffer system is incompatible with the lyophilization process.

[0374] When a pH range is listed, e.g., "a pH between pH 5.5 and 6.0," the range is intended to include the end values ​​of the listed values. Unless otherwise indicated, for lyophilized formulations, pH refers to the pH of the lyophilized formulation of the invention after reconstitution. pH is typically measured at 25°C using a standard glass bulb pH meter. As used herein, a solution containing "histidine buffer at pH X" refers to a solution of pH X and containing a histidine buffer, i.e., pH refers to the pH of the solution.

[0375] In addition to the anti-PD-1 antibody or antigen-binding fragment thereof and rHuPH20 or variant or fragment thereof, the non-reducing disaccharide and the buffer in the above amounts / concentrations, the formulation of the present invention may also include an antioxidant. In an embodiment of the present invention, the antioxidant is methionine. In an embodiment of the present invention, the antioxidant is L-methionine or a pharmaceutically acceptable salt thereof. In a further embodiment, methionine is L-methionine. In other embodiments, the antioxidant is L-methionine HCl.

[0376] In some embodiments, antioxidant (such as L-methionine) is present in the preparation of the present invention with an amount of 1mM to about 20mM. In another embodiment, antioxidant is present in an amount of about 5mM to about 20mM. In a further embodiment, antioxidant is present in an amount of about 5mM to about 15mM. In a further embodiment, antioxidant is present in an amount of about 5mM to about 10mM. In other embodiments, antioxidant is present in an amount of about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM or about 20mM.

[0377] In addition to the anti-PD-1 antibody or antigen-binding fragment thereof, rHuPH20 or variant or fragment thereof, non-reducing disaccharide, buffer and antioxidant in the above amounts / concentrations, the formulation of the present invention may also contain a surfactant. Surfactants that can be used in the formulation of the present invention include, but are not limited to, nonionic surfactants, such as polyoxyethylene sorbitan fatty acid esters (polysorbates, marketed under the trade name (sold by Uniquema Americas LLC, Wilmington, DE), including polysorbate-20 (polyoxyethylene sorbitan monolaurate), polysorbate-40 (polyoxyethylene sorbitan monopalmitate), polysorbate-60 (polyoxyethylene sorbitan monostearate) and polysorbate 80 (polyoxyethylene sorbitan monooleate); poloxamers such as poloxamer 188 or poloxamer 407.

[0378] 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 required to stabilize the active pharmaceutical ingredient (i.e., anti-PD-1 antibody or antigen-binding fragment thereof or rHuPH20 or variant or fragment thereof) in the formulation. 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 invention, the surfactant is present in the formulation in the following amounts: about 0.01% to about 0.04%; about 0.01% to about 0.03%, about 0.01% to about 0.02%, about 0.015% to about 0.04%; about 0.015% to about 0.03%, about 0.015% to about 0.02%, about 0.02% to about 0.04%, about 0.02% to about 0.035%, or about 0.02% to about 0.03%. In a specific embodiment, the surfactant is present in an amount of about 0.02%. In alternative embodiments, the surfactant is present in an amount of about 0.01%, about 0.015%, about 0.025%, about 0.03%, about 0.035%, or about 0.04%.

[0379] 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.

[0380] In a specific embodiment, the formulation of the present invention comprises about 0.01% to about 0.04% PS80. In a further embodiment, the formulation of the present invention comprises 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% PS80. In a specific embodiment, the formulation of the present invention comprises about 0.02% PS80.

[0381] In a specific embodiment, the formulations of the invention comprise from about 0.1% to about 0.4% of poloxamer. In a further embodiment, the formulations of the invention comprise 0.1-0.2% of poloxamer 188 or 407. In a further embodiment, the formulations of the invention comprise 0.2% of poloxamer 188. In a further embodiment, the formulations of the invention comprise about 0.1% of poloxamer 407.

[0382] Freeze-dried pharmaceutical composition

[0383] The lyophilized formulations of therapeutic proteins have several advantages. Lyophilized formulations generally have better chemical stability than solution formulations, thereby extending the shelf life. Depending on clinical factors, such as route of administration or dosage, lyophilized formulations can also be reconstituted at different concentrations. For example, if subcutaneous administration is required, the lyophilized formulation can be reconstituted at high concentrations (i.e., small volumes), or if intravenously administered, reconstituted at lower concentrations. If a specific subject requires a high dose, a high concentration may also be necessary, particularly if subcutaneously administered, where the injection volume must be minimized. One such lyophilized antibody formulation is disclosed in U.S. Patent No. 6,267,958, the entire contents of which are incorporated herein by reference. Another lyophilized formulation of therapeutic proteins is disclosed in U.S. Patent No. 7,247,707, the entire contents of which are incorporated herein by reference.

[0384] Typically, lyophilized formulations are prepared in anticipation of reconstitution at high concentrations of drug product (DP), i.e., are expected to be reconstituted in low volumes of water. Subsequent dilution with water or isotonic buffer can be easily used to dilute the DP to a lower concentration. Typically, the level of excipients included in the lyophilized formulations of the present invention is such that when reconstituted at a high DP concentration, for example, for subcutaneous administration, a substantially isotonic formulation is formed. Reconstitution in a larger volume of water to obtain a lower DP concentration will inevitably reduce the tonicity of the reconstituted solution, but this reduction may not be significant in non-subcutaneous (e.g., intravenous) administration. If isotonicity is required at a lower DP concentration, the lyophilized powder can be reconstituted in a standard low volume of water and then further diluted with an isotonic diluent (e.g., 0.9% sodium chloride).

[0385] In one embodiment of the invention, a formulation comprising a humanized anti-PD-1 antibody (or an antigen-binding fragment thereof) and rHuPH20 or a variant or a fragment thereof is formulated into a lyophilized powder for reconstitution and for subcutaneous administration. In certain embodiments, the lyophilized formulation is reconstituted with sterile water for injection before use. If necessary, 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 is capable of reconstructing the anti-PD-1 antibody at a high concentration, for example, about 50, 60, 75, 100, 125, 130, 150, 165, 175, 185 or 200 mg / mL.

[0386] Lyophilized formulations are essentially dry by definition, so the concept of concentration is not applicable when describing them. It is more useful to describe lyophilized formulations by the weight of the components in the unit dose vial, but it is also problematic because it varies with different doses or vial sizes. When describing the lyophilized formulations of the present invention, it is useful to express the amount of the 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 characteristics of the lyophilized formulations of the present invention and is independent of the vial size, dosage, and reconstitution regimen.

[0387] In other embodiments, the formulation of anti-human PD-1 antibody or antigen-binding fragment thereof and rHuPH20 or variant or fragment thereof is defined according to a pre-lyophilized solution used to prepare a lyophilized formulation, such as a pre-lyophilized solution. In one embodiment, the pre-lyophilized solution comprises an antibody or antigen-binding fragment thereof at a concentration of about 25 mg / mL-200 mg / mL and rHuPH20 or variant or fragment thereof at 150 U / ml-8000 U / ml. The pH of such a pre-lyophilized solution may be about 5.0-6.0, or about pH 5.5.

[0388] In other embodiments, the lyophilized formulation of an anti-human PD-1 antibody or its antigen-binding fragment and rHuPH20 or a variant or a fragment thereof is defined according to a reconstitution solution produced from the lyophilized formulation. The present invention provides a liquid formulation reconstituted by a lyophilized formulation. The reconstitution solution may include an 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 150, 300, 600, 900, 1000, 1500, 2000, 3000, 4000 or 5000 U / ml of rHuPH20 or its variant or fragment. The pH of such a reconstitution solution may be about 5.5, or range from about pH 5.0 to about 6.0.

[0389] The lyophilized formulation of the present invention is formed by freeze drying (freeze drying) of a pre-lyophilized solution. Freeze drying is accomplished by freezing the formulation and then sublimating water at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic point or disintegration temperature of the formulation. Typically, the shelf temperature for primary drying will be in the range of about -30°C to 25°C (assuming that the product remains frozen during primary drying) at a suitable pressure, typically ranging from about 50 to 250 mTorr. The formulation, size and type of container (e.g., glass bottle) holding the sample and the volume of the liquid will determine the time required for drying, ranging from a few hours to a few days (e.g., 40-60 hours). The secondary drying stage can be carried out at about 0°C-40°C, depending primarily on the type and size of the container and the type of protein used. The secondary drying time is determined by the residual moisture content required in the product and typically requires at least about 5 hours. Typically, the moisture content of the lyophilized formulation is less than about 5%, preferably less than about 3%. The pressure may be the same as the pressure used in the primary drying step. Freeze drying conditions may vary depending on the formulation and bottle size.

[0390] In some cases, it may be desirable to lyophilize the protein formulation in a container in which the protein is reconstituted to avoid a transfer step. In this case, the container may be, for example, a 3, 5, 10, 20, 50 or 100 cc vial.

[0391] Reconstitution is typically performed at a temperature of about 25°C to ensure complete hydration, although other temperatures may be used as desired. The time required for reconstitution will depend, for example, on the type of diluent, the amount of excipient(s) and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

[0392] Liquid pharmaceutical composition

[0393] Liquid antibody formulations can be prepared by taking a liquid form of a drug substance (e.g., an anti-human PD-1 antibody, and / or rHuPH20 or a variant or fragment thereof) and exchanging its buffer into a desired buffer. There is no lyophilization step in this embodiment. 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 final formulated drug substance is filtered, for example using a 0.22 μm filter, and filled into a final container (e.g., a glass bottle or a syringe). An example of such a liquid formulation is a final liquid formulation comprising 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).

[0394] Protein expression

[0395] The method for making antibody relates to the mammalian cell of cultivating secretory antibody.The mammalian cell of this type of culture is typically prepared by the recombinant DNA technology related to transient or stable transfection, for example, the plasmid construct (expression vector) collected from the cloning step can be used cationic lipid, polyethyleneimine, LipofectamineTM or ExpiFectamineTM or electroporation transfection to multiple host cells (for example, mammalian cell, HEK 293 or CHO cell, bacterial cell, insect cell, yeast cell) for expression.Technicians know many suitable transfection methods to achieve the expression of recombinant antibodies.Alternately, the method for the stable genome integration of the expression cassette encoding the target protein can be adopted to prepare the production cell line of the mammalian cell of secretory protein. (See, e.g., Zhang, Crispr-Cas Systems and Methods forAltering Expression Of Gene Products, WO2014093661A2; Frendewey et al., Methods and Compositions for the Targeted Modification of a Genome, US9228208 B2; Church et al., Multiplex Automated Genome Engineering, WO2008052101A2, US8153432 B2; Bradley et al., Methods Cells and Organisms, US2015 / 0079680A1; Begemann et al., Compositions and Methods for Modifying Genomes, WO2017141173A2; Gill et al., Nucleic acid-guided nucleases, US9982279 B1; Minshull et al., Enhanced nucleic acid constructs foreukaryotic gene expression, US9428767B2, US9580697B2, US9574209B2; Minshull et al., DNA Vectors, Transposons And Transposases For Eukaryotic Genome Modification, US10041077B2).

