PHARMACEUTICAL COMPOSITIONS OF PROGRAMMED DEATH RECEPTOR 1 (PD-1) ANTIBODIES AND PH20 VARIANTS OR FRAGMENTS THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional patent application No. 63/393,101, filed July 28, 2022, each of which is incorporated by reference in its entirety herein.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the electronic sequence listing (25561-WO-SEQLIST.xml; Size:

22,004 bytes; and Date of Creation: November 28, 2022) are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0003] The invention provides pharmaceutical compositions of anti-PD-1 antibodies or antigen-binding fragments thereof with less than or equal to about 3.0 % oxidation of Metl05 in the CDRH3 heavy chain region and/or about 1.0-12.0% acidic species, and PH20 variants or fragments thereof.

BACKGROUND OF THE INVENTION

[0004] Immune checkpoint therapies targeting the programmed death receptor- 1 (PD-1) axis have resulted in groundbreaking improvements in clinical response in multiple human cancers (Brahmer et al., N Engl J Med 2012, 366: 2455-65; Garon et al. N Engl J Med 2015, 372: 2018-28; Hamid et al., N Engl J Med 2013, 369: 134-44; Robert et al., Lancet 2014, 384: 1109- 17; Robert et al., NEnglJMed 2015, 372: 2521-32; Robert et al., N Engl JMed 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 JMed 2013, 369: 122-33). The interaction of the PD-1 receptor on T-cells with its ligands, PD-L1 and PD-L2, on tumor and immune infiltrating cells regulates T-cell mediated immune responses and may play a role in immune escape by human tumors (Pardoll DM. Nat Rev Cancer 2012,12: 252-64). Binding of PD-1 to either of its ligands results in delivery of an inhibitory stimulus to the T cell. Immune therapies targeting the PD-1 axis include monoclonal antibodies directed to the PD-1 receptor (KEYTRUDA™ (pembrolizumab), Merck and Co., Inc., Kenilworth, NJ and OPDIVO™ (nivolumab), Bristol-Myers Squibb, Princeton, NJ) and also those that bind to the PD-L1 ligand (MPDL3280A; TECENTRIQ™ (atezolizumab), Genentech, San Francisco, CA). Both therapeutic approaches have demonstrated anti-tumor effects in numerous cancer types. [0005] Oxidation of methionine is one of the major degradation pathways in many protein pharmaceuticals. Methionine residues in proteins are susceptible to oxidation, resulting in the formation of methionine sulfoxide and, under extreme conditions, sulfones. An exposed methionine residue or a methionine residue in the CDR of an antibody has the potential of impacting the biological activity of the antibody through oxidation. Major degradation pathways of pembrolizumab include oxidation of methionine 105 (Metl05) in the heavy chain CDR and Fc methionine residues when exposed to light or peroxide stress. The crystal structure of the pembrolizumab Fab fragment in complex with PD-1 shows that Metl05 is part of the paratope, and interacts with Lysl31 in PD-1. Lee, J. Y. et al. Nat. Commun. 7, 13354 (2016). Therefore, it is desirable to obtain pembrolizumab compositions with low oxidation, in particular at the Metl05 position.

[0006] Deamidation of asparagine residues can lead to succinimide formation, which can then be converted to aspartate or isoaspartate. Deamidation in antibodies, in particular in the CDR regions, has the potential of impacting the biological activity of the antibody. The crystal structure of the pembrolizumab Fab fragment in complex with PD-1 shows that N52, N55 and N59 of the 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 interaction with N59. Lee, J. Y. et al. Nat. Commun. 7, 13354 (2016). Therefore, it is also desirable to obtain pembrolizumab compositions with low deamidation variants.

[0007] Hyaluronidases are enzymes that degrade hyaluronic acid present in the extracellular matrix. It is known that there are six types of hyaluronidases in humans: Hyall, Hyal2, Hyal3, Hyal4, HyalPSl, and PH20/SPAM1. PH20/SPAM1 (hereinafter referred to as PH20) is expressed in the sperm plasma membrane and the acrosomal membrane.

[0008] Hyaluronidase hydrolyzes hyaluronic acid, thereby reducing the viscosity of hyaluronic acid in the extracellular matrix and increasing the permeability thereof into tissue (skin). The subcutaneous area 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 examples in which PH20 is used, PH20 is often co-administered with an antibody therapeutic agent which is injected subcutaneously (Bookbinder et al., 2006).

SUMMARY OF THE INVENTION

[0009] The invention provides pharmaceutical compositions comprising anti-human PD- 1 antibodies or antigen-binding fragments thereof with less than or equal to about 3.0 % oxidation of Metl05 in the CDRH3 heavy chain region and PH20 variants or fragments thereof. The invention also provides a composition comprising an anti-human PD-1 antibody main species comprising 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 acidic species of the anti-human PD-1 antibody main species, wherein the amount of acidic species is about 1.0-12.0%, and a PH20 variant or fragment. The invention also provides a composition comprising an anti-human PD-1 antibody main species comprising 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 acidic and basic species of the anti-human PD-1 antibody main species, wherein the amount of main species is about 65- 95%, and a PH20 variant or fragment. The invention also provides pharmaceutical compositions comprising anti -human PD-1 antibodies or antigen-binding fragments thereof and PH20 variants or fragments thereof, wherein the pharmaceutical composition has one or more of the following: (i) less than or equal to about 3.0 % oxidation of Metl05 in the CDRH3 heavy chain region of the anti-human PD-1 antibodies or antigen-binding fragments thereof; (ii) 1.0-12.0% of acidic species of the anti -human PD-1 antibodies or antigen-binding fragments thereof; and (iii) acidic and basic species of the anti-human PD-1 antibody main species, wherein the amount of main species is about 65-85%. In one embodiment, the anti-PD-1 antibodies or antigen-binding fragments were produced or obtainable by a continuous perfusion process.

[0010] The invention also provides a formulation of the above pharmaceutical composition comprising: a) about 20 mg/mL to about 200 mg/mL of the anti -human PD-1 antibodies, or antigen binding fragments thereof; b) about 150 -8000 U/ml of a PH20 variant or fragment thereof; c) a buffer; d) a non-reducing dissacharide; e) a non-ionic surfactant; and, optionally 1) an anti-oxidant.

[0011] In one embodiment, the formulation comprises: a) about 20 mg/mL to about 200 mg/mL of the anti-human PD-1 antibodies, or antigen binding fragments thereof; b) about 150 - 8000 U/ml of the PH20 variant or fragment thereof; c) about 5 mM to about 20 mM buffer; d) about 3% to about 10% weight/volume (w/v) of a non-reducing dissacharide selected from the group consisting of sucrose and trehalose; e) about 0.005 % to about 0. 10% non-ionic surfactant; and, optionally f) about 1 mM to about 30 rnM anti-oxidant.

[0012] Surprisingly, under light stress, certain embodiments of the above formulations have increased hyaluronidase activity of the PH20 variant or fragment thereof compared to the corresponding formulations with pembrolizumab reference produced from a fed batch process. The formulation can be a liquid formulation or a liquid formulation that is reconstituted from a lyophilized formulation.

[0013] In specific embodiments of the invention, the PH20 variant or fragment is PH20 variant fragment 2 set forth in the amino acid sequence of SEQ ID NO: 18.

[0014] Also provided herein are methods of treating cancer in a human patient in need thereof comprising: administering an effective amount of the pharmaceutical compositions of the invention to the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1: Cell culture performance of continuous perfusion culture at 2 L and 50 L scales. Top left: Viable Cell Density (VCD); Top right: Viability; Bottom left: permeate titer; and Bottom right: product sieving.

[0016] Figure 2: The total ion chromatograms were generated by the reduced peptide digestion described in Example 3. Both fed batch produced pembrolizumab formulated drug product reference standard (top) and continuous perfusion produced pembrolizumab (bottom) were loaded similarly and the relative abundance shows identical peptides and retention times for both samples. NL refers to normalization level; m/z refers to mass over charge; FTMS refers to Fourier transform mass spectrometry, ESI refers to electrospray ionization.

[0017] Figure 3:The extracted ion chromatogram of the unmodified Ml 05 peptide for fed batch produced pembrolizumab reference standard (top) and continuous perfusion produced pembrolizumab (bottom) . The retention time of the unmodified peptide containing Ml 05 is ~37.8min. NL refers to normalization level; m/z refers to mass over charge; FTMS refers to Fourier transform mass spectrometry, ESI refers to electrospray ionization; RT refers to retention time.

[0018] Figure 4: The extracted ion chromatogram of the modified M105 peptide for fed batch produced pembrolizumab reference standard (top) and continuous perfusion produced pembrolizumab (bottom) . The retention time of the modified peptide containing Ml 05 is 36.4min. NL refers to normalization level; m/z refers to mass over charge; FTMS refers to Fourier transform mass spectrometry, ESI refers to electrospray ionization. RT refers to retention time.

[0019] Figure 5: Changes in M105 oxidation of pembrolizumab over time in Harvested Cell Culture Fluid (HCCF) samples. The black circles are the results of each sample injection, and the line is the linear fit of the data and is described by y = 0.038x + 0.94, with an R squared value of 0.9833.

[0020] Figure 6: Ion exchange chromatography analytical diagram of pembrolizumab after Protein A chromatography purification of HCCF from the continuous perfusion process. The retention time of each peak is annotated in the figure.

[0021] Figure 7: Ion exchange chromatography analytical diagram of pembrolizumab after Anion exchange chromatography and Protein A chromatography purification of HCCF from the continuous perfusion process. The retention time of each peak is annotated in the figure.

[0022] Figure 8: Overlay of Ion exchange chromatography analytical diagram of pembrolizumab after Anion exchange step of the invention and pembrolizumab reference standard obtained from fed-batch method. The diamonds delineate the start and end of the integrated peak.

[0023] Figure 9: Total % acidic species (acidicl+acidic variants+pre-main) in the cell culture fluid of the bioreactor (BRX), cell-free permeate (PERM), after Protein A chromatography step (PAP), after anion exchange chromatography (AEXP) in a time course by culture days.

[0024] Figure 10: Total % main species (as measured by ion exchange) in the cell-free permeate (PERM), after Protein A chromatography step (PAP), after anion exchange chromatography (AEXP) in a time course by culture days.

[0025] Figure 11: Total % basic species (basic l+basic2+basic variant A+basic variant B) in the cell-free permeate (PERM), after Protein A chromatography step (PAP), after anion exchange chromatography (AEXP) in a time course by culture days.

[0026] Figure 12: % Basicl species in the cell-free permeate (PERM), after Protein A chromatography step (PAP), after anion exchange chromatography (AEXP) in a time course by culture days.

[0027] Figure 13: HIC-HP diagram of pembrolizumab reference from fed-batch process. Pre-peak3 contains antibody with Met! 05 oxidation in both heavy chains; pre-peaks 1 and 2 contain antibody with Metl05 oxidation in one heavy chain. Retention time (x-axis) and absorbance (y-axis) are shown.

[0028] Figure 14. Linearly fit calibration curve for activity assay as described in Example 6.

[0029] Figure 15. Enzymatic activity of PH20 Variant Fragment 2 after light stress when co-formulated with Pembrolizumab CPP. Control is without light stress. Pembrolizumab produced from the continuous perfusion process (CPP) concentration from 0-165 mg/ml was tested.

[0030] Figure 16. Enhanced retention of enzyme activity of PH20 Variant Fragment 2 in Pembrolizumab CPP co-formulations compared to Pembrolizumab reference co-formulation and PH20 Variant Fragment 2 alone after light stress. Pembrolizumab CPP and Pembrolizumab reference was at 165 mg/ml.

[0031] Figure 17. Metl05 oxidation% and acidic species% (peak area%) of Pembrolizumab reference and Pembrolizumab CPP after light stress. Pembrolizumab CPP and Pembrolizumab reference was at 165 mg/ml.

[0032] Figure 18. Enhanced retention of enzyme activity of PH20 Variant Fragment 2 in Pembrolizumab CPP co-formulations after exposure to stainless steel at 3 months 25°C. Pembrolizumab CPP and Pembrolizumab reference was at 165 mg/ml.

[0033] Figure 19. Metl05 % oxidation of Pembrolizumab CPP with or without PH20 Variant Fragment 2 after light stress or exposure to stainless steel (SS exposure).

Pembrolizumab CPP was at 165 mg/ml.

[0034] Figure 20. Acidic species% of Pembrolizumab CPP with or without PH20 Variant Fragment 2 after light stress or exposure to stainless steel (SS). Pembrolizumab CPP was at 165 mg/ml.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions and Abbreviations

[0035] As used throughout the specification and appended claims, the following abbreviations apply:

API active pharmaceutical ingredient

CDR complementarity determining region in the immunoglobulin variable regions CHO Chinese hamster ovary

CI confidence interval

DS drug substance

EC50 concentration resulting in 50% efficacy or binding

ELISA enzyme-linked immunosorbant assay

FFPE formalin-fixed, paraffin-embedded

FR framework region

HC heavy chain

HNSCC head and neck squamous cell carcinoma

HP-HIC high performance hydrophobic interaction chromatography

HP-IEX high performance ion-exchange chromatography

HP-SEC high performance size exclusion chromatography

IC50 concentration resulting in 50% inhibition

IgG immunoglobulin G

IHC immunohistochemistry or immunohistochemical mAb monoclonal antibody

NCBI National Center for Biotechnology Information

NSCLC non-small cell lung cancer

PCR polymerase chain reaction

PD-1 programmed death 1 (a.k.a. programmed cell death- 1 and programmed death receptor 1)

PD-L1 programmed cell death 1 ligand 1

PD-L2 programmed cell death 1 ligand 2

PS80 or PS 80 polysorbate 80

SWFI sterile water for injection

TNBC triple negative breast cancer

VH immunoglobulin heavy chain variable region

VK immunoglobulin kappa light chain variable region

VL immunoglobulin light chain variable region v/v volume per volume

WFI water for injection w/v weight per volume [0036] So that the invention may be more readily understood, 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 one of ordinary skill in the art to which this invention belongs.

[0037] As used throughout the specification and in the appended claims, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.

[0038] Reference to “or” indicates either or both possibilities unless the context clearly dictates one of the indicated possibilities. In some cases, “and/or” was employed to highlight either or both possibilities.

[0039] As used herein, “acidic species” refers to the anti-PD-1 antibody species that is more acidic (e.g. as determined by cation exchange chromatography) than the anti-PD-1 antibody main species. Such acidic species are detected by various chromatography purification methods for separating molecule variants by charge, such as ion exchange, for example, cation exchange chromatography (e.g. the method described in Example 5) or WCX-10 HPLC (a weak cation exchange chromatography), optionally followed by mass spectroscopy. Generally, the acidic species has a lower isoelectric point (pl) than the main species, and can have a more acidic character due to for example, methionine oxidation, sialylation of asparagine residues or deamidated variants of the antibody, or a combination thereof. Examples of the acidic species include but are not limited to the acidic variants, acidic 1 and pre-main peaks identified in Figure 6 or 7 of the invention. Any of the acidic species may also have one or more of CHO N-linked glycans selected from the group consisting of G0-F, Gl-F, G2-F, GO, Gl, G2 and Man5, for example at N297 in the CH2 domain.

[0040] In one embodiment, the anti-PD-1 antibody acidic species is as identified by peak(s) eluted prior to the main peak according to a cation ion exchange method. In another embodiment, the anti-PD-1 antibody acidic species is as identified by peak(s) eluted prior to the main peak according to a weak cation ion exchange method. In an ion exchange chromatography method, the “% acidic species” refers to the total area of acidic species peaks divided by the total area of all peaks in the elution chromatogram.

[0041] As used herein, “acidicl species” refers to an acidic species with the presence of one or more of deamidation, succinimide, aspartate or isoaspartate formation in one or more of N384, N389 and N390 of the heavy chain of the anti-PD-1 antibody main species. Such acidicl species are detected by various chromatography purification methods for separating molecule vanants by charge, such as ion exchange, for example, cation exchange chromatography (e.g. the method described in Example 5) or WCX-10 HPLC (a weak cation exchange chromatography), followed by mass spectroscopy of the acidic species peaks.

[0042] In one embodiment, the anti-PD-1 antibody acidicl species is as identified in acidic 1 peak of Figure 6 or 7, and eluted according to the cation ion exchange method described in Example 5. In an ion exchange chromatography method, the “% acidicl species” refers to the total area of acidicl peak divided by the total area of all peaks in the elution chromatogram.

[0043] As used herein, “acidic variants species” refers to an acidic species with the presence of one or more of deamidation, succinimide, aspartate or isoaspartate formation in one or more of N31, N52, N55, N59, and N61 of the heavy chain; or the presence of M105 oxidation in the heavy chain; or a combination thereof of the anti-PD-1 antibody main species. Such acidic variants species are detected by various chromatography purification methods for separating molecule variants by charge, such as ion exchange, for example, cation exchange chromatography (e.g. the method described in Example 5) or WCX-10 HPLC (a weak cation exchange chromatography), followed by mass spectroscopy of the acidic species peaks.

[0044] In one embodiment, the anti-PD-1 antibody acidic variants species is the anti-PD- 1 antibody species as identified by the acidic variants peak(s) in Figure 6 or 7, and eluted according to the cation ion exchange method described in Example 5. In an ion exchange chromatography method, the “% acidic variants species” refers to the total area of acidic variants peak(s) divided by the total area of all peaks in the elution chromatogram.

[0045] As used herein, “basic species” refers to the anti-PD-1 antibody species that is more basic (e.g. as determined by cation exchange chromatography) than the anti-PD-1 antibody main species. Such basic species are detected by various chromatography purification methods for separating molecule variants by charge, such as ion exchange, for example, cation exchange chromatography (e.g. the method described in Example 5) or WCX-10 HPLC (a weak cation exchange chromatography), optionally followed by mass spectroscopy. Generally, the basic species has a higher pl than the main species, and can have a more basic character due to modifications or differences from the main species including but not limited to the presence of the C-terminal lysine residue (SEQ ID NO: 10 or 12), the presence ofN-terminal glutamine residue (SEQ ID NO: 10 or 13), or alpha-amidation of a C-terminal leucine residue (SEQ ID NO: 14 or 15), truncation of N-termmal amino acid residues 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 the basic species include but are not limited to the basic variant A, basic variant B, basic 1 and basic 2 peaks identified in Figure 6 or 7 of the invention. Any of the basic species may also have one or more of CHO N-linked glycans selected from the group consisting of GO-F, Gl-F, G2-F, GO, Gl, G2 and Man5, for example at N297 in the CH2 domain.