[0396] In other embodiments, WO2020068631 describes expression cassettes that effectively integrate into eukaryotic transcriptional activity hotspots and ensure long-term stable and consistent expression of the target gene (GOI). These expression cassettes can be transfected into various host CHO cell lines, including CHOK1SVTM (Lonza; Slough, UK), HD-BIOP1 (Horizon Discovery, UK), (Sigma-Aldrich, St. Louis, MO) and GS knockout CHO host cell lines. In one embodiment, the expression vector comprises:

[0397] (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 a gene of interest (GOI), IRES, a polynucleotide encoding a eukaryotic selectable marker, a poly A signal, and a second insulator;

[0398] (b) two ITR sequences flanking the first expression cassette;

[0399] (c) a second expression cassette comprising a polynucleotide encoding a bacterial selectable marker; and

[0400] (d) Bacterial plasmid origin of replication.

[0401] 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 a piggyBac ITR. In one embodiment, the insulator is the chicken β-globin HS4 insulator.

[0402] In one embodiment, the eukaryotic selectable marker is neomycin phosphotransferase, histidinol dehydrogenase, hygromycin B phosphotransferase, xanthine guanine phosphoribosyltransferase, dihydrofolate reductase, tryptophan synthetase, puromycin N-acetyltransferase, 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 various expression vectors provided herein, bacterial selectable markers are ampicillin resistance genes, tetracycline resistance genes, hygromycin resistance genes, kanamycin resistance genes, blasticidin resistance genes, etc. In one embodiment, the bacterial selectable marker is an ampicillin resistance gene.

[0403] Upstream cell culture process

[0404] Surprisingly, the continuous perfusion upstream process provided a pembrolizumab composition with lower % Met105 oxidation, lower % acidic species, and / or higher % major species compared to pembrolizumab produced by a fed-batch process.

[0405] The host cells used to produce the antibodies of the present invention can be cultured in a variety of cell culture media, such as CD-CHO liquid or CD-CHO AGT TMCommercially available media such as powder (Life Technologies), Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma)) are suitable for culturing host cells. In addition, Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87 / 00195; or U.S. Reissue No. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented with other components as needed at appropriate concentrations known to those skilled in the art. For example, hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, sodium bicarbonate, calcium, iron, potassium, zinc, copper sulfate, ferric citrate, manganese, magnesium, and phosphate), nucleotides (e.g., adenosine, adenine, thymidine, cytidine, guanosine, uridine, purine), any of the 20 amino acids (tyrosine, cysteine, cystine, glutamic acid), vitamins or supplements (choline, inositol, thiamine, folic acid, biotin, calcium, nicotinamide, p-aminobenzoic acid, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, linoleic acid, selenite, glycine, putrescine, ethanolamine), a selection agent that confers resistance or survival to a selection marker, such as an antibiotic (e.g., geneticin, neomycin, hygromycin B, puromycin, bleomycin, gentamicin TM ), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose, galactose, or an equivalent energy source, so that the physiological conditions of the cells in or on the culture medium promote the host cells to express the protein of interest. Typically, the cell culture medium used for perfusion is more concentrated to allow cells to continue to grow at higher densities. In one embodiment, 2-10 g / L of a nonionic surfactant (e.g. P188) 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 ppb-35 ppb. Depending on the cell density and the uptake of copper by the cells, the copper concentration in the perfusion bioreactor can range from 1 ppb to 35 ppb. The culture medium is preferably serum-free. In some embodiments, the aqueous medium is a liquid so that the host cells are cultured in a cell suspension in the liquid medium.

[0406] Culture conditions, such as temperature (typically about 37°±1°C for mammalian cells), pH (typically, but not necessarily, the cell culture medium is maintained in the range of about pH 6.5-7.5), oxygenation, etc., will be apparent to the skilled artisan. In some embodiments, the dissolved oxygen level is about 15%-100%, and the pH is about 6.7-7.3. Clearly, there will be slight variations in temperature, pH, or other culture conditions over time and at different locations in the culture vessel (i.e., bioreactor), such that there is an operating range for these parameters. (See also, for example, Oguchi et al., pH Condition intemperature 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, RJ 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)). After culturing the transfected or transformed host cells, the antibodies are directly secreted into the cell culture medium (by employing an appropriate secretion targeting signal peptide) and harvested therefrom.

[0407] The perfusion bioreactor can be fluidly connected to the affinity chromatography step with an uninterrupted flow from the bioreactor (directly or indirectly via an intervening unit operation, such as a buffer vessel) to the affinity chromatography system. Some embodiments of the present invention include a first disposable buffer vessel (SUSV1) adapted to receive a volume of cell-free permeate removed from one or more perfusion bioreactors. These permeate volumes can be automatically and fluidly supplied to the SUSV1 from one or more perfusion bioreactors.

[0408] The anti-PD-1 antibody or antigen-binding fragment of the present invention is produced or can be obtained by the following steps:

[0409] a) perfusing mammalian host cells in a cell culture medium in a perfusion bioreactor by applying a perfusion rate of at least about 0.25-6.0 vessel volumes / day (vvd), wherein the host cells comprise a polynucleotide encoding a light chain variable region and a polynucleotide encoding a heavy chain variable region, or a polynucleotide encoding a light chain variable region and a heavy chain variable region of an antibody or antigen-binding fragment of an anti-human PD-1 antibody;

[0410] b) continuously harvesting antibodies from the cell culture fluid to obtain a cell culture harvest fluid;

[0411] c) optionally, transferring the cell culture harvest fluid (HCCF) to a buffer vessel with a residence time of about 0.5 to 8 hours; and

[0412] d) purifying the cell culture harvest successively by affinity chromatography steps to obtain a purified composition.

[0413] In one embodiment, the above method comprises the following steps before step a):

[0414] (i) Using mammalian host cells in cell culture medium at a concentration of about 0.2-0.6 x 10 6 The perfusion bioreactor was inoculated with a cell density of 10 cells / ml.

[0415] ii) growing mammalian cells in a cell culture medium in a perfusion bioreactor to approximately 1.0 to 6.0 x 10 6 The cell density was 10 cells / ml.

[0416] In one embodiment, in step i), the cell density on day 1 is about 0.20 to 0.6 x 10 6 In one embodiment, in step i), the cell density on day 1 is about 0.25 to 0.5 x 10 6 In another embodiment, in step i), the cell density on day 1 is about 0.4 to 0.5 x 10 6 In another embodiment, in step a), perfusion is started on day 3. In another embodiment, in step a), about 2 to 10 x 10 6 In another embodiment, in step a), perfusion is started at a cell density of about 4 to 8 x 10 6In another embodiment, step (ii) grows the mammalian host cells in a cell culture medium to a cell density of about 2.0 to 6.0 x 10 cells / ml in a perfusion bioreactor. 6 The cell density was 10 cells / ml.

[0417] The perfusion rate can start at about 0.5 vvd, and can be increased to 6 vvd continuously, gradually or incrementally from about 0.5 vvd. In one embodiment, in step a), the perfusion rate is about 0.5 vvd to 4 vvd. In one embodiment, in step a), the perfusion rate is about 0.5 vvd to 2 vvd. Preferably, the perfusion rate remains 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 on the 3rd day is about 0.5 vvd, the perfusion rate on the 4th day is about 1 vvd and the perfusion rate on the 5th day is about 2 vvd. In a further embodiment, step d) is performed on or after the 5th day. In a further embodiment, cell expulsion is performed to maintain a specific cell density (e.g., about 80 to 100 x 10 6 In one embodiment, the capacitance value during perfusion is about 70 pF / cm-90 pF / cm or 80 pF / cm. In one embodiment, the permeate titer is 0.2-2 g / L. In one embodiment, the maximum cell density during perfusion is about 80-100 x 10 6 In one embodiment, the maximum cell density during perfusion is about 80-150 x 10 6 In one embodiment, the maximum cell density during perfusion is about 100 x 10 6 In one embodiment, the mammalian host cell is a CHO cell. In one embodiment, the host cell comprises 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 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 ppb-35 ppb. In a further embodiment, the copper concentration in the perfusion bioreactor ranges from 1 ppb to 35 ppb.

[0418] On the other hand, continuous harvesting is performed by setting a constant permeability to obtain cell-free permeate through a hollow fiber membrane connected to the perfusion bioreactor. In one embodiment, the perfusion rate is equal to the sum of the permeability rate and the cell discharge rate. A typical perfusion system with a feed stream, a perfusion stream, and a cell discharge stream is described in Goudar, CT & Chen, C. & Le, H.. (2015). SBE special section: Biopharmaceuticals-Continuous processing in upstream operations. Page 111.

[0419] Downstream Process

[0420] The antibody or antigen-binding fragment composition from the upstream process can be further subjected to a continuous or semi-continuous downstream purification, or a downstream batch purification process.

[0421] Affinity chromatography separates molecules based on highly specific interactions between the target molecule and the functional groups of the resin (e.g., interactions between antigens and antibodies, enzymes and substrates, receptors and ligands, or proteins and nucleic acids). Some commonly used affinity chromatography resins include protein A or protein G resins for purifying antibodies, avidin biotin resins for purifying biotin / avidin and its derivatives, glutathione resins for purifying GST-tagged recombinant proteins, heparin resins for separating plasma coagulation proteins, IMAC resins for purifying proteins that specifically interact with metal ions, etc. The operating conditions of each affinity chromatography depend on the mechanism of interaction and the factors that affect the interaction. Commercial affinity chromatography resins include, but are not limited to, MabSelect Sure, UNOsphere SUPrATM, and In one embodiment, the affinity chromatography step is Protein A chromatography performed in bind and elute mode.

[0422] In one embodiment, the cell culture harvest is purified using protein A affinity chromatography, comprising the following steps:

[0423] a) binding HCCF to a stationary phase;

[0424] b) eluting the antibody or antigen-binding fragment from the protein A stationary phase with an elution solution;

[0425] c) Optionally, cleaning and sterilizing the stationary phase for repeated cycles.

[0426] In one embodiment, prior to step (a), the stationary phase is equilibrated with an equilibration solution. In one embodiment, one or more impurities are present in the flow-through of step a).

[0427] In another aspect of the method, after step a) but before step b), the method further comprises the step of washing the stationary phase with one or more washing solutions. In one embodiment, one or more impurities are removed from the washing step. In one embodiment, the washing solution or elution solution comprises a salt, preferably a monovalent metal ion salt, such as NaCl or KCl. In some embodiments, the washing solution comprises about 400mM-600mM NaCl or KCl. In another embodiment, the washing solution comprises about 500mM NaCl or KCl. In another embodiment, the washing solution comprises about 400mM-500mM NaCl or KCl. In a further embodiment, the first, second and third washing solutions comprise about 5mM-20mM sodium phosphate, and the second washing solution further comprises about 400mM-600mM NaCl or KCl. In a further embodiment, the first, second and third washing solutions comprise about 10mM sodium phosphate, and the second washing solution further comprises about 500mM NaCl or KCl.

[0428] In some embodiments, the pH of the wash or elution solution is about 6-7. In some embodiments, the pH of the wash or elution solution is about 6.5. In another embodiment, the elution solution comprises about 5mM-50mM sodium acetate. In another embodiment, the elution solution comprises about 5mM-30mM sodium acetate. In another embodiment, the elution solution comprises about 20mM sodium acetate.

[0429] In another aspect, the elution solution comprises about 5mM-50mM sodium acetate. In another embodiment, the elution solution comprises about 5mM-30mM sodium acetate. In another embodiment, the elution solution comprises about 20mM sodium acetate. In one embodiment, the elution solution has a pH of about 3.5-3.6. In another embodiment, the elution solution has a pH of about 3-4.

[0430] In one embodiment, the affinity chromatography is performed in a continuous multi-column chromatography (at least two, three or four columns) system, allowing uninterrupted flow of HCCF to the chromatography platform.

[0431] In one embodiment, a perfusion bioreactor or perfusion system comprising HCCF is fluidically connected to the affinity chromatograph indirectly or directly. In one embodiment, the affinity chromatograph is fluidically connected to the perfusion bioreactor.

[0432] In another embodiment, the perfusion bioreactor or system comprising HCCF is fluidically connected to the affinity chromatography via a buffer container. The size of the buffer container is set to control the residence time that the fluid control volume spends in the buffer container. In some embodiments of the present invention, the average residence time is 0.5-30 hours. In some embodiments of the present invention, the average residence time is 0.5-20 hours. In some embodiments of the present invention, the average residence time is 0.5-8 hours. In certain embodiments, the average residence time is 2 hours. In certain embodiments, the average residence time is 1 hour. In certain embodiments, the average residence time is 0.5 hours. HCCF can be stored in a buffer container at 4°C-25°C. In other embodiments, the loading residence time (from leaving 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 HCCF flow rate to the buffer container is equal to the feed rate from the buffer container to the continuous multi-column chromatography system. Alternatively, before being fed to the affinity chromatography, HCCF can be stored in a container at -40°C to -80°C. In one embodiment, the antibody is protected from light during storage in the buffer vessel or container.

[0433] In further embodiments, affinity chromatography is followed by one or more steps of viral inactivation, depth filtration, secondary chromatography, tertiary polishing chromatography, viral filtration, ultrafiltration, diafiltration, single-pass tangential flowfiltration, and an inline diafiltration module, optionally fluidically connected, or fluidically connected through a buffer vessel or connected through a holding vessel.

[0434] In one aspect, the affinity chromatography is fluidly connected to a virus inactivation system with uninterrupted flow, and optionally to a depth filtration system with uninterrupted flow, fluidly connected to a second chromatography system, an optional third polishing chromatography system, a virus filtration system, and an ultrafiltration / diafiltration system, and all connected to the above upstream processing steps with uninterrupted flow using optional intervening buffer containers, and are connected to each other sequentially. See WO2020168315 Figure 1 C. The final ultrafiltration step may consist of a single pass tangential flow filtration step and an online diafiltration step fluidly connected or operated through an optional buffer vessel to maintain continuity from the affinity chromatography step to the online diafiltration product.

[0435] On the other hand, the affinity chromatography is fluidly connected to the virus inactivation system with uninterrupted flow, and optionally to the depth filtration system with uninterrupted flow, a holding vessel (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 are fluidly connected to each other in uninterrupted flow or batch mode, with an optional intervening buffer vessel or holding vessel (i.e., a holding vessel if there are two or more batch steps or operations), as the case may be. See, for example, WO2020168315 Figure 1 D. The final ultrafiltration step may consist of a single pass tangential flow filtration step and an in-line diafiltration step either directly connected or operated through an optional buffer vessel to maintain continuity from the affinity chromatography step to the in-line diafiltration product.

[0436] In some embodiments of the present invention, the affinity chromatography step is followed by a virus inactivation step. For virus inactivation, the collection of affinity chromatography eluates is pooled into one of two virus inactivation collection containers. After collecting a discrete number of eluates in the collection container, the combined volume undergoes automatic pH adjustment from the elution pH to the virus inactivation pH (pH = 3.4-3.7) by adding an acid solution, and is subsequently adjusted to a neutral pH by the addition of 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, new affinity chromatography eluates are collected in a second virus inactivation collection container. Switching between two virus inactivation collection containers allows unit operations to run continuously. In some embodiments of the present invention, the virus inactivation product (VIP) is transferred to a buffer container and continuously supplied to other downstream unit operations.

[0437] In some embodiments of the invention, operations downstream of the virus inactivation system / neutralization system involve continuous processing by transferring the VIP to a buffer container, which can be fed to a second chromatography step or optionally filtered by depth filtration to produce a filtered virus inactivated product pool (FVIP). Semi-continuous flow in these unit operations is maintained by periodically cycling the depth filtration consumables or the second chromatography column. In these cases, flow is suspended during the flushing and regeneration of the phases between consumable switching and during the non-loading steps of the chromatography phase.

[0438] In some embodiments of the present invention, operations downstream of the virus inactivation system / neutralization system involve continuous processing of the virus inactivation product pool (which may also be optionally filtered by depth filtration to produce a filtered virus inactivated product pool (FVIP)); in such embodiments, the virus inactivation product pool is collected in a collection container, and in subsequent batch steps or operations, the purified product pool or virus-free filtrate may optionally be collected in other collection containers between steps. In such discrete operating modes, batch modes, or batch mode processing, one or more collection containers or interchangeable "one or more collection tanks" from one step (which may also be considered as "one or more feed tanks" for subsequent steps in certain embodiments) lack automatic control of a buffer container, and although the collection container (or feed tank) may be physically similar to a buffer container, such a collection container (or interchangeably, a "collection tank") or feed tank is referred to as a "holding vessel", or interchangeably referred to as an "HV" (e.g., HV1, HV2, HV3, HV4, or HV5). A "holding vessel" may be a disposable holding vessel (SUHV) which is different from a disposable collection vessel (SUCV, such as SUCV1 or SUCV2) in a manufacturing process step or operation group in a continuous or semi-continuous format. Figure 1 D.

[0439] The affinity chromatography step may be followed by a second and / or third chromatography step to remove, for example, protein aggregates, host cell proteins or DNA. IEX chromatography separates molecules according to the net charge of the molecule. Separation occurs due to competition between the charged molecule of interest and the counterion for the oppositely charged ligand groups on the IEX chromatography resin. The binding strength of the molecule to the IEX resin depends on the net charge of the molecule, which is affected by operating conditions (e.g., pH and ionic strength). IEX resins include AEX resins and CEX resins. AEX resins may contain substituents, such as diethylaminoethyl (DEAE), trimethylaminoethyl (TMAE), quaternary aminoethyl (QAE) and quaternary amine (O) groups. CEX resins may contain substituents, such as carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). 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-agarose and sepharose can be obtained from GE Healthcare, Piscataway, NJ. In addition, ethylene glycol-methacrylate copolymers derived from DEAE and CM such as TOYOPEARLTM DEAE-650S or M and TOYOPEARLTM CM-650S or M can be obtained from Toso Haas, Philadelphia, PA. POROSTM HS, POROSTMHQ, POROSTM XS can be obtained from Thermo Fisher Scientific, Waltham, MA. In one embodiment, the second chromatographic step is an AEX chromatogram performed in a flow-through mode at a pH of about 6.5-8.0. In one embodiment, the pH value is about 6.5 to 7.5.

[0440] After purification by the chromatography step, the antibody or Fab can be processed by a series of filtering steps, which include nanofiltration for virus removal and ultrafiltration for concentration and buffer exchange. Nanofiltration can be operated in batches using a holding vessel or continuously operated by circulating a nanofiltration membrane with an appropriate frequency. The ultrafiltration step can be routinely performed in batch mode by supplying a unit operation from a holding vessel containing a nanofiltration product filtered by viruses. Alternatively, ultrafiltration can be continuously performed by using a single-pass ultrafiltration, a subsequent direct connection or by using an online diafiltration of an intermediate buffer vessel.

[0441] How to use

[0442] The present invention also relates to a method of treating cancer in a subject, the method comprising administering an effective amount of any pharmaceutical composition of the present invention; that is, administering any composition described herein to a subject. In some embodiments of the method, the pharmaceutical composition is administered to the subject by subcutaneous administration.

[0443] In any of the methods of the 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, squamous cell carcinoma of the skin, lymphoma, kidney 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-refractory prostate cancer), pancreatic cancer, colon cancer, liver cancer, thyroid cancer, glioblastoma, glioma and other malignancies.

[0444] In one embodiment, the cancer is melanoma, non-small cell lung cancer, head and neck cancer, urothelial carcinoma, 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, a tumor mutation high burden, or an MSI-H cancer.

[0445] 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.

[0446] In an alternative embodiment, the lung cancer is small cell lung cancer (SCLC).In one embodiment, the SCLC is metastatic.

[0447] In some embodiments, the lymphoma is Hodgkin lymphoma.

[0448] In other embodiments, the lymphoma is non-Hodgkin's lymphoma. In specific embodiments, the lymphoma is mediastinal large B-cell lymphoma. In some embodiments, the lymphoma is diffuse large B-cell lymphoma (DLBCL).

[0449] In some embodiments, the breast cancer is triple-negative breast cancer.

[0450] In a further embodiment, the breast cancer is ER+ / HER2- breast cancer.

[0451] In some embodiments, the bladder cancer is urothelial carcinoma.

[0452] In some embodiments, the head and neck cancer is nasopharyngeal cancer. In some embodiments, the cancer is thyroid cancer. In other embodiments, the cancer is salivary gland cancer. In other embodiments, the cancer is head and neck squamous cell carcinoma.

[0453] In some embodiments, the cancer is metastatic colorectal cancer with high levels of microsatellite instability (MSI-H).

[0454] In some embodiments, the cancer is a solid tumor with high levels of microsatellite instability (MSI-H).

[0455] In some embodiments, the cancer is a solid tumor with a high mutation load. In one embodiment, the tumor mutation load (TMB) is ≥10 mutations / megabase as determined by an FDA-approved test. In some embodiments, the tumor is metastatic or unresectable.

[0456] In some embodiments, the cancer is selected from the group consisting of melanoma, non-small cell lung cancer, relapsed or refractory classical Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, esophageal cancer, gastric cancer, DLBCL, and hepatocellular carcinoma.

[0457] In other embodiments of the above methods of treatment, the cancer is a heme malignancy. In certain embodiments, the heme 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).

[0458] Malignancies that demonstrate improved disease-free and overall survival associated with the presence of tumor-infiltrating lymphocytes in biopsy or surgical material, such as melanoma, colorectal cancer, liver cancer, kidney cancer, gastric / esophageal cancer, breast cancer, pancreatic cancer, and ovarian cancer are included in the methods and treatments described herein. Such cancer subtypes are known to be susceptible to immune control by T lymphocytes. In addition, refractory or recurrent malignancies are also included, whose growth can be inhibited using the antibodies described herein.

[0459] In some embodiments, the pharmaceutical composition of the present invention is administered to a subject with a cancer characterized by elevated expression of PD-L1 and / or PD-L2 in a test tissue sample, including: ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, breast cancer, liver cancer, gastric cancer, esophageal cancer, and melanoma. Other cancers that may benefit from treatment with anti-PD-1 antibodies (e.g., humanized anti-PD-1 antibody pembrolizumab) include those associated with persistent viral infection, such as human immunodeficiency virus, hepatitis A, B, and C viruses, Epstein Barr virus, human papillomavirus known to be causally associated with Kaposi's sarcoma, liver cancer, nasopharyngeal cancer, lymphoma, cervical cancer, vulvar cancer, anal cancer, penile cancer, and oral cancer.

[0460] In one embodiment, the invention includes a method of treating cancer in a human patient in need thereof, comprising administering to the patient an effective amount of any of the pharmaceutical compositions of the invention.

[0461] In one embodiment, the invention includes a method of treating unresectable or metastatic melanoma in a human patient in need thereof, the method comprising administering to the patient an effective amount of any of the pharmaceutical compositions of the invention.

[0462] In one embodiment, the present invention includes a method for treating metastatic non-small cell lung cancer (NSCLC) in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the present invention. In a specific embodiment, 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 other embodiments, the patient has previously received or has not received platinum-containing chemotherapy. In a specific embodiment, the patient has disease progression during or after receiving platinum-containing chemotherapy. In one embodiment, NSCLC is metastatic or stage III.