[0046] In one embodiment, the anti-PD-1 antibody basic species is as identified by peak(s) eluted after the main peak according to a cation ion exchange method. In another embodiment, the anti-PD-1 antibody basic species is as identified by peak(s) eluted after the main peak according to a weak cation ion exchange method. In an ion exchange chromatography method, the “% basic species” refers to the total area of basic species peak(s) divided by the total area of all peaks in the elution chromatogram.

[0047] As used herein, “main species” refers to the anti-PD-1 antibody species identified as the majority of the antibody species in a mixture with one or more acidic or basic species thereof. Such main species are detected by various chromatography purification methods for separating molecule variants by charge, such as ion exchange, for example, cation exchange chromatography (e.g. the method described in Example 5) or WCX-10 HPLC (a weak cation exchange chromatography), optionally followed by mass spectroscopy. The mixture can be a result of for example, antibody preparations from mammalian cells and post-translational modifications thereof, upstream and downstream processing, or storage. The main species may also have one or more of CHO N-linked glycans selected from the group consisting of G0-F, Gl- F, G2-F, GO, Gl, G2 and Man5, for example at N297 in the CH2 domain.

[0048] In one embodiment, the anti-human PD-1 antibody main species comprises an antibody consisting of two heavy chains and two light chains, each light chain comprises a 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 each heavy' chain comprises a heavy' chain variable region comprising three heavy chain CDRs of CDRH1 of SEQ ID NO:6, CDRH2 of SEQ ID NO:7 and CDRH3 of SEQ ID NO:8. In one embodiment, the main species comprises the anti-PD-1 antibody consisting of two heavy chains and two light chains, each heavy chain consisting of the amino acid sequence of SEQ ID NO: 11, and each light chain consisting of the amino acid sequence of SEQ ID NO: 5. In another embodiment, the anti-PD-1 antibody main species is produced from a Chinese Hamster Ovary cell that comprises a polynucleotide encoding a light chain that consists of the amino acid sequence of SEQ ID NO: 5 and a polynucleotide encoding a heavy chain that consists of the amino acid sequence of SEQ ID NO: 10, 13 or 15, or a polynucleotide encoding the light chain and the heavy chain. [0049] In one embodiment, the mam species is identified as the mam peak according to a cation ion exchange method. In an ion exchange method, the “% main species” refers to the total area of main peak divided by the total area of all peaks in the elution chromatogram.

[0050] As used herein, “basic 1 species” refers to 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: 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 alpha-ami dated, 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 chromatography purification methods for separating molecule variants by charge, such as ion exchange, for example, cation exchange chromatography (e.g. the method described in Example 5) or WCX-10 HPLC (a weak cation exchange chromatography), followed by mass spectroscopy of the basic species peaks.

[0051] In one embodiment, the anti-PD-1 antibody basic 1 species is as identified by basic 1 peak in Figure 6 or 7, and eluted according to the cation ion exchange method described in Example 5. In an ion exchange chromatography method, the “% basicl species” refers to the total area of basicl peak divided by the total area of all peaks in the elution chromatogram.

[0052] As used herein, “deamidated variant” refers to an antibody wherein one or more asparagine residue(s) have been deamidated. The deamidated variant can be in the form of succinimide, aspartate or isoaspartate, i.e., the neutral amide side chain has been converted to a residue with an overall acidic character.

[0053] 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 ion exchange method. In another embodiment, a Thermo Scientific ProPac WCX-10 column is used, with a 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 column temperature of 35 °C. In one embodiment, a non-linear gradient is used with: 22%-22%B for 0-0.6 min; 22%-29%B for 0.6- 15.0 min; 29%-70%B for 15.0-30.0 min; 70%-100%B for 30.0-30.5 min; and 100%-100%B from 30.5-33.0 min. In a further embodiment, the cation ion exchange method is described in Example 5. [0054] As used herein, "express" and "expression" refer to allowing or causing the information in a gene or coding sequence, e.g., an RNA or DNA, to become manifest; for example, producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene. A DNA sequence can be expressed in or by a cell to form an "expression product" such as an RNA (e.g, mRNA) or a protein. The expression product itself may also be said to be “expressed” by the cell.

[0055] As used herein, "expression vector" or "expression construct" refer to a vehicle (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 transcription and translation machinery of the host cell.

[0056] “Expression cassette” as used herein, refers to a polynucleotide that comprises elements sufficient to control expression of a gene, including but not limited to, a promoter operably linked to the gene sequence or operably linked to a multiple cloning site for inserting the gene sequence, and a polyA signal. In some embodiments, the expression cassette further comprises one or more regulatory elements that can regulate the expression of the gene at transcriptional, translational, and/or chromatin levels.

[0057] As used herein, “promoter” or “promoter sequence” refer to a segment of DNA that contains a regulatory region capable of recruiting an RNA polymerase (e.g., directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. Within the promoter sequence may be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the recruiting of RNA polymerase.

[0058] As used herein, “enhancer” or “enhancer sequence” refer to a DNA regulatory region that enhances transcription of a promoter independently of its distance, location, or orientation to the promoter. In certain embodiments, the enhancer is immediately adjacent to the promoter. In some embodiments, the enhancer is distant from the promoter. In other embodiments, the promoter and the enhancer are one combined sequence, referred as a “combo enhancer/promoter” herein.

[0059] As used herein, “internal ribosome entry site” or “IRES” refer to an RNA element or sequence that allows for translation initiation in a cap-independent manner by recruiting ribosomes directly. As used herein, the term “internal ribosome entry site” or “IRES” also encompasses the DNA sequence that can be transcribed into the RNA sequence that allows for translation initiation in a cap-independent manner by recruiting ribosomes directly. IRES can be a wild type IRES from any species or a variant or mutant thereof, whether naturally occurred or man-made. Examples of IRES that can be used include, but are not limited to, the nucleotide sequence of the 5' nontranslated 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.), IRES element described by Bochkov & Palmenberg (Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. Biotechniques. 2006 Sep;41(3):283- 4), IRES element from expression vector plnSRT-GFP (GenBank LC417349. 1), IRES element from expression vector pCeMM-CTAP(SG) (GenBank EF467048.1), 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), IRES element from expression vector pIRESneo3 (Clontech/Takara Bio), IRES elements described in WO 2015/016786, WO 2015/021077, WO 2016/003368, WO 2016/074016, or WO 2013/092743, or variants thereof.

[0060] As used herein, “regulatory element,” “regulatory region,” or “regulatory sequence” refer to a polynucleotide sequence that has the ability to regulate (such as, initiate, activate, enhance, increase, decrease, inhibit, suppress, or silence) expression of a gene. In some embodiments, the regulation is achieved by binding of cellular factors to the polynucleotide sequence. In other embodiments, the regulation is achieved by interaction between cellular factors. The regulation can occur at one or more different levels in the expression process from DNA to protein, including but not limited to transcriptional, translational, or chromatin levels. [0061] As used herein, “insulator” refers to a class of DNA elements or sequences that possess an ability to isolate the proximal DNA region by preventing the positional effect from the surrounding chromosome area. In certain embodiments, the insulator can block enhancer when the insulator is situated between the enhancer and the promoter. In some embodiments, the insulator can act as barriers that prevent the advance of nearby condensed chromatin that might otherwise silence expression. In other embodiments, the insulator can block enhancer and act as barriers.

[0062] As used herein, “expression augmenting sequence element” or “EASE”, refer to a DNA element or sequence that can increase expression of a protein when the DNA element or sequence is placed upstream of the promoter that controls the expression of the protein.

[0063] As used herein, “tripartite leader” or “TPL” refer to an RNA element or sequence in the 5 ’-untranslated region of adenovirus late-expressed mRNA that has an ability to initiate translation of the late-expressed mRNA in a cap-independent manner. As used herein, the term “tripartite leader” or “TPL” also encompasses the DNA sequence that can be transcribed into the RNA sequence in the 5 ’-untranslated region of adenovirus late-expressed mRNA that has an ability to initiate translation of the late-expressed mRNA in a cap-independent manner.

[0064] As used herein, “inverted terminal repeat” or “ITR”, in the context of transposon technology, refers to a DNA element or sequence and its inverted version at either end of a transposon that signals where the breakage and joining should occur.

[0065] As used herein, “selectable marker” or “selection marker” refer to a protein which allows the specific selection of cells that express this protein by the addition of a corresponding selecting agent to the culture medium. In certain embodiments, the selectable marker is a eukaryotic selectable marker, which allows selection of eukaryotic cells that express the marker protein. In some embodiments, the selectable marker is a bacterial selectable marker, which allows selection of bacterial cells that express the marker protein.

[0066] A "polynucleotide sequence", "nucleic acid sequence" or "nucleotide sequence", as used herein, refer to a series of nucleotide bases (also called "nucleotides") in a nucleic acid, such as DNA or RNA, and means any chain of two or more nucleotides.

[0067] As used herein, a “host cell” refers to any cell of any organism that is used for the purpose of producing a recombinant protein encoded by an expression vector or propagating the expression vector introduced into the host cell. A “mammalian recombinant host cell” refers to a mammalian host cell that comprises a heterologous expression vector, which may or may not be integrated into the host cell chromosome. A “bacterial recombinant host cell” refers to a bacterial host cell that comprises a heterologous expression vector, which may or may not be integrated into the host cell chromosome.

[0068] The term "fed-batch culture", as used herein, refers to a method of culturing cells in which additional nutrients are provided to the culture during the cultivation process. A fed- batch culture is typically stopped at some point and the cells and/or components in the medium are harvested. The product accumulates and remains in the bioreactor until the end of the run.

[0069] As used herein, “harvesting” an antibody or antigen-binding fragment involves separating it from particulate matter that can include host cells, cell aggregates, and/or lysed cell fragments, into a cell-free fraction that is substantially free of host cells and cellular debris, i.e., a cell-free “permeate.” Such cells and cellular debris is removed from the cell culture broth, for example, by centrifugation, depth filtration and/or microfiltration. For example, to make the cell- free permeate, one can employ hollow fiber membranes or a series of filtration steps such as depth filtration. “Continuously harvesting” refers to harvesting cell culture broth while antibody production takes place in the bioreactor. Secreted protein products in the bioreactor can be continuously harvested from the cell culture broth by microfiltration during the process of removing medium via the perfusion system, the protein of interest thus being isolated in a microfilter permeate exiting the perfusion system. The microfilter can be a Tangential Flow Filtration (TFF) unit including a hollow fiber module or Alternating Tangential Flow Filtration (ATF) unit. Commercially available TFF units include, but are not limited to, Microza® TFF unit or KrosFlow®Max. Commercially available hollow fiber modules can be obtained for example, from Pall or Repligen. In one embodiment, the perfusion bioreactor has a constant permeate rate for the cell culture broth comprising the antibody or antigen-binding fragment to maintain a consistent flow rate to the affinity chromatography step.

[0070] As used herein, “cell culture broth” refers to broth comprising the host cells, cellular debris, cell culture medium, antibody or antigen-binding fragment during the cell growth and antibody production process.

[0071] As used herein, “harvest cell culture fluid” or “HCCF” refers to the cell culture fluid comprising the antibody or antigen-binding fragment obtained after harvesting the cell culture broth, which is substantially free of host cells and cellular debris. In one embodiment, the HCCF is a cell-free permeate.

[0072] As used herein, “fluidly connected,” “fluidly,” or is “fluidly connected to”, or “fluidly receives material from”, refers to another step of the manufacturing process or from another system, when material containing the protein of interest flows by pipe, tubing, or other closed conduit between steps or systems without manual loading or unloading.

[0073] As used herein in the context of HCCF, “continuously purifying” refers to uninterrupted flow of the HCCF to at least one affinity stationary phase for at least the loading step, and optionally an uninterrupted flow for any washing or elution steps.

[0074] As used herein, “perfusion” or “perfusing” refer to a method of culturing cells in which additional fresh medium is provided continuously over some period of time, to the culture (subsequent to the beginning of the culture process), and simultaneously removing medium while harvesting the antibody or antigen-binding fragment from the medium continuously. The fresh medium typically provides nutritional supplements for the cells that have been depleted during the culturing process.

[0075] “Perfusion rate”, as used herein, refers to the rate at which fresh medium is provided and cell culture fluid is removed. [0076] As used herein, a “perfusion bioreactor” refers to a bioreactor for culturing cells in which equivalent volumes of culture medium can be added to and concurrently removed from the reactor. In one embodiment, the cells are retained in the bioreactor. A perfusion bioreactor includes a bioreactor and an operably attached perfusion system, which provides a steady source of fresh nutrient medium and removal of cell waste products. The bioreactor and the perfusion system of the perfusion bioreactor can be separate mechanical units that operate in coordination. Numerous commercially available examples include, but are not limited to, a variety of Xcellerex® brand single-use bioreactors (SUBs; GE Healthcare Life Sciences) and KrosFlo® brand perfusion flow-path assemblies and systems (Spectrum; Repligen), which bioreactors and perfusion systems can be suitably combined into a perfusion bioreactor by the skilled practitioner. Alternatively, the bioreactor and the perfusion system can be assembled into a single mechanical unit, for example, but not limited to, a 3D Biotek brand perfusion bioreactor (Sigma-Aldrich).

[0077] The term “surge vessel”, as used herein, refers to a well-mixed (providing sufficient mixing such that fluid is homogenous) storage reservoir, mixing vessel, feed tank, or collection vessel (or interchangeably, a “collection tank”), at the downstream end of a conduit, feeder, dam, pipe, or tubing, to absorb discrepant flow rates between two fluidly connected unit operations, e g., the flow rate of a permeate coming from a bioreactor and the flow rate of a first chromatography system under automated control in continuous or semi-continuous format process embodiments of the invention. The surge vessel absorbs changes or differences in flow rates by allowing the volume to surge within pre-set volume range limits between the fluidly connected unit operations.

[0078] The term “residence time”, as used herein, refers to the average time a fluid solution spends inside a vessel. For perfusion this is the inverse of the exchange rate (e.g. 2VVD has a mean residence time of 0.5 day). Sieving of solution components, retention of solution components by the membrane is neglected for residence time.

[0079] As used herein, “M105”, “Metl05”, or “Methionine 105” refers to the methionine in CDRH3 region of the heavy chain in SEQ ID NO: 8 (RDYRFDMGFDY).

[0080] As used herein, “% oxidation of M105” or “% oxidation of Metl05” refer to a) total amount of anti-PD-1 antibody fragment(s) of the invention with oxidized Metl05 versus total amount of anti-PD-1 antibody fragment(s) of the invention with and without oxidized Metl05 as determined by reduced peptide mapping, liquid chromatography and mass spectroscopy; or b) total amount of anti-PD-1 antibody of the invention with oxidized Metl05 versus total amount of anti-PD-1 antibody of the invention with and without oxidized Metl05 as determined in a Hydrophobic Interaction Chromatography (HIC).

[0081] By "binding" an antibody or antigen-binding fragment to a stationary phase, is meant 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 is reversibly associated with the stationary phase by interactions between the antibody or antigen-binding fragment and the ligand immobilized on the stationary phase.

[0082] As used herein, the term “equilibration solution” refers to a solution used to equilibrate the stationary phase prior to loading the antibody or antigen-binding fragment on the stationary phase. The equilibration solution can comprise one or more of a salt and buffering species. In one embodiment, the equilibration solution is the same condition as the loading solution comprising the antibody or antigen-binding fragment.

[0083] As used herein, the term “loading solution” refers to the solution which is used to load the composition comprising the antibody or antigen-binding fragment of interest and one or more impurities onto the stationary phase. The loading solution may optionally further comprise one or more of a buffering species, and salt.

[0084] As used herein, the term "wash solution" refers to a solution used to wash or reequilibrate the 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/are such that the impurities are removed from the stationary phase. For re-equilibration, the wash solution and equilibration solution may be the same, but this is not required. The wash solution can comprise one or more of a salt and buffering species.

[0085] As used herein, the "elution solution" refers to the solution used to elute the antibody or antigen-binding fragment of interest from the stationary phase. The elution solution can comprise one or more of a salt, or buffering species. The presence of one or more of salt, buffering species, pH or conductivity of the elution solution is/are such that the antibody or antigen-binding fragment is eluted from the stationary phase.

[0086] As used herein, the term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, the current flows by ion transport. Therefore, with an increasing amount of ions present in the aqueous solution, the solution will have a higher conductivity. The unit of measurement for conductivity is mS/cm, and can be measured using a conductivity meter sold, e.g., within the GE Healthcare Akta™ System. The conductivity of a solution may be altered by changing the concentration of ions therein. For example, the concentration of a buffering agent and/or concentration of a salt (e.g. NaCl or KC1) in the solution may be altered in order to achieve the desired conductivity. Preferably, the salt concentration of the various buffers is modified to achieve the desired conductivity as in the Examples below.

[0087] As used herein, "purifying" an antibody or antigen-binding fragment of interest or “purified composition” refers to increasing the degree of purity of the antibody or antigenbinding fragment in the composition by removing (completely or partially) at least one impurity from the composition. The impurity can be host cell components such as serum, proteins or nucleic acids, cellular debris, growth medium or antibody aggregates. The term is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical composition that includes the antibody or antigenbinding fragment.

[0088] As used herein, “continuous multi-column chromatography system” refers to a chromatography system containing at least two stationary phases with similar impurity separation function, which allow at least one stationary phase to load the sample, and at least one stationary phase to perform the non-loading steps (one or more of equilibration, washing, elution and regeneration).

[0089] As used herein, "stationary phase" refers to any surface onto which one or more ligands can be immobilized. The stationary phase may be a suspension, a discontinuous phase of discrete particles, plate, sensor, chip, capsule, cartridge, resin, beads, monolith, gel, a membrane, or membrane adsorber etc. Stationary phases may also be packed into a purification column (e.g. packed with resin beads). Examples of materials for forming the stationary phase include mechanically stable matrices such as porous or non-porous beads, inorganic materials (e.g., porous silica, controlled pore glass (CPG) and hydroxyapatite), synthetic organic polymers (e.g., polyacrylamide, polymethylmethacrylate, polystyrene-divinylbenzene, poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles and derivatives of any of the above) and polysaccharides (e.g., cellulose, agarose and dextran). See Jansson, J. C.; Ry den, L. Protein Purification,' Wiley: New York, 1998.

[0090] As used herein, “impurity” refers to a material different from the desired antibody or antigen-binding fragment. The impurity can be Host Cell Protein (HCP), Host Cell DNA (HC-DNA), protein aggregates or clips and other undesired protein modifications (i.e., oxidized species, acid variant species). [0091] "Treat" or "treating" a cancer, as used herein, refers to the administration of a composition of the invention to a subject having an immune condition or cancerous condition, or diagnosed with a cancer or pathogenic infection (e.g. viral, bacterial, fungal), to achieve at least one positive therapeutic effect, such as for example, reduced number of cancer cells, reduced tumor size, reduced rate of cancer cell infiltration into peripheral organs, or reduced rate of tumor metastasis or tumor growth. "Treatment" may include one or more of the following: inducing/increasing an antitumor immune response, stimulating an immune response to a pathogen, toxin, and/or self-antigen, stimulating an immune response to a viral infection, decreasing the number of one or more tumor markers, halting or delaying the growth of a tumor or blood cancer or progression of disease associated with PD-1 binding to its ligands PD-L1 and/or PD-L2 (“PD-1 -related disease”) such as cancer, stabilization of PD-1 -related disease, inhibiting the growth or survival of tumor cells, eliminating or reducing the size of one or more cancerous lesions or tumors, decreasing the level of one or more tumor markers, ameliorating, abrogating the clinical manifestations of PD-1 -related disease, reducing the severity or duration of the clinical symptoms of PD-1 -related disease such as cancer, prolonging the survival of a patient relative to the expected survival in a similar untreated patient, and inducing complete or partial remission of a cancerous condition or other PD-1 related disease.

[0092] “Immune condition” or “immune disorder” encompasses, e.g., pathological inflammation, an inflammatory disorder, and an autoimmune disorder or disease. “Immune condition” also refers to infections, persistent infections, and proliferative conditions, such as cancer, tumors, and angiogenesis, including infections, tumors, and cancers that resist eradication by the immune system. “Cancerous condition” includes, e.g., cancer, cancer cells, tumors, angiogenesis, and precancerous conditions such as dysplasia.

[0093] Positive therapeutic effects in cancer can be measured in a number of ways (See, W. A. Weber, J. Nucl. Med. 50: 1 S-10S (2009)). For example, with respect to tumor growth inhibition, according to NCI standards, a T/C i=42% is the minimum level of anti-tumor activity. A T/C < 10% is considered a high anti-tumor activity level, with T/C (%) = Median tumor volume of the treated/Median tumor volume of the control x 100. In some embodiments, the treatment achieved by administration of a composition of the invention is any of progression free survival (PFS), disease free survival (DFS) or overall survival (OS). PFS, also referred to as “Time to Tumor Progression” indicates the length of time during and after treatment that the cancer does not grow, and includes the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease. DFS refers to the length of time during and after treatment that the patient remains free of disease. OS refers to a prolongation in life expectancy as compared to naive or untreated individuals or patients. While an embodiment of the compositions, treatment methods, and uses of the invention may not be effective in achieving a positive therapeutic effect in every patient, it should do so in a statistically significant number of subjects as determined by any statistical test known in the art such as the Student’s t-test, the chi ² -test, the U-test according to Mann and Whitney, the Kruskal-Wallis test (H-test), Jonckheere-Terpstra-test and the Wilcoxon-test.

[0094] As used herein, the term “patient” (alternatively referred to as “subject” or “individual” herein) refers to a mammal (e.g., rat, mouse, dog, cat, rabbit) capable of being treated with the compositions or compositions of the invention, most preferably a human. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a pediatric patient. Those “in need of treatment” include those patients that may benefit from treatment with the compositions or compositions of the invention, e.g. a patient suffering from cancer or an immune condition.

[0095] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, humanized, fully human antibodies, and chimeric antibodies.

[0096] In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "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 primarily responsible for antigen recognition. The variable regions of each light/heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

[0097] Typically, the variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), which are located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C- terminal, both light and heavy chains variable domains comprise FR1, CDR1, FR2 , CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al. ; National Institutes of Health, Bethesda, Md. ; 5 ^th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) A<7v. 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.

[0098] As used herein, the term "pharmaceutically effective amount" or “effective amount” refers to an amount whereby sufficient therapeutic composition or composition is introduced to a patient to treat a diseased or condition. One skilled in the art recognizes that this level may vary according the patient’s characteristics such as age, weight, etc.

[0099] The term "about", when modifying the quantity (e.g., mM, or M) of a substance or composition, the percentage (v/v or w/v) of a composition component, the pH of a solution/composition, or the value of a parameter characterizing a step in a method, or the like refers to variation in the numerical quantity that can occur, for example, through typical measuring, handling and sampling procedures involved in the preparation, characterization and/or use of the substance or composition; through instrumental error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make or use the compositions or carry out the procedures; and the like. In certain embodiments, "about" can mean a variation of ± 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% of the value.

[0100] The terms “cancer”, “cancerous”, or “malignant”, as used herein, refer to or describe the 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 particular examples of such cancers include squamous cell carcinoma, myeloma, small-cell lung cancer, non-small cell lung cancer, glioma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, gastrointestinal (tract) cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer. [0101] As used herein, the terms “PD-1 binding fragment,” “antigen binding fragment thereof,” “binding fragment thereof’ encompass a fragment or a derivative of an antibody that still substantially retains its 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, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments. Typically, a binding fragment or derivative retains at least 10% of its PD-1 inhibitory activity. In some embodiments, a 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 will be useful. In some embodiments, an antigen binding fragment binds to its antigen with an affinity that is at least two fold greater, preferably at least ten times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with unrelated antigens. In one embodiment the antibody has an affinity that is greater than about 10 ⁹ liters/mol, as determined, e.g., by Scatchard analysis. Munsen et al. (1980) Analyt. Biochem. 107:220-239. It is also intended that a PD-1 binding fragment can include variants having conservative amino acid substitutions that do not substantially alter its biological activity.

[0102] "Humanized antibody," as used herein, refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), ty pically that of a human immunoglobulin. The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.

[0103] The antibodies of the invention also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Pat. No. 5,624,821; W02003/086310; W02005/120571, W02006/0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Such modification can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, and a longer half-life would result in less frequent dosing, with the concomitant increased convenience and decreased use of material. See Presta (2005) J. Allergy Clin. Immunol. l l6 13 at 734-35.

[0104] “Conservatively modified variants” or “conservative substitution,” as used herein, refers to substitutions of amino acids that are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule, even in essential regions of the polypeptide. Such exemplary substitutions are preferably made in accordance with those set forth in Table 1 as follows:

Table 1. Exemplary Conservative Amino Acid Substitutions

[0105] In addition, those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. See, e.g. , Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Edition).

[0106] The phrase "consists essentially of," or variations such as "consist essentially of or "consisting essentially of," as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified dosage regimen, method, or composition. As a nonlimiting example, a binding compound that consists essentially of a recited amino acid sequence may also include one or more ammo acids, including substitutions of one or more ammo acid residues, that do not materially affect the properties of the binding compound.

[0107] “Comprising” or variations such as “comprise”, “comprises” or “comprised of’ are used throughout the specification and claims in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features that may materially enhance the operation or utility of any of the embodiments of the invention, unless the context requires otherwise due to express language or necessary implication.

[0108] "Monoclonal antibody" or “mAb” or “Mab”, as used herein, refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their vanable domains, particularly their CDRs, which are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may 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, for example. See also Presta (2005) J.

Allergy Clin. Immunol. 116:731.

[0109] "Tumor" as it applies to a subject diagnosed with, or suspected of having, a cancer refers to a malignant or potentially malignant neoplasm or tissue mass of any size, and includes primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

[0110] The term "tumor size" refers to the total size of the tumor which can be measured as the length and width of a tumor. Tumor size may be determined by a variety of methods known in the art, such as, e.g. by measuring the dimensions of tumor(s) upon removal from the subject, e.g., using calipers, or while in the body using imaging techniques, e.g., bone scan, ultrasound, CT or MRI scans.

[0111] “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 is interpreted as positive for PD-L1.

[0112] “Mononuclear inflammatory density score (MIDS)” refers to the ratio of the number of PD-L1 expressing mononuclear inflammatory cells (MIC) infiltrating or adjacent to the tumor (small and large lymphocytes, monocytes, and macrophages within the tumor nests and the adjacent supporting stroma) compared to the total number of tumor cells. The MIDS is recorded at a scale from 0 to 4 with 0=none; l=present, but less than one MIC for every 100 tumor cells (<1%); 2=at least one MIC for every 100 tumor cells, but less than one MIC per 10 tumor cells (1-9%); 3=at least one MIC for every 10 tumor cells, but fewer MIC's than tumor cells (10-99%); 4=at least as many MIC's as tumor cells (>100%).

[0113] “Combined positive score (CPS)” refers to the ratio of the number of PD-L1 positive tumor cells and PD-L1 positive mononuclear inflammatory cells (MIC) within the tumor nests and the adjacent supporting stroma (numerator) compared 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 at any intensity is considered positive, i.e., weak (1+), moderate (2+), or strong (3+). [0114] “PD-L1 expression positive” refers to a Tumor Proportion Score, Mononuclear Inflammatory Density Score or Combined Positive Score of at least 1%; AIS is > 5; or elevated level of PD-L1 expression (protein and/or mRNA) by malignant cells and/or by infiltrating immune cells within a tumor compared to an appropriate control.

[0115] “Microsatellite instability (MSI)” refers to the 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, MSI analysis can be carried out using the five National Cancer Institute (NCI) recommended microsatellite markers: 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). Additional markers for example, BAT40, BAT34C4, TGF-|3-RII and ACTC can be used. Commercially available kits for MSI analysis include, for example, the Promega MSI multiplex PCR assay, FoundationOne® CDx (FICDx) next generation sequencing based in vitro diagnostic device using DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens.

[0116] "High frequency microsatellite instability” or “microsatellite instability -high (MSI-H)" refers to if two or more of the five NCI markers indicated above show instability or >30-40% of the total markers demonstrate instability (i.e. have insertion/deletion mutations).

[0117] "Non-MSI-H cancer" as used herein refers to irucrosatelhte stable (MSS) and low frequency MSI (MSI-L) cancer.

[0118] "Microsatellite Stable (MSS)" refers to if none of the five NCI markers indicated above show instability (i.e. have insertion/deletion mutations).

[0119] "Proficient mismatch repair (pMMR) cancer" refers to normal expression of MMR proteins (MLH1, PMS2, MSH2, and MSH6) in tumor specimen by IHC. Commercially available kits for MMR analysis include the Ventana MMR IHC assay.

[0120] "Mismatch repair deficient (dMMR) cancer" refers to low expression of one or more MMR protein(s) (MLH1, PMS2, MSH2, and MSH6) in a tumor specimen by IHC.

[0121] “Variable regions” or “V region” as used herein means the segment of IgG chains which is variable in sequence between different antibodies. It extends to Kabat residue 109 in the light chain and 113 in the heavy chain. [0122] “Pembrolizumab” (formerly known as MK-3475, SCH 900475 and lambrolizumab) alternatively referred to herein as “pembro,” is a humanized IgG4 mAb with the structure described in WHO Drug Information, Vol. 27, No. 2, pages 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 described in the Prescribing Information for KEYTRUDA™ (Merck & Co., Inc., Whitehouse Station, NJ USA; initial U.S. approval 2014).

[0123] As used herein, a “pembrolizumab variant” means a monoclonal antibody that comprises heavy chain and light chain sequences that are substantially identical to those in pembrolizumab, except for having three, two or one conservative amino acid substitutions at positions that are located outside of the light chain CDRs and six, five, four, three, two or one conserv ative amino acid substitutions that are located outside of the heavy chain CDRs, e.g, the variant positions are located in the FR regions or the constant region, and optionally has a deletion of the C-terminal lysine residue of the heavy chain. In other words, pembrolizumab and a pembrolizumab variant comprise identical CDR sequences, but differ from each other due to having a conservative amino acid substitution at no more than three or six other positions in their full length light and heavy chain sequences, respectively. A pembrolizumab variant is substantially the same as pembrolizumab with respect to the following properties: binding affinity to PD-1 and ability to block the binding of each of PD-L1 and PD-L2 to PD-1.

[0124] The term "buffer" encompasses those agents which maintain the solution pH of the compositions of the invention in an acceptable range.

[0125] The term "pharmaceutical composition", “formulation” or “pharmaceutical formulation” refers to preparations with pharmaceutically acceptable excipients which are in such form as to permit the active ingredients to be effective, and which contains no additional components which are toxic to the subjects to which the composition would be administered.

[0126] 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. An excipient may be included in pre-lyophilized formulations to enhance stability of the lyophilized product upon storage.

[0127] "Pharmaceutically acceptable" refers to excipients (vehicles, additives) and compositions that can reasonably be administered to a subject to provide an effective dose of the active ingredient employed and that are "generally regarded as safe" e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and the like, when administered to a human. In another embodiment, this term refers to molecular entities and compositions approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or another generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0128] A "reconstituted" formulation is one that has been 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.

[0129] "Reconstitution time" is the time that is required to rehydrate a lyophilized formulation with a solution to a particle-free clarified solution.

[0130] A "stable" formulation is one in which the protein therein essentially retains its physical stability and/or chemical stability and/or biological activity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected time period. For example, in one embodiment, a stable formulation is a formulation with no significant changes observed at a refrigerated temperature (2-8° C) for at least 12 months. In another embodiment, a stable formulation is a formulation with no significant changes observed at a refrigerated temperature (2-8° C) for at least 18 months. In another embodiment, stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 3 months. In another embodiment, stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 6 months. In another embodiment, stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 12 months. In another embodiment, stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 18 months. The criteria for stability for an antibody formulation are as follows. Typically, no more than 10%, preferably 5%, of antibody monomer is degraded as measured by SEC-HPLC. Typically, the formulation is colorless, or clear to slightly opalescent by visual analysis. Typically, the concentration, pH and osmolality of the formulation have no more than +/-10% change. Potency is typically within 60-140%, preferably 80-120% of the control or reference. Typically, no more than 10%, preferably 5% of clipping of the antibody is observed, i.e., % low molecular weight species as determined, for example, by HP-SEC. Typically, no more than 10%, preferably no more than 5% of aggregation of the antibody is observed, i.e. % high molecular weight species as determined, for example, by HP- SEC.

[0131] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase of aggregation, precipitation and/or denaturation upon visual examination of color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering. The changes of protein conformation can be evaluated by fluorescence spectroscopy, which determines the protein tertiary structure, and by FTIR spectroscopy, which determines the protein secondary structure.

[0132] An antibody "retains its chemical stability" in a pharmaceutical formulation, if it shows no significant chemical alteration. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often alter the protein chemical structure include hydrolysis or clipping (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as by peptide mapping in conjunction with mass spectroscopy or MALDI/TOF/MS), deamidation (evaluated by methods such as ion-exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement), and isomerization (evaluated by measuring the isoaspartic acid content, peptide mapping, etc.).

[0133] 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 at the time the pharmaceutical formulation was prepared. The biological activity of an antibody can be determined, for example, by an antigen binding assay. Formulations of the invention include antibodies and fragments thereof that are biologically active when reconstituted or in liquid form.

[0134] The term "isotonic" means that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmotic pressure from about 270-328 mOsm. Slightly hypotonic pressure is 250-269 and slightly hypertonic pressure is 328-350 mOsm. Osmotic pressure can be measured, for example, using a vapor pressure or ice-freezing type osmometer.

[0135] A “non-reducing dissacharide” is a dissacharide not capable of acting 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 dissachandes include but are not limited to dissacharrides such as sucrose and trehalose.

[0136] “PH 20” refers to the wild-type PH20 hyaluronidase of SEQ ID NO: 16.

[0137] “PH20 variant” as used herein is a variant of PH20 that has amino acid residue substitutions including M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T in SEQ ID NO: 16.

[0138] A “PH20 variant fragment” or “PH20 variant fragment thereof’ “or “fragment of a PH20 variant” is a PH20 variant that has either an N-terminus deletion of amino acid residues 1- 36, 1-37, 1-38, 1-39, 1-40, 1-41, or 1-42 of SEQ ID NO: 16; and/or a C-terminus deletion of ammo acid residues 455-509, 456-509, 457-509, 458-509, 459-509, 460-509, 461-509, 462-509, 463-509, 464-509, 465-509, 466-509, 467-509, 468-509, 469-509, 470-509, 471-509, 472-509, 473-509, 474-509, 475-509, 476-509, 477-509, 478-509, 479-509, 480-509, 481-509, 482-509, 483-509, 484-509, 485-509, 486-509, 487-509, 488-509, 489-509, 490-509, 491-509, 492-509, 493-509, 494-509, 495-509, 496-509, 497-509, 498-509, 499-509, 500-509, 501-509, 502-509, 503-509, 504-509, 505-509, 506-509, 507-509, 508-509, or 509, wherein the numbering is by reference to SEQ ID NO: 16.

[0139] “Unit” or “U” refers to One unit of Hyaluronidase activity: amount of PH20 variant or fragment thereof that causes a change in the optical density at 600 nm at conditions suitable for reaction of hyaluronic acid and the enzyme and calculated according to a calibration curve using an activity standard. An example of the assay is described in Example 4 of US 2022/0089738 as well as Example 6 below. Hyaluronic acid (HA) binds to albumin and the albumin-HA complex develops turbidity. When HA is hydrolyzed by hyaluronidase, turbidity of albumin-HA complex is reduced. As such, this assay measures turbidity to determine hyaluronidase enzy me activity of PH20 variants or fragments thereof. Hyaluronidase activity is based on the following reaction: Hyaluronic acid - > Di- and monosaccharides + smaller hyaluronic acid fragments. One skilled in the art understands that the hyalurodindase activity in Units per mg of hyaluronidase can vary depending on the purity, manufacturing process etc. of the hyaluronidase. In one embodiment, 2000U/ml of PH20 variant fragment 2 is about 0.012 mg/ml, 4000U/ml of PH20 variant fragment 2 is about 0.024 mg/ml, and 5000U/ml of PH20 variant fragment 2 is about 0.030 mg/ml.