[0463] In certain embodiments, the PD-L1 TPS is determined by an FDA-approved test.

[0464] In certain embodiments, the patient's tumor does not have EGFR or ALK genomic aberrations.

[0465] In certain embodiments, the patient's tumor has an EGFR or ALK genomic aberration and has disease progression while or after receiving treatment for one or more EGFR or ALK aberrations prior to receiving a composition of the invention.

[0466] In certain embodiments, the patient has a tumor with a PD-L1 expression CPS ≥ 1%.

[0467] In one embodiment, the present invention includes a method of treating non-squamous non-small cell lung cancer (NSCLC) in a patient in need thereof, wherein the method comprises administering to the patient an effective amount of a pharmaceutical composition of the present invention, pemetrexed, and platinum chemotherapy. In one embodiment, the present invention includes 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 a pharmaceutical composition of the present invention, and (2) administering to the patient an effective amount of pemetrexed and carboplatin. In a specific embodiment, the patient has not previously received anti-cancer treatment prior to initiating a combination treatment regimen using a composition of the present invention, pemetrexed, and carboplatin. In certain embodiments, the patient suffers from metastatic non-squamous non-small cell lung cancer.

[0468] In certain embodiments, pemetrexed is administered at 500 mg / m 2 In a sub-embodiment, pemetrexed is administered to the patient by intravenous infusion every 21 days. In a specific embodiment, the infusion time is about 10 minutes.

[0469] In embodiments of the present invention in which a patient is treated with a pharmaceutical composition of the present invention in combination with pemetrexed, the present invention further comprises administering to the patient about 400 μg to about 1000 μg of folic acid once a day, starting about 7 days before the administration of pemetrexed to the patient and continuing until about 21 days after the last dose of pemetrexed is administered to the patient. In certain embodiments, folic acid is administered orally. In some embodiments, the present invention further comprises administering to the patient about 1 mg of vitamin B12 approximately 1 week before the first administration of pemetrexed and approximately every three pemetrexed administration cycles (i.e., approximately every 9 weeks). 12 In certain embodiments, vitamin B 12 Intramuscular administration. In certain embodiments, the present invention further comprises administering to the patient about 4 mg of dexamethasone twice a day on the day before, on the day of, and on the day after administration of pemetrexed. In certain embodiments, dexamethasone is administered orally.

[0470] In one embodiment, the invention includes a method of treating 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 a pharmaceutical composition of the invention, and (2) administering to the patient effective amounts of paclitaxel or protein-bound paclitaxel and carboplatin. In specific embodiments, the patient has not previously received anti-cancer therapy prior to initiating the combination treatment regimen. In certain embodiments, the patient has metastatic squamous non-small cell lung cancer.

[0471] In one embodiment, the invention includes a method of treating recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) in a human patient in need thereof, the method comprising administering to the patient an effective amount of any pharmaceutical composition of the invention. In certain embodiments, the patient has previously received platinum-containing chemotherapy. In certain embodiments, the patient has disease progression during or after platinum-containing chemotherapy. In specific embodiments, the patient's tumor expresses PD-L1 [combined positive score (CPS) ≥ 1]. In some embodiments, treatment is combined with platinum and FU.

[0472] In one embodiment, the invention includes a method of treating refractory classical Hodgkin lymphoma (cHL) in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the invention. In certain embodiments, the patient has relapsed after 2 or more lines of treatment for cHL. In specific embodiments, the patient is an adult patient. In alternative embodiments, the patient is a pediatric patient.

[0473] In one embodiment, the present invention includes a method for treating locally advanced or metastatic urothelial carcinoma in a human patient in need thereof, the method comprising administering an effective amount of a pharmaceutical composition of the present invention to the patient. In certain embodiments, the patient is not suitable for chemotherapy containing cisplatin. 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 a specific embodiment, the patient's tumor expresses PD-L1 [combined positive score (CPS) ≥ 1]. In one embodiment, the patient suffers from Bacillus Calmette-Guérin (BCG) unresponsive high-risk non-muscle invasive bladder cancer.

[0474] In one embodiment, the invention includes a method of treating an unresectable or metastatic, high microsatellite instability (MSI-H) or mismatch repair deficient solid tumor in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the invention. In a specific embodiment, the patient has disease progression after prior anticancer therapy.

[0475] In one embodiment, the invention includes a method of treating an unresectable or metastatic, high microsatellite instability (MSI-H) or mismatch repair deficient solid tumor or colorectal cancer in a human patient in need thereof, the method comprising administering an effective amount of a pharmaceutical composition of the invention. In a specific embodiment, the patient has disease progression after prior treatment with a fluoropyrimidine, oxaliplatin, and irinotecan.

[0476] In one embodiment, the invention includes a method of treating recurrent locally advanced or metastatic gastric cancer in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the invention.

[0477] 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, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the present invention. In a specific embodiment, the patient's tumor expresses PD-L1 [combined positive score (CPS) ≥ 1]. In a specific embodiment, the patient has disease progression in or after two or more prior lines of treatment, including fluoropyrimidine-containing and platinum-containing chemotherapy. In a specific embodiment, the patient has disease progression in or after two or more prior lines of treatment, including HER2 / neu targeted therapy.

[0478] In one embodiment, the invention includes a method of treating recurrent locally advanced or metastatic esophageal squamous cell carcinoma in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the invention. In a specific embodiment, the patient's tumor expresses PD-L1 [combined positive score (CPS) ≥ 1].

[0479] In one embodiment, the invention includes a method of treating recurrent locally advanced or metastatic cervical cancer in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the invention. In a specific embodiment, the patient's tumor expresses PD-L1 [combined positive score (CPS) ≥ 1].

[0480] In one embodiment, the invention includes a method of treating hepatocellular carcinoma in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the invention. In one embodiment, the invention includes a method of treating recurrent locally advanced or metastatic Merkel cell carcinoma in a human patient, the method comprising administering to the patient a composition of the invention. In one embodiment, the invention includes a method of treating recurrent or metastatic cutaneous squamous cell carcinoma in a human patient, the method comprising administering to the patient a composition of the invention.

[0481] In one embodiment, the invention includes a method of treating advanced renal cell carcinoma in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the invention and axitinib. In one embodiment, the invention includes a method of treating advanced endometrial cancer in a human patient, the method comprising administering to the patient a composition of the invention and lenvatinib. In one embodiment, the endometrial cancer is not MSI-H or dMMR.

[0482] In one embodiment, the invention includes a method of treating cancer in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the invention, wherein the patient has a cancer selected from the group consisting of melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, digestive tract cancer, multiple myeloma, hepatocellular carcinoma, lymphoma, kidney cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, and salivary gland cancer.

[0483] In one embodiment, the invention includes a method of treating small cell lung cancer in a human patient in need thereof, comprising administering to the patient an effective amount of a pharmaceutical composition of the invention.

[0484] In one embodiment, the invention includes a method of treating non-Hodgkin's lymphoma in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the invention. In a specific embodiment, the non-Hodgkin's lymphoma is mediastinal large B-cell lymphoma. In a specific embodiment, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.

[0485] In one embodiment, the present invention includes a method for treating breast cancer in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the present invention. In certain embodiments, the breast cancer is triple-negative breast cancer, optionally in combination with chemotherapy. In certain embodiments, the breast cancer is ER+ / HER2- breast cancer. In a specific embodiment, the patient's tumor expresses PD-L1 [combined positive score (CPS) ≥ 1].

[0486] In one embodiment, the invention includes a method of treating nasopharyngeal carcinoma in a human patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutical composition of the invention.

[0487] In one embodiment, the invention includes a method of treating thyroid cancer in a human patient in need thereof, comprising administering to the patient an effective amount of a pharmaceutical composition of the invention.

[0488] In one embodiment, the invention includes a method of treating salivary gland cancer in a human patient in need thereof, comprising administering to the patient an effective amount of a pharmaceutical composition of the invention.

[0489] As described above, in some embodiments of the method of the present invention, the method further comprises administering an additional therapeutic agent. In a specific embodiment, the additional therapeutic agent is an anti-LAG3 antibody or an antigen-binding fragment thereof, an anti-GITR antibody or an antigen-binding fragment thereof, an anti-TIGIT antibody or an antigen-binding fragment thereof, an anti-CD27 antibody or an antigen-binding fragment thereof. In one embodiment, the additional therapeutic agent is a Newcastle disease virus vector expressing IL-12. In another embodiment, the additional therapeutic agent is dinaciclib. In a further embodiment, the additional therapeutic agent is a STING agonist. In one embodiment, the additional therapeutic agent is Coxsackievirus CVA21.

[0490] Suitable routes of administration of the additional therapeutic agent may, for example, include parenteral delivery, including intramuscular, subcutaneous, and intrathecal, directly intraventricular, intravenous, intraperitoneal. The drug may be administered in a variety of conventional ways, such as intraperitoneal, parenteral, intraarterial or intravenous injection.

[0491] The dosage of the additional therapeutic agent is selected depending on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity and the accessibility of the target cells, tissues or organs in the treated individual. The dosage of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Therefore, the dosage and frequency of administration of each additional therapeutic agent (e.g., a biotherapeutic or chemotherapeutic agent) will depend in part on the specific therapeutic agent, the severity of the cancer being treated and the patient's characteristics. Guidelines for selecting appropriate dosages of antibodies, cytokines and small molecules are available. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 345:783-792. Engl. J. Med. 342: 613-619; Ghosh et al. (2003) New Engl. J. Med. 348: 24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th edition); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). Determination of an appropriate dosage regimen can be made by a clinician, for example, using parameters or factors known or suspected in the art to affect treatment or expected to affect treatment, and will depend on, for example, the patient's clinical history (e.g., previous treatments), the type and stage of cancer to be treated, and biomarkers of response to one or more therapeutic agents in the combination therapy.

[0492] Various references are available to facilitate the selection of pharmaceutically acceptable carriers or excipients for additional therapeutic agents. See, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984); Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, 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.

[0493] The pharmaceutical antibody composition can be administered by continuous infusion or at intervals of, for example, one day, 1-7 times per week, one week, two weeks, three weeks, monthly, every two months, etc. The preferred dosage regimen is a regimen involving the maximum dose or dosage frequency that avoids significant adverse 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, e.g., Yang et al. (2003) New Engl. J. Med. 349:427-434; Herold et al. (2002) New Engl. J. Med. 346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji et al. (20003) Cancer Immunol. Immunother. 52:133-144. The required dosage of small molecule therapeutic agents, e.g., peptide mimetics, natural products, or organic chemicals is about the same as the required dosage of antibodies or polypeptides, on a mole / kg basis.

[0494] In certain embodiments, administration will include administering to the subject an increasing dose of 1.0 mg / kg, 3.0 mg / kg and 10 mg / kg of a pharmaceutical composition or composition of the present invention during the course of treatment. The composition can be a reconstructed liquid composition, or it can be a previously non-lyophilized liquid composition. The time course can vary and can be continued as long as the desired effect is obtained. In certain embodiments, the dose escalation will continue to a dose of about 10 mg / kg. In certain embodiments, the subject will have a histological or cytological diagnosis of a melanoma or other form of solid tumor, and in some cases, the subject may suffer from an unmeasurable disease. In certain embodiments, the subject will have been treated with other chemotherapy, and in other embodiments, the subject will be untreated.