Anti-PD-1 Antibodies and Antigen-Binding Fragments Thereof [0140] In some embodiments, an anti -human PD-1 antibody or antigen binding fragment thereof for use in the compositions of the invention comprises a light chain variable region comprising three light chain CDRs of CDRL1, CDRL2 and CDRL3 and a heavy chain variable region comprising three heavy chain CDRs of CDRHl, CDRH2 and CDRH3.

[0141] 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, CDRHl 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.

[0142] Anti-PD-1 binding fragments of the compositions of the invention comprise a light chain variable region and a heavy chain variable region. In one embodiment of the compositions of the invention, the antibody or antigen binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO:4 and a heavy chain vanable region comprising or consisting of SEQ ID NO: 9.

[0143] In another embodiment, the compositions of the invention comprise an antibody or antigen binding fragment that has a VL domain and/or aVn domain with at least 95%, 90%, 85%, 80%, 75% sequence homology to one of the VL domains or VH domains described above, and exhibits specific binding to PD-1. In another embodiment, the antibody or antigen binding fragment of the compositions of the invention comprises VL and VH domains having up to 1, 2, 3, 4, or 5 or more amino acid substitutions, and exhibits specific binding to PD-1.

[0144] In any of the embodiments above, the anti-PD-1 antibody may be a full-length anti-PD-1 antibody that specifically binds 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 IgGi, IgG2, IgGs, and IgGi. Different constant domains may be appended to the VL and VH regions provided herein. For example, if a particular intended use of an antibody (or fragment) of the invention were to call for altered effector functions, a heavy chain constant domain other than IgGi may be used. Although IgGi antibodies provide for long half-life and effector functions, such as complement activation and antibody -dependent cellular cytotoxicity, such activities may not be desirable for all uses of the antibody. In such instances an IgG4 constant domain, for example, may be used. [0145] In embodiments of the invention, the anti-PD-1 antibody comprises a light chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:5 and a heavy chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO: 10. In some compositions of the invention, the anti-PD-1 antibody is pembrolizumab, or pembrolizumab variant.

[0146] Ordinarily, amino acid sequence variants of the anti-PD-1 antibodies and antigen binding fragments of the invention will have an amino acid sequence having at least 75% ammo acid sequence identity with the amino acid sequence of a reference antibody or antigen binding fragment (e.g. heavy chain, light chain, VH, VL, or humanized sequence), more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and 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 the candidate sequence that are identical with the anti-PD-1 residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity . None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence shall be construed as affecting sequence identity or homology.

[0147] Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned. Sequence identity can be determined using a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. The following references relate to BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul, S.F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, T.L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S.F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, J.C., et al., (1993) Comput. Chem. 17: 149-163; Hancock, J.M. et al., (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., et al., "A model of evolutionary change in proteins." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M.O. Dayhoff (ed ), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, R.M., et al., "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3." M.O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, S.F., (1991) J. Mol. Biol. 219:555-565; States, D.J., et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919; Altschul, S.F., et al., (1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S„ et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S.F.

"Evaluating the statistical significance of multiple distinct local alignments." in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York.

|0148J Likewise, either class of light chain can be used in the compositions and methods herein. Specifically, kappa, lambda, or variants thereof are useful in the present compositions and methods.

Table 2. Exemplary PD-1 Antibody Sequences

PH20 Variants and Fragments thereof

[0149] In one embodiment, the PH20 variant or fragment thereof further comprises an amino acid residue substitution at one or more positions selected from the group consisting of T341, L342, S343, 1344, and N363. In one embodiment, the PH20 variant or fragment thereof further comprises one or more amino acid residue substitutions selected from the group consisting of T341A, T341C, T341D, T341G, T341S, L342W, S343E, I344N and N363G.

[0150] In one embodiment of the PH20 variant or fragment thereof, the amino acid residue substitutions are selected from the following amino acid residue substitution groups:

(a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;

(b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;

(c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T and (d) T341G, L342W, S343E, 1344N, M345T, S347T, M348K, K349E, E352Q, E353A, E3541, D355K, N356E, E359D and I361T;

(e) T341A, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and 1361T;

(f) T341C, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;

(g) T341D, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T;

(h) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; and

(i) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T.

[0151] In one embodiment of the PEI20 variant or fragment thereof, the amino acid residue substitutions consists of T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T.

[0152] In one aspect of the foregoing embodiments of a PH20 variant fragment, the PH20 variant fragment has an N-terminus deletion of amino acid residues 1-36, 1-37, 1-38, 1-39, 1-40, 1-41, or 1-42 of SEQ ID NO: 16. In another embodiment, the PH20 variant fragment has an N- terminus deletion of amino acid residues 1-36 of SEQ ID NO: 16. In another embodiment, the PH20 variant fragment has an N-terminus deletion of amino acid residues 1-37 of SEQ ID NO: 16. In another embodiment, the PH20 variant fragment has an N-terminus deletion of amino acid residues 1-38 of SEQ ID NO: 16.

[0153] In another aspect of the foregoing embodiments of a PH20 variant fragment, the PH20 variant fragment has a C-terminus deletion of amino acid residue(s) 455-509, 456-509, 457-509, 458-509, 459-509, 460-509, 461-509, 462-509, 463-509, 464-509, 465-509, 466-509, 467-509, 468-509, 469-509, 470-509, 471-509, 472-509, 473-509, 474-509, 475-509, 476-509, 477-509, 478-509, 479-509, 480-509, 481-509, 482-509, 483-509, 484-509, 485-509, 486-509, 487-509, 488-509, 489-509, 490-509, 491-509, 492-509, 493-509, 494-509, 495-509, 496-509, 497-509, 498-509, 499-509, 500-509, 501-509, 502-509, 503-509, 504-509, 505-509, 506-509, 507-509, 508-509, or 509, wherein the numbering is in reference to SEQ ID NO: 16. In one embodiment, the PH20 variant fragment thereof has a C-terminus deletion of amino acid residues 455-509, 458-509, 461-509, 464-509, 465-509, 466-509, 467-509, 468-509, 470-509, 471-509, 472-509, 473-509, 474-509, 475-509, 476-509, 478-509, 480-509, 482-509, 484-509, 486-509, 488-509, or 490-509, wherein the numbering is in reference to SEQ ID NO: 16. In one embodiment, the PH20 variant fragment has a C-terminus deletion of amino acid residues 468- 509, wherein the numbering is in reference to SEQ ID NO: 16.

[0154] In one embodiment, the PH20 variant fragment consists of the amino acid sequence set forth in SEQ ID NO: 17 or 18. In other embodiments, the PH20 variant or fragment thereof is any of the sequences disclosed in Table 11 of EP3636752.

Table 3: Hyaluronidase and exemplary variants

Composition comprising anti-PD-1 antibodies or antigen-binding fragments and PH20 variant or fragment

[0155] The invention provides a composition compnsing anti-human PD-1 antibodies or antigen-binding fragments thereof with less than about 3.0% oxidation of Methionine 105, wherein the anti -human PD-1 antibodies or antigen binding fragments thereof comprise a 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 a heavy chain variable region comprising three heavy chain CDRs of CDRH1 of SEQ ID NO:6, CDRH2 of SEQ ID NO:7 and CDRH3 of SEQ ID NO: 8; and a PH20 variant or fragment. In one embodiment, the anti -human PD-1 antibodies or antigen binding fragments thereof comprise a light chain variable region which comprises the amino acid sequence set forth in SEQ ID NON, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9. In another embodiment, a portion of the anti-human PD-1 antibodies comprise 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 antibodies consist of two light chains and two heavy chains, wherein the two light chains consist of the amino acid sequence set forth in SEQ ID NO: 5, wherein 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 antibodies consist of two light chains and two heavy chains, wherein a portion of the anti -human PD-1 antibodies, 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.

[0156] 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%.

[0157] In one embodiment, the % Methionine oxidation is measured by reduced peptide mapping followed by liquid chromatography and mass spectroscopy. In one embodiment, the reduced peptide mapping comprises the steps of denaturing and reducing 100 pg anti-PD-1 antibody in 100 pL 6 M Guanidine-HCl, 50 pM Tris-HCl, 50 pM EDTA and 200 pM DTT; incubating the mixed sample in a thermomixer at 37°C with 300 rpm shaking for 30 min; alkylating with 5 pL of IM iodoacetamide protected from light at 25 °C for 30 min; blocking the unreacted iodoacetamide with 5 pL of 200 pM DTT; adding Lysyl endopeptidase (Lys-C) (Wako, 125-05061) enzyme (1 : 10 (wt: wt)) in 500 pL; incubating the digestion in a thermomixer at 37°C for 60 min; quenching the digestion with 15 pL of 20% TFA.

[0158] In one embodiment, the liquid chromatography and mass spectroscopy comprises the steps of: injecting 20 pL of digestion sample on the Waters Acquity Liquid Chromatography column (UPLC HSS T3 100 , 1.8 pm, 2.1 mm X 150 mm, P/N: 186003540) at 5°C with Mobile phase A 0.02% TFA in water and mobile phase B 0.02% TFA in acetonitrile with a gradient of 0.1% B from 0 to 5min, 0. l%-10% B from 5 to 7 min, followed by a linear increase to 35% B over the next 38 min, collecting MSI data with Q Exactive Orbitrap MS (Thermo Fisher Scientific).

[0159] In another embodiment, the % Methionine oxidation is measured by hydrophobic interaction chromatography (HIC). In one embodiment, the HIC method is performed by an HPLC with 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 0-2 minutes, 0-100% mobile phase A from 2-52 minutes. The % Met 105 oxidation is determined by the percentage of pre peaks 1+2+3 (containing Metl05 oxidation) before the main peak (majority of species) compared to the total peaks.

[0160] In a further aspect, the invention provides compositions comprising an anti -human PD-1 antibody main species comprising an antibody consisting of two heavy chains and two light chains, each light chain comprises a 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 each heavy chain comprises a heavy chain variable region comprising three heavy chain CDRs of 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 main species, wherein the amount of main species is 65-95%; and a PH20 variant or fragment. In one embodiment, the anti-human PD-1 antibody main species comprises a light chain variable region which comprises the amino acid sequence set forth in SEQ ID NON, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9.

[0161] In a further aspect, the invention provides a composition comprising an antihuman PD-1 antibody main species comprising an antibody consisting of two heavy chains and two light chains, each light chain comprises a 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 NON and each heavy chain comprises a heavy chain variable region comprising three heavy chain CDRs of CDRH1 of SEQ ID NO:6, CDRH2 of SEQ ID NO:7 and CDRH3 of SEQ ID NO: 8, and acidic species of the anti-human PD-1 antibody main species, wherein the amount of acidic species is 1.0-12.0%; a PH20 variant or fragment thereof. In one embodiment, the antihuman PD-1 antibody main species comprises a light chain variable region which comprises the amino acid sequence set forth in SEQ ID NON, and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9.

[0162] In a further aspect, the invention provides a composition comprising an anti-PD-1 antibody main species comprising 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 acidic species of the anti-PD-1 antibody main species, wherein the amount of acidic species is about 1.0-12.0%; and a PH20 variant or fragment. In another aspect, the invention provides a composition comprising an anti-PD-1 antibody main species comprising 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 ammo acid sequence of SEQ ID NO: 5, and acidic and basic species of the anti-PD-1 antibody main species, wherein the amount of main species is about 65- 95%; and a PH20 variant or fragment. In a further aspect, the invention provides a composition comprising an anti-PD-1 antibody main species produced from a Chinese Hamster Ovary cell that 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 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 acidic species of the anti-PD-1 antibody main species thereof, wherein the amount of acidic species is 1.0-12.0%; and a PH20 variant or fragment. In one embodiment, the amount of main species is about 65-95%. In one embodiment, the amount of main species is about 65-85%. In one embodiment, the amount of main species is about 65-80%. In another embodiment, the amount of main species is at about 70-80%. In another embodiment, the amount of main species is at about 70-85%. In a further embodiment, the amount of main species is at about 70-75%. In a further embodiment, the amount of main species is at least about 65%.

[0163] 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 acidicl species is about 1-4%. In another embodiment, the amount of acidicl species is about 2- 4%. In another embodiment, the amount of acidicl species is about 2-3%. In a further embodiment, the amount of acidic variants species is about 1-5%. In a further embodiment, the amount of acidic variants species is about 2-5%. In a further embodiment, the amount of acidic variants species is about 2-4%. In yet another embodiment, the composition further comprises basic species of about 12-27%. In yet another embodiment, the composition further comprises basic species of about 15-20%. In yet a further embodiment, the amount of basic 1 species is about 4-12%. In yet a further embodiment, the amount of basic 1 species is about 8-12%.

[0164] 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 acidicl species is 1 - 4%. In another embodiment, the amount of acidicl species is 2-4%. In another embodiment, the amount of acidicl species is 2-3%. In a further embodiment, the amount of acidic variants species is 1-5%. In a further embodiment, the amount of acidic variants species is 2-5%. In a further embodiment, the amount of acidic variants species is 2-4%. In yet another embodiment, the composition further comprises basic species of 12-27%. In yet another embodiment, the composition further comprises basic species of 15-20%. In yet a further embodiment, the amount of basic 1 species is 4-12%. In yet a further embodiment, the amount of basic 1 species is 8- 12%.

[0165] In one aspect of measuring the main species, acidic species or basic species, a cation exchange chromatography is used. In one embodiment, the cation exchange column is ProPac WCX-10, Sepax Proteomix WCX-NP1.7, Thermo MAbPac SCX-10G or Thermo MAbPac SCX50G. One skilled in the art would understand to use any industry equivalent to the foregoing columns. In another embodiment, a weak cation exchange column with a carboxylate functional group is used. In a further embodiment, the weak cation exchange column with a carboxylate functional group has a particle size 10 urn, diameter 4 mm, length 250 mm. In one embodiment, the Thermo Scientific ProPac WCX-10 column is used for the cation ion exchange method. In another embodiment, a Thermo Scientific ProPac WCX-10 column is used at 35 °C, with a Mobile Phase (A) 24 rnM 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 the chromatogram is generated using detection at 280 nm. In one embodiment, a non-linear gradient is used with: 22%-22%B for 0-0.6 mm; 22%-29%B for 0.6-15.0 mm; 29%-70%B for 15.0-30.0 min; 70%-100%B for 30.0-30.5 min; and 100%-100%B from 30.5-33.0 min. In a further embodiment, the cation ion exchange method is described in Example 5. In another aspect of measuring the main species, acidic species or basic species, an anion exchange chromatography is used.

[0166] In one embodiment, the antibodies were produced from mammalian cells.

[0167] In another embodiment, the antibodies were produced from CHO cells.

[0168] In another aspect of the above embodiments, the composition comprises about 5- 200 mg/ml of the antibodies. In one embodiment, the composition comprises about 25-165 mg/ml of the antibodies. In one embodiment, the composition comprises about 25 mg/ml of the antibodies. In one embodiment, the composition comprises about 120 mg/ml of the antibodies. In one embodiment, the composition comprises about 130 mg/ml of the antibodies. In one embodiment, the composition comprises about 165 mg/ml of the antibodies. In another embodiment, the composition comprises 200-800 mg of the antibodies. In another embodiment, the composition comprises 200 mg of the antibodies. In another embodiment, the composition comprises 400 mg of the antibodies.

[0169] In another aspect, the invention provides a pharmaceutical composition or formulation as disclosed therein that comprises about 165 mg/mL of the anti -human PD-1 antibodies, 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 a PH20 variant or fragment. In one embodiment, a pharmaceutical composition comprises about 130 mg/rnL of the anti-human PD-1 antibodies, 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 a PH20 variant or fragment. [0170] In some embodiments of the formulations of the invention, the PH20 variant or fragment thereof is present in a concentration of about 0.006 mg/mL. In another embodiment, the concentration of the PH20 variant or fragment thereof is about 0.009 mg/mL . In another embodiment, the concentration of the PH20 variant or fragment thereof is about 0.012 mg/mL . In another embodiment, the concentration of the PH20 variant or fragment thereof is about 0.018 mg/mL . In another embodiment, the concentration of the PH20 variant or fragment thereof is about 0.024 mg/mL . In another embodiment, the concentration of the PH20 variant or fragment thereof is about 0.030 mg/mL . In another embodiment, the concentration of the PH20 variant or fragment thereof is about 0.036 mg/mL . In a further embodiment, the concentration of the PH20 variant or fragment thereof is about 0.006-0.036 mg/mL . In a further embodiment, the concentration of the PH20 variant or fragment thereof is about 0.012-0.030 mg/mL . In a further embodiment, the concentration of the PH20 variant or fragment thereof is about 0.006-0.030 mg/mL . In a further embodiment, the concentration of the PH20 variant or fragment thereof is about 0.006-0.012 mg/mL.

[0171] In some embodiments of the formulations of the invention, the PH20 variant or fragment thereof is present in a concentration of about 1000 U/ml. In another embodiment, the concentration of the PH20 variant or fragment thereof is about 1500 U/ml. In another embodiment, the concentration of the PH20 variant or fragment thereof is about 2000 U/ml. In another embodiment, the concentration of the PH20 variant or fragment thereof is about 3000 U/ml. In another embodiment, the concentration of the PH20 variant or fragment thereof is about 4000 U/ml. In another embodiment, the concentration of the PH20 variant or fragment thereof is about 5000 U/ml. In another embodiment, the concentration of the PH20 variant or fragment thereof is about 6000 U/ml. In a further embodiment, the concentration of the PH20 variant or fragment thereof is about 1000-6000 U/ml. In a further embodiment, the concentration of the PH20 variant or fragment thereof is about 2000-5000 U/ml.

[0172] In some embodiments of the formulations of the invention, the PH20 variant or fragment thereof is present in a concentration of about 0.0009 mg/mL. In another embodiment, the concentration of the PH20 variant or fragment thereof is about 0.0018 mg/mL . In another embodiment, the concentration of the PH20 variant or fragment thereof is about 0.0036 mg/mL . In another embodiment, the concentration of the PH20 variant or fragment thereof is about 0.0045 mg/mL . In a further embodiment, the concentration of the PH20 variant or fragment thereof is about 0.0009-0.030 mg/mL . [0173] In some embodiments of the formulations of the invention, the PH20 variant or fragment thereof is present in a concentration of about 150 U/ml. In another embodiment, the concentration of the PH20 variant or fragment thereof is about 300 U/ml. In another embodiment, the concentration of the PH20 variant or fragment thereof is about 600 U/ml. In another embodiment, the concentration of the PH20 variant or fragment thereof is about 750 U/ml. In a further embodiment, the concentration of the PH20 variant or fragment thereof is about 150-5000 U/ml.

Specific Aspects and Embodiments of the Invention

[0174] In one embodiment, the invention provides a pharmaceutical composition comprising: a) about 25 mg/mL to about 175 mg/mL of the anti-human PD-1 antibodies, or antigen binding fragments thereof; b) about 2000 U/ml of PH20 variant or fragment thereof; c) about 5 mM to about 20 mM histidine buffer; d) about 6% to about 8% w/v sucrose; e) about 0.01 % to about 0.04% w/v polysorbate 80; and

I) about 5 mM to about 20 mM L-methionine, or a pharmaceutically acceptable salt thereof.

[0175] In one embodiment, the invention provides a pharmaceutical composition comprising: a) about 25 mg/mL to about 175 mg/mL of the anti-human PD-1 antibodies, or antigen binding fragments thereof; b) about 2000 U/ml of PH20 variant or fragment thereof; c) about 5 mM to about 20 mM histidine buffer; and d) about 6% to about 8% w/v sucrose.

[0176] In one embodiment, the invention provides a pharmaceutical composition comprising: a) about 25 mg/mL to about 175 mg/mL of the anti-human PD-1 antibodies, or antigen binding fragments thereof; b) about 2000 U/ml of PH20 variant or fragment thereof; c) about 5 mM to about 20 mM histidine buffer; d) about 6% to about 8% w/v sucrose; and e) about 0.01 % to about 0.04% w/v polysorbate 80.

[0177] In one embodiment, the invention provides a pharmaceutical composition comprising: a) about 25 mg/mL to about 175 mg/mL of the anti-human PD-1 antibodies, or antigen binding fragments thereof; b) about 2000 U/ml of PH20 variant or fragment thereof; c) about 5 mM to about 20 mM histidine buffer; d) about 6% to about 8% w/v sucrose; and e) about 5 mM to about 20 mM L-methionine, or a pharmaceutically acceptable salt thereof.

[0178] In one embodiment, the invention provides a pharmaceutical composition comprising: a) about 25 mg/mL to about 175 mg/mL of the anti-human PD-1 antibodies, or antigen binding fragments thereof; b) about 2000 U/ml of PH20 variant or fragment thereof; c) about 10 mM histidine buffer; d) about 10 mM L-methionine, or a pharmaceutically acceptable salt thereof; e) about 7% w/v sucrose; and f) about 0.02 % w/v polysorbate 80.

[0179] In one embodiment, the invention provides a pharmaceutical composition comprising: a) about 25 mg/mL to about 175 mg/mL of the anti-human PD-1 antibodies, or antigen binding fragments thereof; b) about 2000 U/ml of PH20 variant or fragment thereof; c) about 10 mM histidine buffer; and d) about 7% w/v sucrose.

[0180] In one embodiment, the invention provides a pharmaceutical composition comprising: a) about 25 mg/mL to about 175 mg/mL of the anti-human PD-1 antibodies, or antigen binding fragments thereof; b) about 2000 U/ml of PH20 variant or fragment thereof; c) about 10 mM histidine buffer; d) about 7% w/v sucrose; and e) about 0.02% w/v polysorbate 80.

[0181] In one embodiment, the invention provides a pharmaceutical composition comprising: a) about 25 mg/mL to about 175 mg/mL of the anti-human PD-1 antibodies, or antigen binding fragments thereof; b) about 2000 U/ml of PH20 variant or fragment thereof; c) about 10 mM histidine buffer; d) about 7% w/v sucrose; and e) about 10 mM L-methionine, or a pharmaceutically acceptable salt thereof.

[0182] In the foregoing embodiments, in one aspect, the anti-human PD-1 antibodies, or antigen binding fragments thereof is about 25-165 mg/ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibodies, or antigen binding fragments thereof is about 50-1 5 mg/ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibodies, or antigen binding fragments thereof is about 75-165 mg/ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibodies, or antigen binding fragments thereof is about 75-175 mg/ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibodies, or antigen binding fragments thereof is about 100-165 mg/ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibodies, or antigen binding fragments thereof is about 130-165 mg/ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibodies, or antigen binding fragments thereof is about 150-165 mg/ml. In the foregoing embodiments, in one aspect, the anti-human PD-1 antibodies, or antigen binding fragments thereof is about 130 mg/ml. In the foregoing embodiments, in one aspect, the anti -human PD-1 antibodies, or antigen binding fragments thereof is about 165 mg/ml.

[0183] In any of the specific aspects and embodiments described herein, one of the anti- PD-1 antibodies, or antigen binding fragments thereof, described in the section “Composition comprising anti-PD-1 antibodies or antigen-binding fragments and PH20 variant or fragment” or “Anti-PD-1 antibody or antigen-binding fragment” is used. In any of the specific aspects and embodiments described herein, one of the PH20 variant or fragment thereof, e.g. described in the section entitled “PH20 variant or fragment thereof’, is used.

[0184] In some embodiments of the invention, any of the formulations described herein is in aqueous solution. In alternative embodiment, the invention provides lyophilized formulations made by lyophilizing an aqueous formulation to provide a reconstituted formulation of the invention, as discussed more fully, infra.

Formulation Excipients

[0185] In embodiments of the invention, the non-reducing dissacharide is sucrose. In additional embodiments, the non-reducing dissacharide is trehalose.

[0186] In some embodiments, the non-reducing dissacharide is about 6% to about 8% w/v sucrose. In some embodiments, the non-reducing dissacharide is about 6% to about 8% w/v trehalose.

[0187] In still further embodiments, the 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.

[0188] In addition to the anti-PD-1 antibodies or antigen binding fragments thereof and PH20 variant or fragment thereof, and anon-reducing dissacharide in the amounts/concentrations specified above, the formulations of the invention may also comprise a buffer. In some embodiments the buffer is present in an amount of about 5 mM to about 20 mM. In further embodiments, the buffer has a pH in a range of about 5.0 to about 6.0. In still further embodiments, the pH is from about 5.3 to about 5.8. In other embodiments, the pH is from about 6.0 to about 6.4.

[0189] In particular 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. In embodiments where the formulation is lyophilized, it is preferred that the buffer is not acetate because acetate buffer systems are not compatible with the lyophilization process.

[0190] When a range of pH values is recited, such as “a pH between pH 5.5 and 6.0,” the range is intended to be inclusive of the recited values. Unless otherwise indicated, for a lyophilized formulation, the pH refers to the pH after reconstitution of the lyophilized formulations of the invention. The pH is typically measured at 25°C using standard glass bulb pH meter. As used herein, a solution compnsmg “histidine buffer at pH X” refers to a solution at pH X and comprising the histidine buffer, i.e. the pH is intended to refer to the pH of the solution.

[01 1] In addition to the anti-PD-1 antibodies or antigen binding fragments thereof and PH20 variant or fragment thereof, a non-reducing dissacharide, and a buffer in the amounts/concentrations specified above, the formulations of the invention may also comprise an anti-oxidant. In embodiments of the invention, the anti-oxidant is methionine. In embodiments of the invention, the anti-oxidant is L-methionine, or a pharmaceutically acceptable salt thereof. In further embodiments, the methionine is L-methionine. In other embodiments, the antioxidants is L-methionine HC1.

[0192] In some embodiments, the anti-oxidant (e.g. L-methionine) is present in the formulations of the invention in an amount of 1 mM to about 20 mM. In another embodiment, the anti-oxidant is present in an amount of about 5 mM to about 20 mM. In a further embodiment, the anti-oxidant is present in about 5 mM to about 15 mM. In a further embodiment, the anti-oxidant is present in about 5 mM to about 10 mM. In additional embodiments, the anti-oxidant is present in an amount of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM or about 20 mM.

[0193] In addition to the anti-PD-1 antibodies or antigen binding fragments thereof, a PH20 variant or fragment thereof, a non-reducing dissacharide, a buffer, and an anti-oxidant in the amounts/concentrations specified above, the formulations of the invention may also comprise a surfactant. Surfactants that may be useful in the formulations of the invention include, but are not limited to: nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Polysorbates, sold under the trade name Tween® (Uniquema Americas LLC, Wilmington, DE)) including Polysorbate-20 (polyoxyethylene sorbitan monolaurate), Polysorbate-40 (polyoxyethylene sorbitan monopalmitate), Polysorbate-60 (polyoxyethylene sorbitan monostearate), and Polysorbate-80 (polyoxyethylene sorbitan monooleate).

[0194] The amount of surfactant to be included in the formulations of the invention is an amount sufficient to perform the desired function, i.e. a minimal amount necessary to stabilize the active pharmaceutical ingredient (i.e. the anti-PD-1 antibodies or antigen binding fragments thereof or PH20 variant or fragment thereof) in the formulation. Typically, the surfactant is present in a concentration of from 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 an amount from about 0.01% to about 0.04%; from about 0.01% to about 0.03%, from about 0.01% to about 0.02%, from about 0.015% to about 0.04%; from about 0.015% to about 0.03%, from about 0.015% to about 0.02%, from about 0.02% to about 0.04%, from about 0.02% to about 0.035%, or from about 0.02% to about 0.03%. In specific embodiments, the surfactant is present in an amount of about 0.02%. In alternative embodiments, the surfactant is present in an amount of about 0.01%, about 0.015%, about 0.025%, about 0.03%, about 0.035%, or about 0.04%.

[0195] In exemplary embodiments of the invention, the surfactant is a nonionic surfactant selected from the group consisting of: Polysorbate 20, and Polysorbate 80. In preferred embodiments, the surfactant is Polysorbate 80.

[0196] In specific embodiments, the formulations of the invention comprise about 0.01% to about 0.04% PS80. In further embodiments, the formulations of the invention comprise PS80 in an amount of about 0.008%, about 0.01%, about 0.015%, about 0.02%, about 0.025%, about 0.03%, about 0.035%, about 0.04% or about 0.045%. In particular embodiments, the formulations of the invention comprise about 0.02% PS80.

Lyophilized Pharmaceutical Compositions

[0197] Lyophilized formulations of therapeutic proteins provide several advantages. Lyophilized formulations in general offer better chemical stability than solution formulations, and thus increased shelf-life. A lyophilized formulation may also be reconstituted at different concentrations depending on clinical factors, such as route of administration or dosing. For example, a lyophilized formulation may be reconstituted at a high concentration (i.e. in a small volume) if necessary for subcutaneous administration, or at a lower concentration if administered intravenously. High concentrations may also be necessary if high dosing is required for a particular subject, particularly if administered subcutaneously where injection volume must be minimized. One such lyophilized antibody formulation is disclosed at U.S. Pat. No. 6,267,958, which is hereby incorporated by reference in its entirety. Lyophilized formulations of another therapeutic protein are disclosed at U.S. Pat. No. 7,247,707, which is hereby incorporated by reference in its entirety.

[0198] Typically, the lyophilized formulation is prepared in anticipation of reconstitution at high concentration of drug product (DP), i.e. in anticipation of reconstitution in a low volume of water. Subsequent dilution with water or isotonic buffer can then readily be used to dilute the DP to a lower concentration. Typically, excipients are included in a lyophilized formulation of the invention at levels that will result in a roughly isotonic formulation when reconstituted at high DP concentration, e.g. for subcutaneous administration. Reconstitution in a larger volume of water to give a lower DP concentration will necessarily reduce the tonicity of the reconstituted solution, but such reduction may be of little significance in non-subcutaneous, e.g. intravenous, administration. If isotonicity is desired at lower DP concentration, the lyophilized powder may be reconstituted in the standard low volume of water and then further diluted with isotonic diluent, such as 0.9% sodium chloride.

[0199] In an embodiment of the invention, a formulation comprising the anti-PD-1 antibodies (or antigen binding fragments thereof) and PH20 variant or fragment thereof is formulated as a lyophilized powder for reconstituting and utilizing for subcutaneous administration. In certain embodiments, the lyophilized formulation is reconstituted with sterile water for injection prior to use. If desired, the reconstituted solution may 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 invention enables reconstitution of the anti-PD-1 antibody to high concentrations, such as about 50, 60, 75, 100, 125, 130, 150, 165, 175, 185 or 200 mg/mL.

[0200] Lyophilized formulations are by definition essentially dry, and thus the concept of concentration is not useful in describing them. Describing a lyophilized formulation in the terms of the weight of the components in a unit dose vial is more useful, but is problematic because it varies for different doses or vial sizes. In describing the lyophilized formulations of the invention, it is useful to express the amount of a component as the ratio of the weight of the component compared to the weight of the drug substance (DS) in the same sample (e.g. a vial). This ratio may be expressed as a percentage. Such ratios reflect an intrinsic property of the lyophilized formulations of the invention, independent of vial size, dosing, and reconstitution protocol.

[0201] In other embodiments, the formulation of anti-human PD-1 antibodies, or antigen binding fragments and PH20 variant or fragment thereof, is defined in terms of the prelyophilization solution used to make the lyophilized formulation, such as the pre-lyophilization solution. In one embodiment the pre-lyophilization solution comprises antibody, or antigenbinding fragment thereof, at a concentration of about 25-200 mg/mL and 150-8000 U/ml of PH20 variant or fragment thereof. Such pre-lyophilization solutions may be at pH about 5.0-6.0, or about pH 5.5. [0202] In yet other embodiments, the lyophilized formulation of anti-human PD-1 antibodies, or antigen binding fragments and PH20 variant or fragment thereof, is defined in terms of the reconstituted solution generated from the lyophilized formulation. The invention provides a liquid formulation that is reconstituted from a lyophilized formulation. Reconstituted solutions may comprise antibody, or antigen-binding fragment thereof, at concentrations of about 25, 30, 40, 50, 60, 75, 80, 90, 100, 120, 130, 150, 165, 167, 185 or 200 mg/mL, and 0.0009-0.050 mg/ml (150, 300, 600, 900, 1000, 1500, 2000, 3000, 4000 or 5000 U/ml) of PH20 variant or fragment thereof. Such reconstituted solutions may be at about pH 5.5, or range from about pH 5.0 to about 6.0.

[0203] The lyophilized formulations of the invention are formed by lyophilization (freeze-drying) of a pre-lyophilization solution. Freeze-drying is accomplished by freezing the formulation and subsequently subliming water at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic point or the collapse temperature of the formulation. Typically, the shelf temperature for the primary drying will range from about -30 to 25°C (provided the product remains frozen during primary drying) at a suitable pressure, ranging typically from about 50 to 250 mTorr. The formulation, size and type of the container holding the sample (e.g., glass vial) and the volume of liquid will dictate the time required for drying, which can range from a few hours to several days (e g. 40-60 hrs). A secondary drying stage may be carried out at about 0-40°C, depending primarily on the type and size of container and the ty pe of protein employed. The secondary drying time is dictated by the desired residual moisture level in the product and typically takes at least about 5 hours.

Typically, the moisture content of a lyophilized formulation is less than about 5%, and preferably less than about 3%. The pressure may be the same as that employed during the primary drying step. Freeze-drying conditions can be varied depending on the formulation and vial size.

[0204] In some instances, it may be desirable to lyophilize the protein formulation in the container in which reconstitution of the protein is to be carried out in order to avoid a transfer step. The container in this instance may, for example, be a 3, 5, 10, 20, 50 or 100 cc vial.

[0205] Reconstitution generally takes place at a temperature of about 25°C to ensure complete hydration, although other temperatures may be employed as desired. The time required for reconstitution will depend, e.g., on the type of diluent, amount of excipient(s) and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g. phosphate-buffered sahne), sterile saline solution, Ringer's solution or dextrose solution. Liquid Pharmaceutical Compositions

[0206] A liquid antibody formulation can be made by taking the drug substance (e.g., anti -human PD-1 antibody, and/or PH20 variant or fragment thereof) which is in liquid form and buffer exchanging it into the desired buffer. There is no lyophilization step in this embodiment. The drug substance in the final buffer is concentrated to a desired concentration. Excipients such as sucrose, methionine and polysorbate 80 are added to the drug substance and it is diluted using the appropriate buffer to final protein concentration. The final formulated drug substance is filtered, e.g. using 0.22pm filters, and filled into a final container (e.g. glass vials or syringes). Such a liquid formulation is exemplified by a final liquid formulation comprising 10 mM histidine pH 5.5, 7% sucrose, 0.02% polysorbate 80, 25-200 mg/mL anti-PD-1 antibodies of the invention, and 150-8000 U/ml of PH20 variant fragment 1 or 2.

Protein expression

[0207] The methods for manufacturing the antibody involves culturing antibody-secreting mammalian cells. Such cultured mammalian cells are typically made by recombinant DNA technology involving transient or stable transfection, e.g., the pooled plasmid constructs (expression vectors) from the cloning step can be transfected into a plurality of host cells (e.g., mammalian, HEK 293 or CHO, bacterial, insect, yeast cells) for expression using a cationic lipid, polyethylenimine, LipofectamineTM, or ExpiFectamineTM, or electroporation. The skilled practitioner is aware of numerous suitable means for transfecting to achieve expression of recombinant antibodies. Alternatively, methods for stable genomic integration of expressions cassettes encoding the protein of interest can be employed to make a production cell line of protein-secreting mammalian cells. (See, e.g., Zhang, Crispr-Cas Systems and Methods for Altering Expression Of Gene Products, WO2014093661 A2; Frendewey et al., Methods and Compositions for the Targeted Modification of a Genome, US9228208 B2; Church et al., Multiplex Automated Genome Engineering, W02008052101A2, US8153432 B2; Bradley et al., Methods Cells and Organisms, US2015/0079680 Al; Begemann et al., Compositions and Methods for Modifying Genomes, WO2017141173A2; Gill et al., Nucleic acid-guided nucleases, US9982279 Bl; Minshull et al., Enhanced nucleic acid constructs for eukaryotic gene expression, US9428767B2, US9580697B2, US9574209B2; Minshull et al., DNA Vectors, Transposons And Transposases For Eukaryotic Genome Modification, US10041077B2). [0208] In other embodiments, expression cassettes that efficiently integrate into eukaryotic transcriptionally active hot spots and ensure long term stable and consistent expression of the gene of interest (GOI) is described in W02020068631. These expression cassettes can be transfected into various host CHO cell lines, including CHOK1SVTM (Lonza; Slough, U.K.), HD-BIOP1 (Horizon Discovery, U.K.), CHOZN® (Sigma- Aldrich, St. Louis, MO) and GS knock-out CHO host cell lines. In one embodiment, the expression vector comprises:

(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a first insulator, an EASE, a promoter, a TPL, an insertion site for a Gene of Interest (GOI), an IRES, a polynucleotide encoding a eukaryotic selectable marker, a polyA signal, and a second insulator;

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

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

(d) a bacterial plasmid origin of replication.