[0495] In other embodiments, the dosage regimen will include administering any of the pharmaceutical compositions described herein or compositions of 1 mg / kg, 3 mg / kg or 10 mg / kg throughout the treatment process. For such a dosage regimen, the interval between dosages will be approximately 14 days (± 2 days). In certain embodiments, the interval between dosages will be approximately 21 days (± 2 days).

[0496] In certain embodiments, the administration regimen will include administration of a dose of about 0.005 mg / kg to about 10 mg / kg with dose escalation in the patient. In certain embodiments, a dose of 5 mg / kg or 10 mg / kg will be administered at intervals of every 3 weeks or every 2 weeks. In other 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 should have unresectable disease; however, the patient may have previously undergone surgery.

[0497] In certain embodiments, the subject will be administered a 30-minute IV infusion of any pharmaceutical composition or composition described herein. In certain embodiments of ascending doses, the administration interval between the first and second doses is about 28 days (± 1 day). In certain embodiments, the interval between the second and third doses will be about 14 days (± 2 days). In certain embodiments, for doses after the second dose, the dosing interval will be about 14 days (± 2 days). In certain embodiments, for doses after the second dose, the administration interval will be about 3 weeks. In certain embodiments, for doses after the second dose, the administration interval will be about 6 weeks.

[0498] In certain embodiments, the use of cell surface markers and / or cytokine markers (as described in WO2012 / 018538 or WO2008 / 156712) will be used in bioassays for monitoring, diagnosis, patient selection and / or treatment regimens involving blocking the PD-1 pathway. Subcutaneous administration can be performed using a syringe or using other injection devices (e.g. injection device; injection pen; or needle-free device (such as MediJector and ).

[0499] Embodiments of the present invention also include one or more pharmaceutical compositions or formulations described herein for (i) use in a medicament or composition, (ii) use as a medicament or composition for, or (iii) use in the preparation of a medicament for: (a) therapy (e.g., human therapy); (b) medicine; (c) inducing or increasing an anti-tumor immune response; (d) reducing the amount of one or more tumor markers in a patient's body; (e) stopping or slowing the growth of a tumor or blood cancer; (f) stopping or slowing the progression of a PD-1-related disease; (g) stopping or slowing 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) prolonging a patient's survival relative to the expected survival of a similar untreated patient, n) inducing complete or partial remission of a cancerous condition or other PD-1-related disease, or o) treating cancer.

[0500] General approach

[0501] Standard methods in molecular biology are described in Sambrook, Fritsch and Maniatis (1982 & 1989 2nd edition, 2001 3rd edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0502] Methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described (Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modifications, post-translational modifications, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) Bio Directory, Piscataway, NJ, pp. 384-391). The production, purification and fragmentation of polyclonal and monoclonal antibodies are described (Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand / receptor interactions are available (see, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0503] Monoclonal, polyclonal and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Harbor, NY, pp. 139-243; Carpenter et al. (2000) J. Immunol. 165:6205; He et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

[0504] While various embodiments of the present invention have been described herein with reference to the accompanying drawings, it should be understood that the invention is not limited to those precise embodiments and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

[0505] Example 1: Continuous perfusion process

[0506] The following procedures and examples use glutamine synthetase knockout CHO host cells (expressing pembrolizumab) comprising 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.

[0507] 3L Bioreactor Operation

[0508] A glass bioreactor (3 L, Sartorius Stedim, NE) with a marine impeller (70 mm diameter) and a drilled sparger (14 x 0.5 mm diameter holes) was used. Germany). The bioreactor was 0.5×10 6Inoculate at a target cell density of 10 cells / mL. Control dissolved oxygen (DO) at 30%-60% of air saturation using pure O2 and gradually increase the agitation speed in the range of 260–450 rpm to control the O2 sparging rate to <0.30 vvm to avoid excessive foam formation. Add as needed for foam control Defoamer (Sigma-Aldrich, St. Louis, MO). Overlay air was controlled at 0.1 L / min. The temperature was maintained at 36.5°C throughout the culture period. The pH of the bioreactor was controlled at 7.0 ± 0.3 during the culture period.

[0509] Commercially available basal medium was used for initial bioreactor batch growth and perfusion medium was subsequently used for medium exchange. The perfusion medium had a copper concentration of 10 ppb-35 ppb. The bioreactor started with a working volume of 1.6 L and was maintained at the same level during the perfusion period. On day 3, when the cell density reached 2 to 4 x 10 6 When the perfusion rate was between 10 cells / mL, medium replacement was started and the perfusion rate was gradually increased from 0.5 vvd according to a predetermined schedule. On day 4, a perfusion rate of 1 vvd was applied and a maximum perfusion rate of 2 vvd was reached between day 5 and the end of production. TFF system with magnetic levitation pump ( The TFF system (Repligen, Waltham, MA) was used for cell retention and medium exchange. The TFF system continuously circulates the cell culture medium through the hollow fiber module while collecting the permeate at a predetermined rate. The hollow fiber module specifications 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 crossflow rate of 1.0 L / min was used in all experiments. Once the cell density reached 100 × 10 6 Cells / mL or a capacitance target of 80 pF / cm, the cell ejection pump is turned on to maintain the cell density or biovolume (capacitance, pF / cm). Cell ejection is automatically controlled by a capacitance probe (Incyte DN12, Hamilton Bonaduz AG, Switzerland). The perfusion culture duration was 28-32 days. The copper concentration in the bioreactor ranged from 1 ppb to 35 ppb. After day 5, continuous harvesting was started when the bioreactor had reached a perfusion rate of 2 vvd.

[0510] 50L Bioreactor Operation

[0511] Xcellerx XDR 50 single-use bioreactors (GE Healthcare, Marlborough, MA) were used as production bioreactors for this study. 6 The target cell density was 10 cells / mL, and the dissolved oxygen (DO) was controlled at 30%-60% of air saturation using pure O2 supply. The agitation of the bioreactor was controlled at 86 rpm-93 rpm to ensure proper mixing and mass transfer. Add as needed for foam control. Antifoam (Sigma-Aldrich, St. Louis, MO). Cover air was controlled at 0.1 L / min-0.5 L / min. Temperature was maintained at 36.5°C throughout the culture period. The pH of the bioreactor was controlled at 7.0±0.3 during the culture period.

[0512] Commercially available basal medium was used for initial bioreactor batch growth and perfusion medium was subsequently used for medium exchange. The perfusion medium had a copper concentration of 32.5 ppb. The bioreactor started with a working volume of 50 L and was maintained at the same level during the perfusion period. On day 3, when the cell density reached 2 to 4 x 10 6 When the perfusion rate was between 10 cells / mL, the medium was replaced and the perfusion rate was gradually increased from 0.5 vvd on the 3rd day to 1 vvd on the 4th day and reached a maximum perfusion rate of 2 vvd on the 5th day (average residence time of 0.5 days). TFF system with magnetic levitation pump ( The TFF system (Repligen, Waltham, MA) was used for cell retention and media exchange. The TFF system continuously circulates the cell culture medium through the 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 cell retention devices in this study. A TFF crossflow rate of 10.5 L / min was used in this study. Once the cell density reached 100-105×10 6 Cells / mL or a capacitance target of 80-85 pF / cm, the cell ejection pump was turned on to maintain the cell density or biovolume (capacitance, pF / cm). Cell ejection was automatically controlled by a capacitance probe (Incyte DN12, Hamilton Bonaduz AG, Switzerland). The perfusion culture duration was 28 days. After day 5, continuous harvesting was started when the bioreactor had reached a perfusion rate of 2 vvd.

[0513] Samples were taken from the bioreactor and permeate pipeline every day. Viable cell density (VCD) and viability were measured on Cedex Hi-Res cell counter (Roche Diagnostics GmbH, Mannheim, Germany) using trypan blue exclusion. Cell diameter was measured and reported on the same cell counter. Off-line pH, pO2 and pCO2 were measured using ABL80 blood gas analyzer (Radiometer, Denmark). Glucose, lactic acid, ammonium and lactate dehydrogenase (LDH) were measured using RX Daytona+ or Imola analyzer (Randox Laboratories, Ltd., Crumlin, UK). Bioreactor supernatant and permeate antibody titer were analyzed using Agilent 1100 high performance liquid chromatographs (HPLC, Agilent Technologies, Santa Clara, CA) equipped with protein A columns.

[0514] Example 2: Downstream purification

[0515] Protein A affinity chromatography

[0516] Protein A affinity chromatography was used as the primary capture step using GE Healthcare TM MabSelect SuRe resin is used for overall purification. The product is bound to the resin, while impurities such as culture medium components and host cell proteins are not bound and remain in the column flow-through fraction. The protein A step is operated in a continuous manner, using a continuous multi-column chromatography platform to allow the HCCF from the bioreactor to flow uninterruptedly to supply the platform (BioSMB PD, Sartorius Stedim Biotech GmbH Gottingen Germany). This is achieved by utilizing 4 comparable columns equipped with the same protein A resin on the platform. At any one time, three columns are dedicated to loading, and the fourth column carries out the non-loading steps (equilibrium step, washing step, elution step, regeneration step) in the protein A method.

[0517] The HCCF from the bioreactor was connected to the continuous multi-column chromatography platform via a 3 L disposable buffer container (Cercell, Herlev, Denmark). The flow rate of the HCCF to the disposable buffer container matched the feed rate from the buffer container to the continuous multi-column chromatography system, thereby maintaining a consistent volume in the container. The operating volume range in the buffer container gave an average residence time of 15-45 minutes for the fluid in the container, with a target value of 30 minutes. Figure 5 As shown, due to oxidative instability, the residence time of HCCF loaded onto the Protein A column is preferably less than 24 hours.

[0518] Operationally, multi-column protein A chromatography follows the same steps as single-column batch mode chromatography, including column equilibration, loading, washing, and regeneration phases. After equilibration of the column with 10 mM sodium phosphate (pH 6.5), the loading phase begins. The protein is loaded into a 50 g / L capacity protein A resin. The loading phase of the protein A step is followed by three washing steps, including: 10 mM sodium phosphate (pH 6.5) and 10 mM sodium phosphate with 0.5 M sodium chloride, to elute loosely bound impurities and improve the purity of the antibody in the product stream. The product is eluted with 20 mM sodium acetate (pH 3.6) via a low pH transition and monitored by online spectrophotometry at an absorbance of 280 nm. The steps in the buffer vessel and the protein A step are performed at ambient temperature (20 ± 5 ° C).

[0519] After Protein A chromatography, the protein is processed through additional purification and filtration steps to obtain an ultrafiltration product in a continuous process connected by intermediate buffer vessels, including: viral inactivation, depth filtration, anion exchange chromatography, viral filtration, single-pass tangential flow filtration and in-line diafiltration.

[0520] Virus inactivation / deep filtration

[0521] The protein A affinity pool was adjusted to a low pH (target pH 3.6) with 1 M acetic acid to inactivate any viruses that might be present. This step was performed at ambient temperature (20 ± 5 °C). The pool after virus inactivation was adjusted to a target pH of 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 neutralized virus inactivated product (FNVIP) can be stored at 2-8 °C and then further processed in a subsequent anion exchange chromatography step.

[0522] Anion Exchange

[0523] Anion exchange chromatography (AEX) was performed using a POROS HQ 50 from Applied Biosystems and was the first polishing step in the process. The pembrolizumab antibody flowed through the column, while residual impurities, such as possible host cell proteins (HCPs), DNA, aggregates, and viruses, were bound to the resin. The column was balanced and washed with 25 mM sodium phosphate (pH 7.2). Before starting this step, the pH of the pool after virus inactivation was adjusted to a target pH of 7.2 with 1 M Tris. The column effluent UV absorbance 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 with 1 M acetic acid for continued processing in the post-step buffer container.