[0209] In one embodiment, the promotor is a SV40 promoter (Nature 273(5658):113-20 (1978), Proc. Natl. Acad. Sci. USA 81 (l):23-27 (1984), GenBank: J02400.1. In another embodiment, the promotor is hCMV immediate-early enhancer/promoter (GenBank XI 7403.1). In another embodiment, the ITR is piggyBac ITR. In one embodiment, the insulator is a Chicken 0-globin HS4 insulator.

[0210] In one embodiment, the eukaryotic selectable marker is a neomycin phosphotransferase, a histidinol dehydrogenase, a hygromycin B phosphotransferase, a xanthine- guanine phosphoribosyltransferase, a dihydrofolate reductase, a tryptophan synthetase, a puromycin N-acetyl-transferase, a thymidine kinase, an adenine phosphoribosyl transferase, a glutamine synthetase, an adenosine deaminase, or metallothionein-1. In one embodiment, the eukaryotic selectable marker is a neomycin phosphotransferase. In another embodiment, the eukaryotic selectable marker is a glutamine synthetase. In certain embodiments of the various expression vectors provided herein, the bacterial selectable marker is an ampicillin resistance gene, a tetracycline resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a blasticidin resistance gene, or the like. In one embodiment, the bacterial selectable marker is an ampicillin resistance gene.

Upstream Cell Culture Process [0211] Surprisingly, a continuous perfusion upstream process provided pembrolizumab compositions with lower % Metl05 oxidation, lower % acidic species and/or higher % main species compared to pembrolizumab produced by a fed batch process.

[0212] The host cells used to produce the antibody in the invention can be cultured in a variety of cell culture media. Commercially available media such as CD-CHO liquid or CD-CHO AGT™ Powder (Life Technologies), Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; W090103430; WO 87/00195; or U.S. Patent Re. No. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented with other ingredients as necessary at appropriate concentrations that would be known to those skilled in the art. For example, hormones and/or other grow th factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, sodium bicarbonate, calcium, iron, potassium, zinc, copper sulfate, ferric citrate, manganese, magnesium, and phosphate), nucleotides (such as adenosine, adenine, thymidine, cytidine, guanosine, uridine, purine), any of the 20 amino acids (tyrosine, cysteine, cystine, glutamic acid), vitamin or supplements (choline, inositol, thiamine, folic acid, biotin, calcium, niacinamide, p-aminobenzoic acid, pyridoxine, riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, linoleic acid, selenite, glycine, putrescine, ethanolamine), selection agents that confer resistance or survival to selectable markers such as antibiotics (such as geneticin, neomycin, hygromycin B, puromycin, zeocin, Gentamycin™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose, galactose or an equivalent energy source, such that the physiological conditions of the cell in, or on, the medium promote expression of the protein of interest by the host cell. Typically, cell culture medium for perfusion is more concentrated to allow for sustained growth of cells at higher density. In one embodiment, nonionic surfactants such as Kolliphor® P188 at 2-10 g/L can be added to prevent cell death during the perfusion process and maintain high cell density and viability. See Xu et al., Bioprocess Biosyst Eng (2017) 40: 1317-1326. In another embodiment, the copper concentration in the perfusion cell culture medium is 10-35 ppb. Depending on the cell density and cell uptake of copper, the copper concentration in the perfusion bioreactor can range from 1 to 35 ppb. The culture medium is preferably serum-free. In some embodiments, the aqueous medium is liquid, such that the host cells are cultured in a cell suspension within the liquid medium. [0213] The culture conditions, such as temperature (for mammalian cells, typically, about 37° ± 1°C), pH (typically, but not necessarily, the cell culture medium is maintained within the range of about pH 6.5-7.5), oxygenation, and the like, will be apparent to the ordinarily skilled artisan. In one embodiment, the dissolved oxygen level is about 15-100%, and the pH is about 6.7-7.3. Clearly, there will be small variations of the temperature, pH, or other culture condition over time, and from location to location through the culture vessel (i.e., the bioreactor) such that there is an operating range for these parameters. (See, also, e.g., Oguchi et al., pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture, Cytotechnology. 52(3): 199-207 (2006); Al-Fageeh et al., The cold-shock response in cultured mammalian cells: Harnessing the response for the improvement of recombinant protein production, Biotechnol. Bioeng. 93:829-835 (2006); Marchant, R.J. et al., Metabolic rates, growth phase, and mRNA levels influence cell-specific antibody production levels from in vitro cultured mammalian cells at sub-physiological temperatures, Mol. Biotechnol. 39:69-77 (2008)). [0214] Upon culturing the transfected or transformed host cells, the antibody is directly secreted into the cell culture medium (by employing appropriate secretory-directing signal peptides) and are harvested therefrom.

[0215] The perfusion bioreactor can be fluidly connected to an affinity chromatography step in an uninterrupted flow coming from the bioreactor (directly or indirectly via intervening unit operations, for example a surge vessel) to the affinity chromatography system. Some embodiments of the invention include a first single-use surge vessel (SUSV1) adapted to receive volumes of cell-free permeate removed from the perfusion bioreactor(s). These permeate volumes can be automatically and fluidly fed from the one or more perfusion bioreactor(s) into the SUSV1.

[0216] The anti-PD-1 antibodies or antigen-binding fragments of the invention are produced or obtainable by steps comprising: a) perfusing mammalian host cells in cell culture medium in a perfusion bioreactor by applying a perfusion rate of at least about 0.25-6.0 vessel volume per day (vvd), wherein the host cell comprises a polynucleotide encoding the light chain variable region and a polynucleotide encoding the heavy chain variable region, or a polynucleotide encoding the light chain variable region and the heavy chain variable region of the antibody or antigen binding fragment of the anti -human PD-1 antibody; b) continuously harvesting the antibody from the cell culture broth to obtain a harvest cell culture fluid; c) optionally, transferring the harvest cell culture fluid (HCCF) to a surge vessel for a residence time of about 0.5 to 8 hours; and d) continuously purifying the harvest cell culture fluid with an affinity chromatography step to obtain a purified composition.

[0217] In one embodiment, the above method comprises the following steps before step a):

(i) inoculating a perfusion bioreactor with mammalian host cells in a cell culture medium at a cell density of about 0.2-0.6 xlO ⁶ cells/ml, ii) growing the mammalian cells in a cell culture medium to a cell density of about 1.0 to 6.0 xlO ⁶ cells/ml in a perfusion bioreactor.

[0218] In one embodiment, in step i) the cell density is about 0.20 to 0.6 xlO ⁶ cells/ml on Day 1. In one embodiment, in step i) the cell density is about 0.25 to 0.5 xlO ⁶ cells/ml on Day 1. In another embodiment, in step i) the cell density is about 0.4 to 0.5 xlO ⁶ cells/ml on Day 1. In another embodiment, in step a) the perfusion is initiated on Day

3. In another embodiment, in step a) the perfusion is inflated at a cell density of about 2 to 10 xlO ⁶ cells/ml. In another embodiment, in step a) the perfusion is inflated at a cell density of about 4 to 8 xlO ⁶ cells/ml. In another embodiment, step (ii) is growing the mammalian host cells in a cell culture medium to a cell density of about 2.0 to 6.0 xlO ⁶ cells/ml in a perfusion bioreactor.

[0219] The perfusion rate may initiate at about 0.5 vvd, and can be continuously, gradually or incrementally increased from about 0.5 vvd to 6 vvd. In one embodiment, in step a) the perfusion rate is at about 0.5 vvd to 4 vvd. In one embodiment, in step a) the perfusion rate is at about 0.5 vvd to 2 vvd. Preferably, the perfusion rate is kept constant after reaching the target. Alternatively, the perfusion rate can be adjusted throughout the continuous manufacturing process depending on the continuously measured viable cell density. In one embodiment, the perfusion rate is about 0.5 vvd on Day 3, about 1 vvd on Day 4 and about 2 vvd on Day 5. In a further embodiment, step d) is performed on or after Day 5. In a further embodiment, cell bleeding is performed to maintain a specific cell density, for example about 80 to 100 xlO ⁶ cells/ml, or capacitance value (about 70-90 or 80 pF/cm). In one embodiment, capacitance value is about 70-90 pF/cm during perfusion. In one embodiment, the permeate titer is 0.2-2 g/L. In one embodiment, the maximum cell density is at about 80-100 xlO ⁶ cells/ml during perfusion. In one embodiment, the maximum cell density is at about 80-150 xlO ⁶ cells/ml during perfusion. In one embodiment, the maximum cell density is at about 100 xlO ⁶ cells/ml during perfusion. In one embodiment, the mammalian host cell is CHO cell. In one embodiment, the host cell comprises a polynucleotide that encodes a light chain and a polynucleotide that encodes a heavy chain, or a polynucleotide that encodes a light chain and a heavy chain, wherein the light chain consists of the amino acid sequence set forth in SEQ ID NO: 5, and the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 10. In another embodiment, the perfusion cell culture medium has a copper concentration of 10-35 ppb. In a further embodiment, the copper concentration in the perfusion bioreactor ranges from 1 to 35 ppb.

[0220] In another aspect, continuous harvesting is performed by setting a constant permeate rate to obtain a 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 permeate rate and cell bleed rate. A typical perfusion system with feed stream, perfusion stream and cell bleeding stream is described in Goudar, C.T. & Chen, C. & Le, H.. (2015). SBE special section: Biopharmaceuticals - Continuous processing in upstream operations, pi ll.

Downstream process

[0221] The antibody or antigen-binding fragment compositions from the upstream process may further undergo continuous or semi-continuous downstream purification, or a downstream batch purification process.

[0222] Affinity chromatography separates molecules based on a highly specific interaction between the molecule of interest and the functional group of the resin, such as interaction between antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid, etc. Some commonly used affinity chromatographic resins include protein A or protein G resin to purify antibodies, avidin biotin resin to purify' biotin/avidin and their derivatives, glutathione resin to purify GST-tagged recombinant proteins, heparin resin to separate plasma coagulation proteins, IMAC resin to purify proteins that specifically interact with the metal ions, etc. Operating conditions of each affinity chromatography depend on the mechanism of the interaction and factors that affect the interaction. Commercial affinity chromatographic resins include but are not limited to MabSelect Sure, UNOsphere SUPrATM, Affi-Gel®, and Affi-Prep®. In one embodiment, the affinity chromatography step is protein A chromatography performed in bind and elute mode.

[0223] In one embodiment, the harvest cell culture fluid is purified with a Protein A affinity chromatography comprising the steps of: a) binding the HCCF to a stationary phase; b) eluting the antibodies or antigen binding fragments from the Protein A stationary phase with an elution solution; c) optionally, cleaning and sanitizing the stationary phase for repeated cycling.

[0224] In one embodiment, prior to step (a), equilibrating the stationary phase with an equilibration solution is performed. In one embodiment, one or more impurities are in the flow- through of step a).

[0225] In another aspect of the method, after step a) but prior to step b), the method further comprises the step of washing the stationary phase with one or more wash solutions. In one embodiment, one or more impurities are removed from the wash step. In one embodiment, the wash solution or elution solution comprises a salt, preferably a monovalent metal ion salt, such as NaCl or KC1. In one embodiment, the wash solution comprises about 400-600 mM NaCl or KC1. In another embodiment, the wash solution comprises about 500 mM NaCl or KC1. In another embodiment, the wash solution comprises about 400-500 mM NaCl or KC1. In a further embodiment, a first, second and third wash solution comprises about 5-20 mM Sodium Phosphate and the second wash solution further comprises about 400-600 mM NaCl or KC1. In a further embodiment, a first, second and third wash solution comprises about 10 mM Sodium Phosphate and the second wash solution further comprises about 500 mM NaCl or KC1.

[0226] In one embodiment, the pH of the wash or elution solution is about 6-7. In one embodiment, the pH of the wash or elution solution is about 6.5. In another embodiment, the elution solution comprises about 5-50 mM Sodium Acetate. In another embodiment, the elution solution comprises about 5-30 mM Sodium Acetate. In another embodiment, the elution solution comprises about 20 mM Sodium Acetate.

[0227] In another aspect, the elution solution comprises about 5-50 mM Sodium Acetate. In another embodiment, the elution solution comprises about 5-30 mM Sodium Acetate. In another embodiment, the elution solution comprises about 20 mM 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.

[0228] In one embodiment, the affinity chromatography is operated in a continuous multi-column chromatography (at least two, three or four columns) system allowing for uninterrupted flow of the HCCF to the chromatography skid. [0229] In one embodiment, the perfusion bioreactor or perfusion system comprising the HCCF is fluidly connected indirectly or directly to the affinity chromatography. In one embodiment, the affinity chromatography is fluidly connected to the perfusion bioreactor.

[0230] In another embodiment, the perfusion bioreactor or system comprising the HCCF is fluidly connected through a surge vessel to the affinity chromatography. The surge vessel is sized in such a manner to control the residence time a fluid control volume spends in the surge vessel. In some embodiments of the invention, the mean residence time is 0.5-30 hours. In some embodiments of the invention, the mean residence time is 0.5-20 hours. In some embodiments of the invention, the mean residence time is 0.5-8 hours. In some embodiments, the mean residence time is 2 hrs. In some embodiments, the mean residence time is 1 hr. In some embodiments, the mean residence time is 0.5 hr. The HCCF can be stored at 4-25°C in the surge vessel. In other embodiments, the loading residence time (from exiting the perfusion bioreactor to loading on 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 surge vessel equals the feed rate from the surge vessel to the continuous multi-column chromatography system. Alternatively, the HCCF can be stored in a container at -40 to -80 °C before being fed to the affinity chromatography. In one embodiment, the antibody is protected from light during storage in the surge vessel or container.

[0231] In a further embodiment, the affinity chromatography is followed by one or more steps of a viral inactivation, depth filtration, a second chromatography, a third polishing chromatography, viral filtration, ultrafiltration, diafiltration, single pass-tangential flow filtration and an in-line diafiltration, optionally fluidly connected, or fluidly connected through a surge vessel or connected through a holding vessel.

[0232] In one aspect, the affinity chromatography is fluidly connected in an uninterrupted flow to, a viral inactivation system, and optionally, in an uninterrupted flow to depth filtration, fluidly connected to a second chromatography system, an optional third polishing chromatography system, viral filtration system, and ultrafiltration/diafiltration system, all in an uninterrupted flow to the afore-mentioned upstream processing steps and successively to each other, with optional intervening surge vessels. See Figure 1C of WO2020168315. The final ultrafiltration step may be comprised of a single pass-tangential flow filtration step and an in-line diafiltration step operated either fluidly connected or through an optional surge vessel to maintain continuity from the affinity chromatography step through the in-line diafiltration product. [0233] In another aspect, the affinity chromatography is fluidly connected in an uninterrupted flow to, a viral inactivation system, and optionally in an uninterrupted flow to depth filtration system, a holding vessel (HV1) for temporary storage of virally inactivated product pool; a second chromatography system, an optional third polishing chromatography system, and ultrafiltration/ diafiltration system, successively fluidly connected to each other in an uninterrupted flow or in batch mode, with optional intervening surge vessels or holding vessels (i.e., holding vessels if there are two or more batch steps or operations), as the case may be. See for example Figure ID of WO2020168315. The final ultrafiltration step may be comprised of a single pass-tangential flow filtration step and an in-line diafiltration step operated either directly connected or through an optional surge vessel to maintain continuity from the affinity chromatography step through the in-line diafiltration product.

[0234] In some embodiments of the invention, the affinity chromatography step is followed by a viral inactivation step. For viral inactivation, a collection of affinity chromatography elutions are pooled into one of two viral inactivation pooling vessels. After a discrete number of elutions have been collected in the pooling vessel, the pool volume undergoes an automated pH adjustment from elution pH, to the viral inactivation pH (pH=3.4-3.7) by adding an acid solution, followed by an adjustment to a neutral pH through 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 automated adjustment cycle, new affinity chromatography elutions are collected in the second viral inactivation pooling vessel. Switching between the two viral inactivation pooling vessel allows for the unit operation to operate continuously. In some embodiments of the invention the viral inactivated product (VIP) is transferred into a surge vessel feeding other downstream unit operations continuously.

[0235] In some embodiments of the invention, operations downstream of the viral inactivation system/neutralization system involve continuous processing by transferring the VIP into a surge vessel which can feed a second chromatography step or optionally be filtered by depth filtration to yield a filtered virally inactivated product pool (FVIP). Semi-continuous flow in these unit operations is maintained by cycling either depth filtration consumables or the second chromatography column at regular intervals. In these instances, flow is paused dunng flushing and regeneration of the phases between consumable switch outs and during non-load steps of the chromatography phase.

[0236] In some embodiments of the invention, operations downstream of the viral inactivation system/neutralization system involve continuous processing of the virally inactivated product pool (which can optionally also be filtered by depth filtration to yield a filtered virally inactivated product pool (FVIP)); in such embodiments, the virally inactivated product pool is collected in a collection vessel, and in subsequent batch-wise steps or operations, the purified product pool or virus-free filtrate can optionally be collected in other collection vessels between steps. In such discrete operation, batch-wise, or batch mode, processing, the collection vessel(s) or interchangeably “collection tank(s),” from one step (which in certain embodiments may also be deemed a “feed tank(s)” for the subsequent step) lack the automated controls of a surge vessel, and although the collection vessel (or feed tank) may physically resemble a surge vessel, such a collection vessel (or interchangeably, “collection tank”) or feed tank, is called a “holding vessel” or, interchangeably an “HV” (e.g., HV1, HV2, HV3, HV4, or HV5). A “holding vessel” can be a single-use holding vessel (SUHV), distinct from a single-use collection vessel (SUCV, e.g., SUCV1 or SUCV2) in a continuous or semi-continuous format set of manufacturing process steps or operations. See Figure ID of WO2020168315.