[0524] Virus Filtering

[0525] The AEX product was filtered through a 0.1 μm prefilter or an equivalent filter in series with a Planova 20N (mean pore size 19 nm) virus reduction filter (Asahi Kasei Bioprocess, Glenview, IL). The prefilter and filter were connected for autoclaving and in series with a nanofilter in a biosafety cabinet. The filter was then continuously rinsed in a closed manner with 10 mM histidine, 10 mM methionine (pH 5.4).

[0526] Ultrafiltration concentration and diafiltration

[0527] First, by the single pass tangential flow filtration (SPTFF) module (Pall Corporation Westboro MA Port Washington, NY) with 30kDa molecular weight cut-off, the virus filtration product is concentrated 8x.Then the product from the SPTFF step is diafiltered with 10mM histidine, 10mM methionine (pH 5.4), thereby causing the buffer exchange of concentrated protein stream.Utilize six-stage series diafiltration module (ILDF) device containing 30kDa MW cut-off membrane (Pall Corporation Westboro MA Port Washington, NY) to perform buffer exchange.The diafiltration product, also referred to as ultrafiltration product, is further filtered and collected in storage container by sterile filter.Once the ultrafiltration product of enough quality has been accumulated, the ultrafiltration product is brought together and further concentrated to the ultimate concentration of 190-200g / L by standard batch ultrafiltration step using Ultracel 30kDa molecular weight cut-off membrane (Millipore, Burlington, MA). Stock excipients were added to obtain 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.

[0528] Example 3: Method for determining the oxidation level of M105

[0529] Reduced peptide mapping, liquid chromatography, and mass spectrometry methods

[0530] Sample preparation

[0531] In-process samples or bulk drug (DS) samples are diluted with water to 5mg / mL. A total of 20 μL diluted samples (containing 100 μg) are denatured and reduced in a final solution (100 μL) containing 6M guanidine hydrochloride, 50 μM Tris-HCl, 50 μM EDTA and 200 μM DTT. The mixed samples are incubated with 300rpm vibrations at 37 ° C for 30min in a thermomixer. After mixing and centrifugation, each sample is alkylated at 25 ° C for 30min with 5 μL iodoacetamide (IAM) (1M) protected from light. A total of 5 μL DTT (200 μM) is added to block unreacted iodoacetamide (IAM). 500 μL lysyl endopeptidase (Lys-C) (Wako, 125-05061) enzyme (1: 10 (wt: wt)) is added to the protein sample, and the liquid is slowly pipetted up and down 3 times to fully mix. The digests were incubated in a thermomixer at 37°C for 60 min. The digests were quenched with 15 μL of 20% TFA. The digested samples were analyzed by LC-MS within 24 h after sample digestion. Otherwise, the digested samples were stored at -80°C for future analysis.

[0532] LC-MS methods and data analysis

[0533] 20 μL of sample was injected into the column using Waters Acquity liquid chromatography (UPLC HSS T3 1.8 μm, 2.1 mm x 150 mm, P / N: 186003540). The autosampler was set at 5°C. Mobile phase A was 0.02% TFA in water and mobile phase B was 0.02% TFA in acetonitrile with a gradient of 0.1% B from 0 to 5 min, 0.1%-10% B from 5 to 7 min, followed by a linear increase to 35% B over the next 38 min. A Q Exactive Orbitrap MS (Thermo) was used to collect MS1 data. Chromeleon and Xcalibur were used for data analysis. The extracted ion chromatograms (EICs) of M105 unmodified and modified peptides (each peptide has two charged states (+3 and +4) and each with 3 isotopic ions) were used to determine the M105 oxidation % using the following formula: [extracted ion chromatogram (EIC) peak area of ​​modified peptide] / [EIC peak area of ​​modified peptide+EIC peak area of ​​unmodified peptide]×100. See also Figure 2-4 The %CV (coefficient of variation; calculated as (standard deviation / mean) x 100) of M105 oxidation was less than 20%.

[0534] Table 3

[0535]

[0536] The formulation of the pembrolizumab drug product reference standard for fed-batch production was 25 mg / mL pembrolizumab in 10 mM histidine buffer containing 7% (w / v) sucrose and 0.02% (w / v) polysorbate 80, pH 5.5. The formulation of the pembrolizumab formulated drug substance for continuous perfusion production was 165 mg / mL pembrolizumab, 10 mM L-histidine, 7% (w / v) sucrose, 0.02% (w / v) PS80, and 10 mM L-methionine, pH 5.5.

[0537] The oxidized M105% of the pembrolizumab formulated drug product reference standard for fed-batch production was about 4.9%, while the pembrolizumab samples produced by continuous perfusion contained about 0.5%-3.0% oxidized M105. Some representative samples from protein A purification, ultrafiltration product (unformulated DS), and formulated DS are shown in Table 3.

[0538] Hydrophobic Interaction Chromatography (HP-HIC)

[0539] For the determination of % Met105 oxidation, high performance hydrophobic interaction chromatography (HP-HIC) was used to separate and quantify the oxidation products from the non-oxidized molecules. Peak 3 contained antibodies with Met105 oxidation in both heavy chains; Peaks 1 and 2 contained antibodies with Met105 oxidation in either heavy chain ( Fig.13 ). The percentage of the pre-peak as well as the percentage of the main peak and the percentage of the post-peak were determined. The HP-HIC method was performed by diluting the sample to 5.0 mg / mL in purified water. The sample (10 μL) was then injected into an HPLC equipped with a Tosoh Phenyl-5PW column (30 °C) and a 280 nm UV detector. For HIC analysis, the mobile phase used contained a gradient of 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). The gradient was 0% mobile phase A from 0-2 min, 0%–100% mobile phase A from 2 min-52 min, followed by column washing and equilibration. The pembrolizumab reference produced by a fed-batch process was subjected to batch protein A, virus inactivation, depth filtration, and AEX purification, and tested for Met105 oxidation %. A representative value for the % Met105 oxidation of the unfiltered sample after viral inactivation and AEX purification of the pembrolizumab reference was approximately 5.5%.

[0540] Example 4: Measuring M105 oxidation of pembrolizumab in HCCF over time

[0541] The oxidation rate was determined by repeatedly measuring the amount of oxidation in the HCCF sample according to Example 1 using ProA-HIC 2D-LC as described in Example 3 and below.

[0542] Samples were removed from the bioreactor and filtered to remove cells, yielding HCCF. Under sterile conditions, HCCF samples were divided into 200 μL aliquots across 48 wells of a 96-well plate. The plates were covered with foil covers to maintain sterility and protect the samples from light. The samples were immediately placed in an Agilent 1290 autosampler set to 25 °C with the internal lights off. Samples from each well were analyzed sequentially over the time course by 2D-LC separation.

[0543] The resulting chromatograms for each sample were integrated to determine the percent oxidation, and the data were summarized as Figure 5 Linear regression of the data showed that the percent oxidation started at 0.94% and increased at a rate of approximately 0.038% per hour over the next 26 hours to a maximum of 1.95% oxidation.

[0544] Example 5: Ion Exchange (IEX) Method to Measure Acidic Species of Anti-PD-1 Antibodies

[0545] For the IEX method, a Waters Alliance LC system (Milford, MA, USA) was used, and the ThermoScientific ProPac WCX-10 (p / n: 054993, particle size 10 μm, diameter 4 mm, length 250 mm) was selected, and 80 μg of sample was loaded. The mobile phase (A) was 24 mM MES pH 6.1 with 4% acetonitrile and the mobile phase (B) was 20 mM sodium phosphate, 95 mM NaCl pH 8.0 with 4% acetonitrile as a nonlinear, sigmoidal, pH gradient, and the flow rate was 0.5 mL min over 34 min. –1 The separation was monitored at a flow rate of 1.5 % and the column temperature was 35 °C. The gradient used was: 22%–22% B for 0 min–0.6 min; 22%–29% B for 0.6 min–15.0 min; 29%–70% B for 15.0 min–30.0 min; 70%–100% B for 30.0 min–30.5 min; and 100%–100% B from 30.5–33.0 min. The mobile phase (C) was 10 mM CHES pH 8.0, 40 mM Tris, 15 mM EDTA, 200 mM NaCl, and 4% acetonitrile at 0.5 mL min –1 The column was stripped from 33.1 min–34.0 min and then treated with 22% B at 1.0 mL min –1Re-equilibrate from 34.5 min to 44.5 min. From 44.5 to 45 min, the flow rate is reduced to 0.5 mL min –1 Elution was monitored at 280 nm for peak detection. Assay variability was determined to be within 1%.

[0546] The chemical composition of each identified peak of the pembrolizumab drug substance reference sample (produced from a fed-batch process) was determined by collecting samples of each peak and performing peptide mapping and reversed phase liquid chromatography, followed by mass spectrometry, according to the same methods as those analyzed by MS / MS in Example 3. It was determined that the main peak contained mainly antibodies with light chains and heavy chains having the amino acid sequences shown in SEQ ID NO:5 and SEQ ID NO:11, 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 having the following were detected: a heavy chain consisting of the amino acid sequence of SEQ ID NO:11, a 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 a heavy chain consisting of the amino acid sequence of SEQ ID NO:11, a heavy chain consisting of the amino acid sequence of SEQ ID NO:14 (wherein the C-terminal leucine is α-amidated), and two light chains consisting of the amino acid sequence of SEQID NO:5.

[0547] The ion exchange chromatogram and the acidic species %, main peak % and basic species % of the sample after protein A chromatography (PAP) and anion exchange chromatography (AEXP) according to the procedures in Example 1-2 are shown in Figure 6-7 and Table 4. The sum of acidic species (acidic variant, acid 1 and pre-main peak) for PAP and AEXP samples was 7.86% and 8.76%, respectively. The sum of basic species (basic 1, basic variant A, basic 2, basic variant B) for PAP and AEXP samples was 16.95%, 18.19%, respectively. The ion exchange chromatogram of the pembrolizumab reference obtained by the fed-batch method and the acidic species %, main peak % and basic species % are shown in Figure 8and Table 5. The sum of the acidic species (acidic variants, acidic 1, and before the main peak) of the reference sample is 16.56%. The sum of the basic species (basic 1, basic variant A, basic 2, basic variant B) of the reference sample is 23.76%. The sum of the basic variants (basic variant A and basic variant B) of the reference sample is 6.23%. In summary, the pembrolizumab samples prepared by the continuous perfusion process of the present invention have a higher percentage of main species due to the lower percentage of acidic species in the mixture. In other batches of pembrolizumab samples prepared by a continuous perfusion process substantially similar to Examples 1-2, the main peak is about 74%-80%.

[0548] Table 4: Pembrolizumab obtained from the method of the present invention: acidic, major and basic species

[0549]

[0550]

[0551] Table 5: Acidic, primary and basic species of pembrolizumab reference product

[0552] name Retention time area% Acidic variant 9.546 8.1 Acid 1 10.513 6.41 In front of the main peak 11.79 2.05 Main Peak 13.269 59.68 Alkalinity 1 14.726 8.07 Basic variant A 16.782 1.36 Alkaline 2 17.743 9.46 Basic variant B 21.795 4.87

[0553] Figure 9-12 Also provided is the time course of total acidic species %, major species %, total basic species %, and basic 1 species % in the cell-free permeate (PERM) prepared according to the procedures in Examples 1-2, after the protein A chromatography step (PAP), after anion exchange chromatography (AEXP), over the time course in days of culture.

[0554] Example 6: Hyaluronidase activity assay

[0555] Enzyme activity was determined by turbidity assay on a Molecular Devices SpectraMax M5e microplate reader. Calibration curve standards and test samples were prepared by diluting with cold enzyme diluent (20 mM sodium phosphate, 77 mM sodium chloride, 0.01% bovine serum albumin (BSA), pH 7.0, 25°C) to the working concentrations listed in Table 6.

[0556] Table 6. Example working concentrations used in activity assays

[0557]

[0558]

[0559] 50 μ L of diluted standard and sample are transferred to transparent bottom 96-well plates (plate 1) in triplicate. 50 μ L enzyme dilution solution is added to the special wells on the plate as blank control. The plate is sealed and incubated at 37 ℃ for 10 minutes. After incubation, 50 μ L 37 ℃ of hyaluronic acid solutions (0.06% hyaluronic acid 300mM phosphate, pH 5.35, 37 ℃) are added to each well containing solution with a multichannel pipette. The plate is sealed, then incubated at 37 ℃ for 45 minutes with 600rpm shaking. Before plate 1 is taken out from incubation, in each well, 200 μ L acidic albumin solution (24mM sodium acetate, 79mM acetic acid, 0.1% BSA, pH 3.75, 25 ℃) is prepared for the second plate (plate 2), producing the same hole layout as plate 1. After incubation for exactly 45 minutes on plate 1, remove 40 μL of solution from each well using a multichannel pipette and add to the corresponding wells in plate 2 (containing the acidic albumin solution). Plate 2 is incubated at 25°C for 20 minutes in the plate chamber of the microplate reader. The microplate reader is set to read the absorbance at 600 nm after 5 seconds of shaking. After incubation for 20 minutes, read the absorbance at 600 nm for each well.

[0560] Generate calibration curve

[0561] The triplicate absorbance values ​​from the calibration curve standards were averaged and the average absorbance of the blank was subtracted. The resulting absolute values ​​of the corrected absorbance were plotted against the calibration curve standard volume activity values ​​(e.g., 15, 12, 10, 7.5, 5, and 2 units / mL). The plot was fitted with a first order polynomial ( Fig.14 ), and the obtained fitting equation was used to determine the enzymatic activity of the samples.

[0562] Calculate the enzymatic activity of the test sample

[0563] The triplicate absorbance values ​​were averaged and the average absorbance of the blank was subtracted. The absolute value of the corrected absorbance obtained was entered into the fitting equation of the calibration curve to determine the calculated activity of the test sample. Any corrected absorbance value that fell outside the calibration curve was discarded. The dilution value was corrected by multiplying the assay volume activity by the dilution factor used to prepare the solution. For example, if the estimated enzymatic activity of the preparation or standard stock solution is 1500 units / mL and is diluted to 12, 10, and 7.5 units / mL for analysis, the dilution factors are 125, 150, and 200, respectively. The final calculated enzyme activity values ​​were averaged and reported in units / mL.

[0564] Example 7: Evaluation of the stability of rHuPH20 variant 1 after heat stress using pembrolizumab CPP

[0565] Co-formulated samples containing pembrolizumab manufactured by a continuous perfusion process (CPP) and rHuPH20 variant 1 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), pH 5.5, and were filled in 2R vials with a 1.6 mL fill volume.

[0566] Table 7. Co-formulations of pembrolizumab CPP and rHuPH20 variant 1 used in heat stress studies

[0567]

[0568] The enzyme activity was measured after incubating each sample at 35 °C for 1 week. Fig.15 Activity data for heat stressed samples are shown, which have been normalized to the activity values ​​observed for control (initial) samples. Surprisingly, the data indicate that at concentrations of 5–165 mg / mL pembrolizumab CPP, enhanced rHuPH20 variant 1 enzyme activity and stability following heat stress were observed in the presence of pembrolizumab CPP compared to rHuPH20 variant 1 alone. Retention of enzyme activity following heat stress demonstrated a dependence on pembrolizumab CPP concentration. The data indicate that at concentrations greater than 50 mg / mL pembrolizumab CPP, the enhancement in rHuPH20 variant 1 enzyme activity was unexpectedly greater than that observed at lower concentrations of pembrolizumab CPP. Furthermore, no effect on rHuPH20 variant 1 enzyme activity was observed following heat stress at concentrations greater than 100 mg / mL pembrolizumab CPP compared to control samples.

[0569] Example 8: Evaluation of the stability of recombinant human hyaluronidase PH20 variant 1 in the presence of pembrolizumab CPP after stainless steel exposure

[0570] Test formulations of rHuPH20 variant 1 co-formulated with pembrolizumab CPP were prepared in polyethylene terephthalate copolyester diol modified (PETG) bottles (125 mL) with compositions as shown in Table 8. Each formulation was exposed to SS solid cylinders at 25°C (protected from light) for 72 hours. All formulations were filtered using a 0.22 μm PES filter. Control samples were similarly prepared in the absence of stainless steel exposure. All samples were stably stored at 25°C (protected from light) for 4 weeks to evaluate rHuPH20 variant 1 activity.

[0571] Table 8. Co-formulations of pembrolizumab CPP and rHuPH20 variant 1 exposed to stainless steel

[0572]

[0573] Fig.16 Enzyme activity data for samples exposed to stainless steel are shown, normalized to the activity values ​​for unexposed samples. Interestingly, the enzyme activity of rHuPH20 variant 1 co-formulated with pembrolizumab CPP showed enhanced activity and stability compared to rHuPH20 variant 1 alone after stainless steel exposure followed by storage at 25°C for 4 weeks. After storage at 25°C for 4 weeks, enzyme-only samples exposed to stainless steel showed reduced activity relative to control samples. Enzyme activity in samples co-formulated with pembrolizumab CPP was not affected after storage, indicating enhanced enzyme stability and activity in the presence of pembrolizumab CPP.

[0574] Example 9: Evaluation of the Effect of Sucrose (Thermostater) in Co-formulations of Pembrolizumab Reference and rHuPH20 Variant 1

[0575] Co-formulations of pembrolizumab reference and rHuPH20 variant 1 (165 mg / mL pembrolizumab, 2000 units / mL rHuPH20 variant 1 in 0.2 mg / mL PS-80, 10 mM methionine in 10 mM histidine buffer, 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. Samples were heat stressed at 35°C for up to 2 weeks.

[0576] Table 9. Co-formulations of pembrolizumab and rHuPH20 variant 1

[0577]

[0578] The stressed samples were analyzed by microfluidics imaging (MFI) to determine the effect of the thermal stabilizer on the subvisible particle population. Table 10 shows the concentration of particles greater than or equal to 2 μm (particles / mL). The data show that the co-formulations prepared without the thermal stabilizer had increased particle counts after incubation at 35°C for 2 weeks compared to the co-formulations prepared with the thermal stabilizer.

[0579] Table 10. Subvisible particle distribution based on MFI

[0580]

[0581] All references cited herein 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 was specifically and individually indicated to be incorporated by reference. Each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application or patent is each clearly identified in accordance with 37 CFR § 1.57 (b) (2), even if such reference is not followed by a specific statement of incorporation by reference. The specific statement of incorporation by reference contained in the specification, if any, does not in any way weaken the general statement of incorporation by reference. The references cited herein are not intended to admit that the reference is relevant prior art, nor do they constitute any admission of the content or date of these publications or documents. If the definition of the term claimed for protection provided by the reference conflicts with the definition provided in this specification, the definition provided in this specification should be used to interpret the claimed invention.

Claims

1. A pharmaceutical composition comprising: an anti-human PD-1 antibody comprising a light chain variable region comprising three light chain CDRs and a heavy chain variable region comprising three heavy chain CDRs, wherein the three light chain CDRs comprise CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the three heavy chain CDRs comprise CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8, wherein the pharmaceutical composition comprises about 0.1% to 3.0% oxidation of methionine 105; rHuPH20 or a variant or fragment thereof, wherein the rHuPH20 or a variant or fragment thereof is SEQ ID NO: Amino acid residues of NO:16: 36-464, 36-465, 36-466, 36-467, 36-468, 36-469, 36-470, 36-471, 36-472, 36-473, 36-474, 36-475, 36-476, 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, 36-483, 37-464, 37-465, 37-466, 37-467, 37 -468, 37-469, 37-470, 37-471, 37-472, 37-473, 37-474, 37-475, 37-476, 37-477, 37-478, 37-479, 37-480, 37-481, 37-482, 37-483, 38-464, 38-465, 38-466, 38-467, 38-468, 38-469, 38-470, 38-471, 38-472, 38-473, 38-474 8-474, 38-475, 38-476, 38-477, 38-478, 38-479, 38-480, 38-481, 38-482, 38-483, 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, 39-483, 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, 40-482, 40-483, 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, 41-483, 42-464, 42-465 5, 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 of claim 1, wherein the anti-human PD-1 antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

9.

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

4. The pharmaceutical composition of claim 1, wherein the anti-human PD-1 antibody consists of two light chains and two heavy chains, wherein in a portion of the anti-PD-1 antibodies, 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 of claim 1, wherein the antibody is a pembrolizumab variant.

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

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

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

9. A pharmaceutical composition comprising: an anti-human PD-1 antibody comprising a major species and an acidic species of the major species, wherein the amount of the acidic species is 1.0%-12.0%, the major species comprising an antibody consisting of two heavy chains and two light chains, each light chain comprising a light chain variable region comprising three light chain CDRs, the three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and each heavy chain comprising a heavy chain variable region comprising three heavy chain CDRs, the three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8; rHuPH20 or a variant or fragment thereof, wherein the rHuPH20 or a variant or fragment thereof is SEQ ID NO: Amino acid residues of NO:16: 36-464, 36-465, 36-466, 36-467, 36-468, 36-469, 36-470, 36-471, 36-472, 36-473, 36-474, 36-475, 36-476, 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, 36-483, 37-464, 37-465, 37-466 6. 37-467, 37-468, 37-469, 37-470, 37-471, 37-472, 37-473, 37-474, 37-475, 37-476, 37-477, 37-478, 37-479, 37-480, 37-481, 37-482, 37-483, 38-464, 38-465, 38-466, 38-467, 38-468, 38-469, 38-470, 3 8-471, 38-472, 38-473, 38-474, 38-475, 38-476, 38-477, 38-478, 38-479, 38-480, 38-481, 38-482, 38-483, 39-464, 39-465, 39-466, 39-467, 39-468, 39-469, 39-470, 39-471, 39-472, 39-473, 39-474, 39-475 75, 39-476, 39-477, 39-478, 39-479, 39-480, 39-481, 39-482, 39-483, 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, 40-482, 40-483, 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, 41-48 2, 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.