[0237] The affinity chromatography step can be followed by a second and/or third chromatography steps to remove for example, protein aggregates, host cell protein or DNA. IEX chromatography separates molecules based on net charge of the molecules. Separation occurs as a result of competition between the charged molecule of interest and counter ions for oppositely charged ligand groups on the IEX chromatographic resin. Strength of the binding of the molecule to the IEX resin depends on the net charge of the molecules, which is affected by operating conditions, such as pH and ionic strength. IEX resins include AEX resins and CEX resins. AEX resins may contain substituents such as di ethyl aminoethyl (DEAE), trimethy al aminoethyl (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). Cellulosic IEX resins such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent, U.K. Sephadex-based and crosslinked IEX resins are also known. For example, DEAE-, QAE-, CM-, and SP-Sephadex, and DEAE-, Q-, CM- and S-Sepharose, and Sepharose are all available from GE Healthcare, Piscataway, NJ. Further, both DEAE and CM derived ethylene glycol-methacrylate copolymer such as TOYOPEARLTM DEAE-650S or M and TOYOPEARLTM CM-650S or M are available from Toso Haas Co., Philadelphia, PA. POROSTM HS, POROSTM HQ, POROSTM XS are available from Thermo Fisher Scientific, Waltham, MA. In one embodiment, the second chromatography step is AEX chromatography performed in flow-through mode at pH about 6.5- 8.0. In one embodiment, the pH is about 6.5 to 7.5. [0238] Following purification through the chromatography steps, the antibody or antigenbinding fragment may be processed through a series of filtration steps including nanofiltration for virus removal and ultrafiltration for concentration and buffer exchange. The nanofiltration may be operated batchwise through the use of holding vessels or continuously by cycling nanofiltration membranes at an appropriate frequency. The ultralfiltration steps may be performed conventionally in batch mode by feeding the unit operation from a holding vessel containing virally filtered nano-filtration product. Alternatively, the ultrafiltration may be conducted continuously by using single pass ultrafiltration followed by in-line diafiltration either directly connected or through the use of an intermediate surge vessel.

Methods of Use

[0239] The invention also relates to a method of treating cancer in a subject, the method comprising administering an effective amount of any of the pharmaceutical compositions of the invention; i.e., any composition described herein, to the subject. In some embodiments of this method, the pharmaceutical composition is administered to the subject by subcutaneous administration.

[0240] 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 cancer, merkel cell carcinoma, cutaneous squamous cell carcinoma, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, endometrial cancer, cervical cancer, thyroid cancer, salivary cancer, prostate cancer (e.g. hormone refractory prostate adenocarcinoma), pancreatic cancer, colon cancer, liver cancer, thyroid cancer, glioblastoma, glioma, and other neoplastic malignancies.

[0241] In one embodiment, the cancer is melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, multiple myeloma, hepatocellular cancer, merkel cell carcinoma, renal cell carcinoma, endometrial carcinoma, cutaneous squamous cell carcinoma, non-Hodgkm lymphoma, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary cancer, prostate cancer, glioblastoma, Tumor Mutational Burden-High or MSI-H cancer. [0242] 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.

[0243] In alternate embodiments, the lung cancer is small-cell lung cancer (SCLC). In one embodiment, the SCLC is metastatic.

[0244] In some embodiments, the lymphoma is Hodgkin lymphoma.

[0245] In other embodiments, the lymphoma is non-Hodgkin lymphoma. In particular embodiments, the lymphoma is mediastinal large B-cell lymphoma. In some embodiments, the lymphoma is diffuse large B-cell lymphoma (DLBCL).

[0246] In some embodiments, the breast cancer is triple negative breast cancer.

[0247] In further embodiments , the breast cancer is ER+/HER2- breast cancer.

[0248] In some embodiments, the bladder cancer is urothelial cancer.

[0249] In some embodiments, the head and neck cancer is nasopharyngeal cancer. In some embodiments, the cancer is thyroid cancer. In other embodiments, the cancer is salivary cancer. In other embodiments, the cancer is squamous cell carcinoma of the head and neck.

[0250] In some embodiments, the cancer is metastatic colorectal cancer with high levels of microsatellite instability (MSI-H).

[0251] In some embodiments, the cancer is a solid tumor with a high level of microsatellite instability (MSI-H).

[0252] In some embodiments, the cancer is a solid tumor with a high mutational burden. In one embodiment, the Tumor Mutation Burden (TMB) is >10 mutations/megabase as determined by an FDA approved test. In one embodiment, the tumor is metastatic or unresectable.

[0253] 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 cancer, esophageal cancer, gastric cancer, DLBCL and hepatocellular cancer.

[0254] In other embodiments of the above treatment methods, 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’s 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).

[0255] Malignancies that demonstrate improved disease-free and overall survival in relation to the presence of tumor-infiltrating lymphocytes in biopsy or surgical material, e.g. melanoma, colorectal, liver, kidney, stomach/esophageal, breast, pancreas, and ovarian cancer are encompassed in the methods and treatments described herein. Such cancer subtypes are known to be susceptible to immune control by T lymphocytes. Additionally, included are refractory or recurrent malignancies whose growth may be inhibited using the antibodies described herein.

[0256] In some embodiments, the pharmaceutical compositions of the invention are administered to a subject having a cancer characterized by elevated expression of PD-L1 and/or PD-L2 in tested tissue samples, including: ovarian, renal, colorectal, pancreatic, breast, liver, gastric, esophageal cancers and melanoma. Additional cancers that can benefit from treatment with anti-PD-1 antibodies such as humanized anti-PD-1 antibody pembrolizumab include those associated with persistent infection with viruses such as human immunodeficiency viruses, hepatitis viruses class A, B and C, Epstein Barr virus, human papilloma viruses that are known to be causally related to for instance Kaposi’s sarcoma, liver cancer, nasopharyngeal cancer, lymphoma, cervical, vulval, anal, penile and oral cancers.

[0257] In one embodiment, the invention comprises a method of treating cancer in a human patient in need thereof comprising administering an effective amount of any pharmaceutical composition of the invention to the patient.

[0258] In one embodiment, the invention comprises a method of treating unresectable or metastatic melanoma in a human patient in need thereof comprising administering an effective amount of any pharmaceutical composition of the invention to the patient.

[0259] In one embodiment, the invention comprises a method of treating metastatic nonsmall cell lung cancer (NSCLC) in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient. In specific embodiments, the patient has a tumor with high PD-L1 expression [(Tumor Proportion Score (TPS) >50%)]. In other embodiments, the patient has a tumor with PD-L1 expression (TPS >1%). In still other embodiments, the patient was or was not previously treated with platinum- containing chemotherapy. In specific embodiments, the patient had disease progression on or after receiving platinum-containing chemotherapy. In one embodiment, the NSCLC is metastatic or Stage III.

[0260] In certain embodiments, the PD-L1 TPS is determined by an FDA-approved test.

[0261] In certain embodiments, the patient’s tumor has no EGFR or ALK genomic aberrations.

[0262] In certain embodiments, the patient’s tumor has an EGFR or ALK genomic aberration and had disease progression on or after receiving treatment for the EGFR or ALK aberration(s) prior to receiving the compositions of the invention.

[0263] In certain embodiments, the patient has a tumor with PD-L1 expression CPS >1%.

[0264] In one embodiment, the invention comprises a method of treating nonsquamous non-small cell lung cancer (NSCLC) in a patient in need thereof, wherein the method comprises administering an effective amount of a pharmaceutical composition of the invention, pemetrexed and platinum chemotherapy to the patient. In one embodiment, the invention comprises a method of treating nonsquamous non-small cell lung cancer (NSCLC) in a human patient in need thereof comprising: (1) administering an effective amount of a pharmaceutical composition of the invention to the patient, and (2) administering effective amounts of pemetrexed and carboplatm to the patient. In specific embodiments, the patient was not previously treated with an anticancer therapeutic prior to starting the combination treatment regimen with the composition of the invention, pemetrexed and carboplatin. In a certain embodiments, the patient has metastatic nonsquamous non-small cell lung cancer.

[0265] In certain embodiments, pemetrexed is administered to the patient in an amount of 500 mg/m ² . In sub-embodiments, pemetrexed is administered to the patient via intravenous infusion every 21 days. In specific embodiments, the infusion time is about 10 minutes.

[0266] In embodiments of the invention where the patient is treated with a pharmaceutical composition of the invention in combination with pemetrexed, the invention further comprises administering about 400 pg to about 1000 pg of folic acid to the patient once per day, beginning about 7 days prior to administering pemetrexed to the patient and continuing until about 21 days after the patient is administered the last dose of pemetrexed. In certain embodiments the folic acid is administered orally. In some embodiments, the invention further comprises administering about 1 mg of vitamin B12 to the patient about 1 week prior to the first administration of pemetrexed and about every three cycles of pemetrexed administration (i.e., approximately every 9 weeks). In certain embodiments the vitamin B12 is administered intramuscularly. In certain embodiments, the invention further comprises admmistenng about 4 mg of dexamethasone to the patient twice a day on the day before, the day of, and the day after pemetrexed administration. In certain embodiments the dexamethasone is administered orally . [0267] In one embodiment, the invention compnses a method of treating squamous nonsmall cell lung cancer (NSCLC) in a human patient in need thereof comprising: (1) administering an effective amount of a pharmaceutical composition of the invention to the patient, and (2) administering effective amounts of paclitaxel or protein-bound paclitaxel and carboplatin to the patient. In specific embodiments, the patient was not previously treated with an anti-cancer therapeutic prior to starting the combination treatment regimen. In certain embodiments, the patient has metastatic squamous non-small cell lung cancer.

[0268] In one embodiment, the invention comprises a method of treating recurrent or metastatic head and neck squamous cell cancer (HNSCC) in a human patient in need thereof comprising administering an effective amount of any pharmaceutical composition of the invention to the patient. In certain embodiments, the patient was previously treated with platinum-containing chemotherapy. In certain embodiments, the patient had disease progression on or after platmum-containing chemotherapy. In specific embodiments, the patient’s tumor expresses PD-L1 [Combined Positive Score (CPS) >1]. In one embodiment, the treatment is in combination with platinum and FU.

[0269] In one embodiment, the invention comprises a method of treating refractory classical Hodgkin lymphoma (cHL) in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient. In certain embodiments, the patient has relapsed after 2 or more lines of therapy for cHL. In specific embodiments, the patient is an adult patient. In alternative embodiments, the patient is a pediatric patient.

[0270] In one embodiment, the invention comprises a method of treating locally advanced or metastatic urothelial carcinoma in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient. In certain embodiments, the patient is not eligible for cisplatin-containing chemotherapy. In certain embodiments, the patient has disease progression during or following platinum- containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy. In specific embodiments, the patient’s tumor expresses PD- L1 [Combined Positive Score (CPS) >1]. In one embodiment, the patients have Bacillus Calmette-Guerin (BCG)-unresponsive, high risk, non-muscle invasive bladder cancer. [0271] In one embodiment, the invention comprises a method of treating unresectable or metastatic, microsatellite instability -high (MSI-H) or mismatch repair deficient solid tumors in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient. In specific embodiments, the patient had disease progression following prior anti-cancer treatment.

[0272] In one embodiment, the invention comprises a method of treating unresectable or metastatic, microsatellite instability -high (MSI-H) or mismatch repair deficient solid tumors or colorectal cancer in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention. In specific embodiments, the patient had disease progression following prior treatment with a fluoropyrimidine, oxaliplatin, and irinotecan.

[0273] In one embodiment, the invention comprises a method of treating recurrent locally advanced or metastatic gastric cancer in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient.

[0274] In one embodiment, the invention comprises a method of treating recurrent locally advanced or metastatic gastroesophageal junction adenocarcinoma in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient. In specific embodiments, the patient’s tumor expresses PD-L1 [Combined Positive Score (CPS) >1], In specific embodiments, the patient has disease progression on or after two or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy. In specific embodiments, the patient has disease progression on or after two or more prior lines of therapy including HER2/neu-targeted therapy .

[0275] In one embodiment, the invention comprises a method of treating recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient. In specific embodiments, the patient’s tumor expresses PD-L1 [Combined Positive Score (CPS) >1],

[0276] In one embodiment, the invention comprises a method of treating recurrent locally advanced or metastatic cervical cancer in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient. In specific embodiments, the patient’s tumor expresses PD-L1 [Combined Positive Score (CPS) >1]. [0277] In one embodiment, the invention comprises a method of treating hepatocellular carcinoma in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient. In one embodiment, the invention comprises a method of treating recurrent locally advanced or metastatic merkel cell carcinoma in a human patient comprising administering a composition of the invention to the patient. In one embodiment, the invention comprises a method of treating recurrent or metastatic cutaneous squamous cell carcinoma in a human patient comprising administering a composition of the invention to the patient.

[0278] In one embodiment, the invention comprises a method of treating advanced renal cell carcinoma in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient and axitinib. In one embodiment, the invention comprises a method of treating advanced endometrial carcinoma in a human patient comprising administering a composition of the invention to the patient and lenvatinib. In one embodiment, the endometrial carcinoma is not MSI-H or dMMR.

[0279] In one embodiment, the invention comprises a method of treating cancer in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient, wherein the patient has a cancer selected from the group consisting of: melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, lymphoma, renal cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, and salivary cancer.

[0280] In one embodiment, the invention comprises a method of treating small cell lung cancer in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient.

[0281] In one embodiment, the invention comprises a method of treating non-Hodgkin lymphoma in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient. In specific embodiments, the nonHodgkin lymphoma is mediastinal large B-cell lymphoma. In specific embodiments, the nonHodgkin lymphoma is diffuse large B-cell lymphoma.

[0282] In one embodiment, the invention comprises a method of treating breast cancer in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient. 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 specific embodiments, the patient's tumor expresses PD-L1 [Combined Positive Score (CPS) >1],

[0283] In one embodiment, the invention comprises a method of treating nasopharyngeal cancer in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient.

[0284] In one embodiment, the invention comprises a method of treating thyroid cancer in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient.

[0285] In one embodiment, the invention comprises a method of treating salivary cancer in a human patient in need thereof comprising administering an effective amount of a pharmaceutical composition of the invention to the patient.

[0286] As noted above, in some embodiments of the methods of the invention, the method further comprises administering an additional therapeutic agent. In particular embodiments, the additional therapeutic agent is an anti-LAG3 antibody or antigen binding fragment thereof, an anti-GITR antibody, or antigen binding fragment thereof, an anti-TIGIT antibody, or antigen binding fragment thereof, an anti-CD27 antibody or antigen binding fragment thereof. In one embodiment, the additional therapeutic agent is a Newcastle disease viral vector expressing IL-12. In a further embodiment, the additional therapeutic agent is dinaciclib. In still further embodiments, the additional therapeutic agent is a STING agonist. In one embodiment, the additional therapeutic agent is Coxsakievirus CVA21.

[0287] Suitable routes of administration for the additional therapeutic agents may, for example, include parenteral delivery, including intramuscular, subcutaneous, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal. Drugs can be administered in a variety of conventional ways, such as intraperitoneal, parenteral, intraarterial or intravenous injection.

[0288] Selecting a dosage of the additional therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissue or organ in the individual being treated. The dosage of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Accordingly, the dose amount and dosing frequency of each additional therapeutic agent (e.g. biotherapeutic or chemotherapeutic agent) will depend in part on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Guidance in selecting appropriate doses 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 Y ork, NY ; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341 : 1966-1973; Slamon eKti. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed);

Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). Determination of the appropriate dosage regimen may be made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment, and will depend, for example, the patient's clinical history (e.g., previous therapy), the type and stage of the cancer to be treated and biomarkers of response to one or more of the therapeutic agents in the combination therapy.

[0289] Various literature references are available to facilitate selection of pharmaceutically acceptable carriers or excipients for the additional therapeutic agent. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984); Hardman et al. (2001) Goodman and Gilman ’s The Pharmacological Basis of Therapeutics , McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0290] A pharmaceutical antibody composition can be administered by continuous infusion, or by doses at intervals of, e.g., one day, 1-7 times per week, one week, two weeks, three weeks, monthly, bimonthly, etc. A preferred dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. A total weekly dose is generally at least 0.05 pg/kg, 0.2 pg/kg, 0.5 pg/kg, 1 pg/kg, 10 pg/kg, 100 pg/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) Neurol. Neurosurg. Psych. 67:451-456; Portielji et al. (20003) Cancer Immunol. Immunother. 52:133-144. The desired dose of a small molecule therapeutic, e.g., a peptide mimetic, natural product, or organic chemical, is about the same as for an antibody or polypeptide, on a moles/kg basis.

[0291] In certain embodiments, dosing will comprise administering to a subject escalating doses of 1.0, 3.0, and 10 mg/kg of the pharmaceutical composition or a composition of the invention, over the course of treatment. The composition can be a reconstituted liquid composition, or it can be a liquid composition not previously lyophilized. Time courses can vary, and can continue as long as desired effects are obtained. In certain embodiments, dose escalation will continue up to a dose of about lOmg/kg. In certain embodiments, the subject will have a histological or cytological diagnosis of melanoma, or other form of solid tumor, and in certain instances, a subject may have non-measurable disease. In certain embodiments, the subject will have been treated with other chemotherapeutics, while in other embodiments, the subject will be treatment naive.

[0292] In yet additional embodiments, the dosing regimen will comprise administering a dose of 1, 3, or 10 mg/kg of any of the pharmaceutical compositions or compositions described herein, throughout the course of treatment. For such a dosing regimen, the interval between doses will be about 14 days (± 2 days). In certain embodiments, the interval between doses will be about 21 days (± 2 days).

[0293] In certain embodiments, the dosing regimen will comprise administering a dose of from about 0.005mg/kg to about lOmg/kg, with intra-patient dose escalation. 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 yet additional embodiments, a dose of 3mg/kg will be administered at three week intervals for melanoma patients or patients with other solid tumors. In these embodiments, patients should have non-resectable disease; however, patients may have had previous surgery [0294] In certain embodiments, a subject will be administered a 30 minute IV infusion of any of the pharmaceutical compositions or compositions described herein. In certain embodiments for the escalating dose, the dosing interval will be about 28 days ((± 1 day) between the first and second dose. In certain embodiments, the interval between the second and third doses will be about 14 days (± 2 days). In certain embodiments, the dosing interval will be about 14 days (± 2 days), for doses subsequent to the second dose. In certain embodiments, the dosing interval will be about 3 weeks, for doses subsequent to the second dose. In certain embodiments, the dosing interval will be about 6 weeks, for doses subsequent to the second dose. [0295] 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, diagnostic, patient selection, and/or treatment regimens involving blockade of the PD-1 pathway. Subcutaneous administration may performed by injected using a syringe, or using other injection devices (e.g. the Inject-ease® device); injector pens; or needleless devices (e.g. MediJector and BioJector®).