10. A pharmaceutical composition comprising: an anti-human PD-1 antibody comprising a major species and an acidic species and a basic species of the major species, wherein the amount of the major species is 65%-95%, the major species comprising an antibody consisting of two heavy chains and two light chains, each light chain comprising a light chain variable region comprising three light chain CDRs, the three light chain CDRs comprising CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and each heavy chain comprising a heavy chain variable region comprising three heavy chain CDRs, the three heavy chain CDRs comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, and CDRH3 of SEQ ID NO: 8; rHuPH20 or a variant or fragment thereof, wherein the rHuPH20 or a variant or fragment thereof is SEQ ID NO: Amino acid residues of NO:16: 36-464, 36-465, 36-466, 36-467, 36-468, 36-469, 36-470, 36-471, 36-472, 36-473, 36-474, 36-475, 36-476, 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, 36-483, 37-464, 37-465, 37-476. 66, 37-467, 37-468, 37-469, 37-470, 37-471, 37-472, 37-473, 37-474, 37-475, 37-476, 37-477, 37-478, 37-479, 37-480, 37-481, 37-482, 37-483, 38-464, 38-465, 38-466, 38-467, 38-468, 38-469, 38-47 0, 38-471, 38-472, 38-473, 38-474, 38-475, 38-476, 38-477, 38-478, 38-479, 38-480, 38-481, 38-482, 38-483, 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、39-483、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, 40-482, 40-483, 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, 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.

11. The pharmaceutical composition of 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 pharmaceutical composition comprising an anti-human PD-1 antibody comprising a major species produced by Chinese hamster ovary cells and an acidic species of the major species, wherein the amount of the acidic species is 1.0%-12.0%, wherein the Chinese hamster ovary cells comprise a polynucleotide encoding a light chain and a polynucleotide encoding a heavy chain, or polynucleotides 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; rHuPH20 or a variant or fragment thereof, wherein the rHuPH20 or a variant or fragment thereof is SEQ ID NO: Amino acid residues of NO:16: 36-464, 36-465, 36-466, 36-467, 36-468, 36-469, 36-470, 36-471, 36-472, 36-473, 36-474, 36-475, 36-476, 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, 36-483, 37-464, 37-465, 37-466, 37-467, 37-468, 37-479. 69, 37-470, 37-471, 37-472, 37-473, 37-474, 37-475, 37-476, 37-477, 37-478, 37-479, 37-480, 37-481, 37-482, 37-483, 38-464, 38-465, 38-466, 38-467, 38-468, 38-469, 38-470, 38-471, 38-472, 38-473, 38-474, 38-475, 38-476 6. 38-477, 38-478, 38-479, 38-480, 38-481, 38-482, 38-483, 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, 39-483 ,40-464,40-465,40-466,40-467,40-468,40-469,40-470,40-471,40-472,40-473,40-474,40-475,40-476,40-477,40-478,40-479,40-480,40-481,40-482,40-483,41-464,41-465,41-466,41-467,41-468,41-469,41-470,41-471, 41-472, 41-473, 41-474, 41-475, 41-476, 41-477, 41-478, 41-479, 41-480, 41-481, 41-482, 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.

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

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

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

16. The pharmaceutical composition of any one of claims 9-15, comprising an acidic 1 species, wherein the amount of the acidic 1 species is about 1%-4%.

17. The pharmaceutical composition of any one of claims 9-16, comprising an acidic variant species, wherein the amount of the acidic variant species is about 1%-5%.

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

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

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

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

22. The pharmaceutical composition of 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 MAbPacSCX50G is used.

23. The pharmaceutical composition of claim 21, wherein a weak cation exchange column with carboxylic acid functional groups is used.

24. A pharmaceutical composition as described in claim 21, wherein the major species, acidic species, acidic 1 species, acidic variant species or basic species are determined by the following: ProPac WCX-10, using mobile phase (A) 24mM MES pH 6.1, with 4% acetonitrile, and mobile phase (B) 20mM sodium phosphate, 95mM NaCl pH 8.0, with 4% acetonitrile, a column temperature of 35°C, applying a gradient: 22%-22% B for 0 min-0.6 min; 22%-29% B for 0.6 min-15.0 min; 29%-70% B for 15.0 min-30.0 min; 70%-100% B for 30.0 min-30.5 min; and 100%-100% B for 30.5 min-33.0 min, and generating a chromatogram using detection at 280 nm.

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

26. The pharmaceutical composition of any one of claims 1-8 and 13-24, wherein methionine 105 oxidation % is measured by hydrophobic interaction chromatography (HIC) using a Tosoh Phenyl-5PW column and a mobile phase containing a gradient of 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 of 0% mobile phase A from 0 to 2 minutes, 0%-100% mobile phase A from 2 to 52 minutes, and a detection wavelength of 280 nm.

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

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

29. The pharmaceutical composition of claim 28, wherein the pharmaceutical composition has a pH of about 5.2 to about 5.

8.

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

31. The pharmaceutical composition of any one of claims 28-30, wherein the buffer is a histidine buffer present at a concentration of about 8 mM to about 12 mM at a pH of about 5.

5.

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

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

34. A pharmaceutical composition as described in any one of claims 28-33, wherein the non-ionic surfactant is polysorbate 80, 60, 40 or 20.

35. The pharmaceutical composition of claim 34, wherein the nonionic surfactant is present at about 0.005%-0.02% w / v.

36. The pharmaceutical composition of claim 35, wherein the nonionic surfactant is present at about 0.02% w / v.

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

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

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

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

41. A pharmaceutical composition as described in any one of claims 28-40, wherein the concentration of the rHuPH20 or its variant or fragment is 2000U / ml.

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

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

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

45. The pharmaceutical composition of claim 28, comprising: a) about 5 mg / mL to about 165 mg / mL of the anti-human PD-1 antibody; b) about 2000 U / ml of rHuPH20 or a variant or fragment thereof; c) about 10 mM histidine buffer, pH 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.

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

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

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

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

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

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

52. A pharmaceutical composition as described in any one of claims 1-51, wherein the rHuPH20 or a variant or fragment thereof consists of amino acid residues 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:

16.

53. A pharmaceutical composition as described in any one of claims 1-51, comprising rHuPH20 of SEQ ID NO:

18.

54. A pharmaceutical composition as described in any one of claims 1-51, comprising the rHuPH20 variant of SEQ ID NO:

17.

55. The pharmaceutical composition of any one of claims 1 to 54, wherein the anti-human PD-1 antibody is produced or obtainable by a method comprising the following steps: a) perfusing Chinese hamster ovary cells in a cell culture medium in a perfusion bioreactor by applying a perfusion rate of at least about 0.5-6.0 vascular volumes / day (vvd) and maintaining a capacitance value of 70-90 pF / cm, wherein the host cells comprise a polynucleotide encoding the light chain variable region and a polynucleotide encoding the heavy chain variable region, or both; b) continuously harvesting the antibody from the cell culture fluid to obtain a cell culture harvest fluid; c) purifying the cell culture harvest by successive affinity chromatography steps to obtain a purified composition.

56. The pharmaceutical composition of claim 55, wherein in step a), when the cell density reaches about 2 to 10 x 10 6 The perfusion was started when the concentration of 1 μg / mL cells / ml was 0.1 μg / mL.

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

58. A pharmaceutical composition as described in any one of claims 55-57, 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) on day 5 or after day 5.

59. The pharmaceutical composition of any one of claims 55-58, wherein the copper concentration in the perfusion bioreactor ranges from 1 ppb to 35 ppb.

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

61. The pharmaceutical composition of any one of claims 55-60, 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. pharmaceutical compositions as described in any one in claim 55-61, wherein said host cell comprises polynucleotides encoding light chains and polynucleotides encoding heavy chains, or polynucleotides encoding light chains and heavy chains, wherein said light chain is comprised of the amino acid sequence shown in SEQ ID NO:5, and said heavy chain is comprised of the amino acid sequence shown in SEQ ID NO:

10.

63. A pharmaceutical composition comprising: a) about 5 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody, wherein the antibody comprises a light chain variable region comprising three light chain CDRs and a heavy chain variable region comprising three heavy chain CDRs, wherein the three light chain CDRs comprise CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the three heavy chain CDRs comprise 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) about 5 mM to about 20 mM histidine buffer; pH between about 5.0 and about 6.0; d) about 6% to about 8% w / v sucrose; f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

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

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

66. The pharmaceutical composition of claim 63, comprising: a) about 5 mg / mL to about 165 mg / mL of the anti-human PD-1 antibody; b) about 2000 U / ml of said rHuPH20 or variant; c) about 10 mM histidine buffer, pH 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.

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

68. A pharmaceutical composition comprising: a) about 50 mg / mL to about 165 mg / mL of an anti-human PD-1 antibody, wherein the antibody comprises a light chain variable region comprising three light chain CDRs and a heavy chain variable region comprising three heavy chain CDRs, wherein the three light chain CDRs comprise CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, and CDRL3 of SEQ ID NO: 3, and the three heavy chain CDRs comprise 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 having a pH between about 5.0 and about 6.0; d) about 3% to about 10% w / v non-reducing disaccharide; e) from about 0.005% to about 0.4% w / v of a nonionic surfactant; and f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

69. The pharmaceutical composition of claim 68, comprising a), b), c) a histidine or acetate buffer at a pH between about 5.0 and about 6.0; d) about 6% to about 8% w / v sucrose or trehalose; e) optionally, from about 0.02% to about 0.2% w / v of a nonionic surfactant; and f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

70. The pharmaceutical composition of claim 68, comprising a), b), c) about 1 mM to about 30 mM histidine or acetate buffer, pH between about 5.0 and about 6.0; d) about 6% to about 8% w / v sucrose or trehalose; e) optionally, from about 0.01% to about 0.05% w / v polysorbate 80; f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

71. The pharmaceutical composition of claim 68, comprising a), b), c) about 1 mM to about 30 mM histidine or acetate buffer, pH between about 5.0 and about 6.0; d) about 6% to about 8% w / v sucrose or trehalose; e) about 0.1% to about 0.4% w / v poloxamer; f) optionally, about 5 mM to about 20 mM L-methionine or a pharmaceutically acceptable salt thereof.

72. The pharmaceutical composition of claim 68, comprising a), b), c) about 1 mM to about 30 mM histidine or acetate buffer, pH between about 5.0 and about 6.0; d) about 6% to about 8% w / v 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 L-methionine or a pharmaceutically acceptable salt thereof.

73. The pharmaceutical composition of any one of claims 63-72, wherein the concentration of the anti-human PD-1 antibody is about 75 mg / mL to about 165 mg / mL.

74. The pharmaceutical composition of any one of claims 63-72, wherein the concentration of the anti-human PD-1 antibody is about 100 mg / mL to about 165 mg / mL.

75. The pharmaceutical composition of any one of claims 63-72, wherein the concentration of the anti-human PD-1 antibody is about 130 mg / mL to about 165 mg / mL.

76. The pharmaceutical composition of any one of claims 63-72, wherein the concentration of the anti-human PD-1 antibody is about 130 mg / mL.

77. The pharmaceutical composition of any one of claims 63-72, wherein the concentration of the anti-human PD-1 antibody is about 165 mg / mL.

78. A pharmaceutical composition as described in any one of claims 63-77, wherein the anti-human PD-1 antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

9.

79. A pharmaceutical composition as described in any one of claims 63-77, wherein the anti-human PD-1 antibody consists of two light chains and two heavy chains, wherein the two light chains consist of the amino acid sequence shown in SEQ ID NO: 5, and wherein the two heavy chains consist of the amino acid sequence shown in any one of SEQ ID NOs: 10-15 or a combination thereof.

80. The pharmaceutical composition of any one of claims 1-79, which is a liquid.

81. The pharmaceutical composition of any one of claims 1-79, which is a reconstituted solution from a lyophilized formulation.

82. The pharmaceutical composition of any one of claims 1-81, wherein the pharmaceutical composition is contained in a glass vial or an injection device.

83. A method of treating cancer in a human patient in need thereof, the method comprising administering to the patient an effective amount of the pharmaceutical composition of any one of claims 1-82.

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