[0296] Embodiments of the invention also include one or more of the pharmaceutical compositions or formulations described herein (i) for use in, (ii) for use as a medicament or composition for, or (iii) for use in the preparation of a medicament for: (a) therapy (e.g., of the human body); (b) medicine; (c) induction of or increasing of an antitumor immune response (d) decreasing the number of one or more tumor markers in a patient; (e) halting or delaying the growth of a tumor or a blood cancer; (f) halting or delaying the progression of PD-l-related disease; (g) halting or delaying the progression of cancer; (h) stabilization of PD-l-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) reduction of the progression, onset or severity of PD-l-related disease; (1) reducing the severity or duration of the clinical symptoms of PD-l- related disease such as cancer (m) prolonging the survival of a patient relative to the expected survival in a similar untreated patient n) inducing complete or partial remission of a cancerous condition or other PD-1 related disease, or o) treatment of cancer.

GENERAL METHODS

[0297] Standard methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 1989 2 ^nd Edition, 2001 3 ^rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3 ^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DN A, 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 in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0298] Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, 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 inMolecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89;

Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY ; Harlow and Lane, supra). Standard techniques 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).

[0299] 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, pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang el n/. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) 7. Biol. Chem. 272:10678-10684; Chothia eln/. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

[0300] Having described different embodiments of the invention herein with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

EXAMPLE 1 : Continuous Perfusion Process

[0301] Glutamine Synthetase knock-out CHO host cell comprising a polynucleotide encoding a light chain with the ammo acid sequence set forth in SEQ ID NO:5 and a polynucleotide encoding a heavy chain with the amino acid sequence set forth in SEQ ID NOTO (expressing pembrolizumab) was used in the below procedures and Examples.

3 L bioreactor operation [0302] Glass bioreactors (3 L, Sartorius Stedim, Gottingen, Germany) with a marine impeller (70 mm diameter) and a drilled hole sparger (14 x holes with 0.5 mm diameter) were used. The bioreactors were inoculated at a target cell density of 0.5 x io ⁶ cells/mL. Dissolved oxy gen (DO) was controlled at 30-60 % of air saturation using pure O2 and agitation speed which was gradually increased in the range of 260 - 450 rpm to control O2 sparge rate at <0.30 vvm to avoid excess foam formation. EX-CELL® antifoam (Sigma- Aldrich, St. Louis, MO) was added as needed for foam control. Overlay air was controlled at 0. 1 L/min. Temperature was maintained at 36.5 °C throughout the culture duration. The pH of the bioreactor was controlled at 7.0±0.3 during the cultivation period.

[0303] A commercially available basal media was used for the initial bioreactor batch growth and subsequently a perfusion media was used for medium exchange. The perfusion media has a copper concentration of 10-35 ppb. Bioreactors started with a working volume of 1.6 L and maintained at the same level during perfusion. On day 3, when cell density reached between 2 to 4 xlO ⁶ cells/mL, medium exchange was started and the perfusion rate ramped up according to a predetermined schedule starting from 0.5 vvd. On day 4, the 1 vvd perfusion rate was applied and reached the maximum perfusion rate of 2 vvd between day 5 and end of the production. A TFF system with a magnetic levitating pump was used (KrosFlo® KML System, Repligen, Waltham, MA) for cell retention and medium exchange. The TFF system continuously circulates the cell culture fluid through a hollow fiber module while permeate was collected at the pre-determined rates. 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 pm (Pall, Port Washington, NY). A TFF cross flow rate of 1.0 L/min was used in all experiments. Once the cell density reaches 100 x io ⁶ cells/mL or capacitance target of 80 pF/cm , a cell bleed pump turns on to maintain the cell density or biovolume (capacitance, pF/cm Cell bleed was automatically controlled by a capacitance probe (Incyte DN12, Hamilton Bonaduz AG, Switzerland). Perfusion culture duration was 28 - 32 days. The copper concentration in the bioreactor ranged from 1 to 35 ppb. Continuous harvesting is started after day 5 when 2vvd perfusion rate has been achieved for the bioreactor.

50 L bioreactor operation

[0304] The Xcellerx XDR 50 single use bioreactor (GE healthcare, Marlborough, MA) was used as a production bioreactor for this study. The bioreactor was inoculated at a target cell density of 0.5 x io ⁶ cells/mL which dissolved oxygen (DO) controlled at 30-60 % of air saturation using pure O2 supply. The agitation of the bioreactor was controlled at the 86-93 rpm to ensure proper mixing and mass transfer. EX-CELL® antifoam (Sigma- Aldrich, St. Louis, MO) was added as needed for foam control. Overlay air was controlled at 0. 1-0.5 L/min. Temperature was maintained at 36.5 °C throughout the culture duration. The pH of the bioreactor was controlled at 7.0±0.3 during the cultivation period.

[0305] A commercially available basal media was used for the initial bioreactor batch growth and subsequently a perfusion media was used for medium exchange. The perfusion media has a copper concentration of 32.5 ppb. Bioreactors started with a working volume of 50 L and maintained at the same level during perfusion. On day 3, when cell density reached between 2 to 4 xlO ⁶ cells/mL, medium exchange was started and the perfusion rate ramped up according to a predetermined schedule starting from 0.5 vvd at day 3, to 1 vvd at day 4 and reached the maximum perfusion rate of 2 vvd (0.5 day mean residence time) at day 5. A TFF system with a magnetic levitating pump was used (KrosFlo® KML System, Repligen, Waltham, MA) for cell retention and medium exchange. The TFF system continuously circulates the cell culture fluid through a hollow fiber module while permeate was collected at the pre-determined rates. The hollow fiber with pore size of 0.2 um ((Pall, Port Washington, NY) was used as a cell retention device for this study. A TFF cross flow rate of 10.5 L/min was used in this study. Once the cell density reaches 100-105 x io ⁶ cells/mL or capacitance target of 80-85 pF/cm , a cell bleed pump turns on to maintain the cell density or biovolume (capacitance, pF/cm Cell bleed was automatically controlled by a capacitance probe (Incyte DN12, Hamilton Bonaduz AG, Switzerland) Perfusion culture duration was 28 days. Continuous harvesting started after day 5 when 2 vvd perfusion rate has been achieved for the bioreactor.

[0306] Samples were taken from the bioreactor and the permeate line daily. Viable cell density (VCD) and viability were measured using the trypan blue exclusion method on a Cedex Hi-Res cell counter (Roche Diagnostics GmbH, Mannheim, Germany). Cell diameter was determined on the same cell counter and reported. Offline pH, pO2, and pCO2 were measured using an ABL80 blood gas analy zer (Radiometer, Denmark). Glucose, lactate, ammonium, and lactate dehydrogenase (LDH) were measured using a RX Daytona+ or a Imola analyzer (Randox Laboratories, Ltd., Crumlin, UK). Bioreactor supernatant and permeate antibody titers were analyzed using an Agilent 1100 high-performance liquid chromatography (HPLC, Agilent Technologies, Santa Clara, CA) equipped with a Protein A column.

EXAMPLE 2: Downstream Purification

Protein A affinity chromatography [0307] Protein A affinity chromatography serves as the primary capture step, using MabSelect SuRe resin from GE Healthcare™ for the bulk of purification. The product binds to the resin, while impurities such as media components and host cell proteins are not bound and remain in the column flowthrough fraction. The protein A step is operated in a continuous manner using a continuous multi-column chromatography skid to allow for uninterrupted flow of HCCF from the bioreactor to feed the skid (BioSMB PD, Sartorius Stedim Biotech GmbH Goettingen Germany). This is achieved by utilizing 4 comparable columns packed with the same Protein A resin on the skid. At any one time, three columns are dedicated to loading while the fourth is undergoing the non-loading steps (equilibration step, washing steps, elution steps, regeneration steps) in the protein A method.

[0308] The HCCF from the bioreactor is connected to a continuous multi-column chromatography skid through a 3L single-use surge vessel (Cercell, Herlev Denmark). The HCCF flow rate to the single use surge vessel matched the feed rate from the surge vessel to the continuous multi-column chromatography system maintaining a consistent volume in the vessel. The operational volume range in the surge vessel gave a 15-45 minute mean residence time of the fluid in the vessel with a target value of 30 minutes. As shown in Figure 5, due to the oxidative instability, the residence time of the HCCF loading to the protein A columns is optimal at less than 24 hours.

[0309] Operationally, the same steps are performed in multi-column Protein A chromatography as in single-column batch-mode chromatography including phases for column equilibration, loading, washing, and regeneration. After equilibrating the column with 10 mM Sodium Phosphate, pH 6.5 the loading phase was initiated. The protein was loaded to a capacity of 50g/L of Protein A resin. The loading phase of the Protein A step was followed by three wash 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 increase the purity of the antibody in the product stream. The product was eluted with 20 mM Sodium Acetate pH 3.6 via low pH shift and monitored by on-line spectrophotometry at an absorbance of 280 nm. The step in the surge vessel and the Protein A step was performed at ambient temperature (20 ± 5 °C).

[0310] After Protein A chromatography, the protein was processed through additional purification and filtration steps to achieve the ultrafiltration product in a continuous process connected though intermediate surge vessels including: viral inactivation, depth filtration, anion exchange chromatography, virus filtration, single-pass tangential flow filtration, and in-line diafiltration. Viral Inactivation/Depth filtration

[0311] The Protein A affinity pool was adjusted with 1 M Acetic Acid to a low pH (target pH 3.6) to inactivate viruses that might be present. This step was performed at ambient temperature (20 ± 5°C). The pool after viral inactivation was adjusted with 1 M Tris to a target pH of 5.5 and is filtered through a charged depth filter (A1HC) and 0.22 pm filter for clarification (Millipore Sigma, Burlington MA). The filtered neutralized viral inactivation product (FNVIP) may be stored at 2-8°C prior to further processing in the subsequent anion exchange chromatography step.

Anion Exchange

[0312] Anion exchange chromatography (AEX), performed with POROS HQ 50 from Applied Biosystems, is the first polishing step in the process. The pembrolizumab antibody flows through the column whereas residual impurities, such as host cell protein (HCP), DNA, aggregates, and virus that may be present are bound to the resin. The column was equilibrated and washed with 25 mM sodium phosphate pH 7.2. Prior to the start of the step, the post viral inactivation pool was pH adjusted with IM Tris to a target pH of 7.2. The column effluent UV absorbance was monitored online at a wavelength of 280 nm and used to collect the AEX pool. The pool was pH adjusted with IM Acetic Acid to pH 5.5 for continued processing in the post step surge vessel.

Viral Filtration

[0313] The AEX product was filtered through a 0. 1 pm pre-filter or equivalent in-line with a Planova 20N (19 nm mean pore size) virus reduction filter (Asahi Kasei Bioprocess, Glenview, IE). The prefilter and filter connections were autoclaved and placed in line with the nanofilter in a biosafety cabinet. The filters were then flushed in series with 10 mM Histidine, 10 mM Methionine, pH 5.4 in a closed manner.

Ultrafiltration Concentration and Diafiltration

[0314] The viral filtration product was first concentrated 8x by a single-pass tangential flow filtration (SPTFF) module with a 30 kDa molecular weight cutoff (Pall Corporation Westboro MA Port Washington, NY). The product from the SPTFF step is then diafiltered with 10 mM Histidine, 10 mM Methionine, pH 5.4 resulting in a buffer exchange of the concentrated protein stream. A six-stage in-line diafiltration module (ILDF) unit containing 30kDa MW cutoff membrane (Pall Corporation Westboro MA Port Washington, NY) was utilized to perform the buffer exchange. The diafiltration product, also referred to as the ultrafiltration product was further filtered through a sterile filter and collected into the storage containers. Once a sufficient mass of ultrafiltration product has been accumulated, the ultrafiltration product was pooled and was further concentrated by a standard batch ultrafiltration step using an Ultracel 30kDa molecular weight cutoff membrane (Millipore, Burlington, MA) up to a final concentration of 190-200g/L. Stock excipients were added to achieve a final drug substance formulation of 165mg/mL in a 10 mM Histidine, 10 mM Methionine, 7% (w/v) sucrose, and 0.02% (w/v) PS-80 buffer pH 5.5.

EXAMPLE 3: Method for determining Ml 05 oxidation levels

Reduced peptide mapping, liquid chromatography and mass spectrometry method

Sample preparation

[0315] In-process or Drug Substance (DS) samples were diluted with water to 5 mg/mL. A total of 20 pL of the diluted sample (contains 100 pg) was denatured and reduced in final solution (100 pL) containing 6 M Guanidine-HCl, 50 pM Tris-HCl, 50 pM EDTA and 200 pM DTT. The mixed sample was incubated in thermomixer at 37°C with 300 rpm shaking for 30 min. After mix and spin down, each sample is alkylated with 5 pL of iodoacetamide (IAM) (1 M) protected from light at 25 °C for 30 min. A total of 5 pL of DTT (200 pM) was added to block the unreacted iodoacetamide (IAM). Lysyl endopeptidase (Lys-C) (Wako, 125-05061) enzyme (1 :10 (wt: wt)) in 500 pL was added to the protein sample with slowly pipette up/down 3 times to mix well. The digestion was incubated in a thermomixer at 37°C for 60 min. The digestion is quenched with 15 pL of 20% TFA. Digested sample was analyzed by LC-MS within 24 h after sample digestion. Otherwise, digested samples were stored at -80 °C for future analysis.

LC-MS methods and data analysis

[0316] Waters Acquity Liquid Chromatography was used to inject 20 pL of sample on the column (UPLC HSS T3 100A, 1.8 pm, 2.1 rnrn X 150 mm, P/N: 186003540). 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 5min, 0. 1 %-l 0% B from 5 to 7 min, followed by a linear increase to 35% B over the next 38 min. Q Exactive Orbitrap MS (Thermo) was used to collect the MSI data. Chromeleon and Xcalibur was used for data analysis. The extracted ion chromatogram (E1C) of Ml 05 non-modified and modified peptides (two charged states (+3 and +4) for each peptide and each with 3 isotopic ions) were used to determine the % Ml 05 oxidation using the formula [peak area of the extracted ion chromatogram (EIC) of the modified peptide] / [peak area of E1C of the modified peptide + peak area of E1C of the unmodified peptide] x too. See Figures 2-4. The %CV (Coefficient of Variation; calculated as (standard deviation/mean) x 100) for the Ml 05 oxidation was less than 20%.

Table 3

[0317] The formulation for fed batch produced pembrolizumab drug product reference standard is 25 mg/rnL pembrolizumab in 10 mM histidine buffer containing 7% (w/v) sucrose and 0.02% (w/v) polysorbate 80, pH 5.5. The formulation for continuous perfusion produced pembrolizumab formulated Drug Substance is 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.

[0318] The % oxidized Ml 05 for fed batch produced pembrolizumab formulated drug product reference standard was about 4.9% while the continuous perfusion produced pembrolizumab samples contain about 0.5%-3.0% oxidized M105. Some representative samples from protein A purification, ultrafiltered product (unformulated DS) and formulated DS are shown in Table 3.

Hydrophobic interaction chromatography (HP-HIC)

[0319] For determination of % Metl05 oxidation, high performance hydrophobic interaction chromatography (HP-HIC) was used to separate and quantitate oxidized products from the non-oxidized molecule. Pre-peak3 contains antibody with Metl05 oxidation in both heavy chains; pre-peaks 1 and 2 contain antibody with Metl05 oxidation in either heavy chain (Figure 13). The percentage of pre-peaks, as well as the percentage of the main and percentage of the post peaks were determined. A HP-HIC method was performed by diluting the sample to 5.0 mg/mL in purified water. The sample was then injected (10 pL) into an HPLC equipped with a Tosoh Phenyl-5PW column at 30 °C and a UV detector at 280 nm. For the HIC analysis, 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;) was used. The gradient was 0% mobile phase A from 0 -2 minutes, 0% - 100% mobile phase A from 2 min - 52min followed by column washing and equilibration. Pembrolizumab reference produced by a fed-batch process which underwent batch Protein A, viral inactivation, depth filtration and AEX purification was tested for % Metl05 oxidation. Representative values of the % Metl05 oxidation for unfiltered sample after viral inactivation and, and after AEX purification of the Pembrolizumab reference is about 5.5%.

Example 4: Measure changes in M105 oxidation of pembrolizumab over time in HCCF [0320] The rate of oxidation was determined by repeatedly measuring the amount of oxidation in a HCCF sample according to Example 1 by ProA-HIC 2D-LC as described in Example 3 and below.

[0321] A sample was taken from the bioreactor and filtered to remove the cells, giving HCCF. Under sterile conditions, the HCCF sample was split into 200 pL aliquots across 48 wells of a 96 well plate. The plate was covered with an aluminum foil lid to retain sterility and shield the samples from light. The samples were immediately placed into an Agilent 1290 autosampler set at 25 °C, with the internal light turned off. Each well of sample was analyzed in sequence by a two-dimensional liquid chromatography separation according to the time course.

[0322] The resultant chromatogram of each sample was integrated to determine the percent oxidation, and the summary of the data is shown in Figure 5. The linear regression of the data shows the percent oxidation starting 0.94% and increasing at approximately 0.038% per hour over the next 26 hours to a maximum of 1.95% oxidation.

Example 5: Ion Exchange (IEX) method to measure acidic species of anti-PD-1 antibodies [0323] For the 1EX method, using a Waters Alliance LC system (Milford, MA, U.S.A.), the Thermo Scientific’s ProPac WCX-10 (p/n: 054993, particle size 10 um, diameter 4 mm, length 250 mm) was chosen with a loading of 80 pg sample. 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 was used as a non-linear, sigmoidal shape, pH gradient, and the separation was monitored over 34 min with a flow rate of 0.5 mL min '. with the column temperature being 35 °C. The gradient used was: 22%-22%B for 0-0.6 min; 22%-29%B for 0.6-15.0 min; 29%- 70%B for 15.0-30.0 min; 70%-100%B for 30.0-30.5 min; and 100%-100%B from 30.5-33.0 min. Mobile phase (C) 10 mM CHES pH 8.0, 40 mM Tris, 15 mM EDTA, 200 mM NaCl, and 4% acetonitrile was used to strip the column at 0.5 mL min ¹ from 33. 1-34.0 min, followed by re-equilibration with 22%B from 34.5-44.5 min at 1.0 mL min ¹ . From 44.5-45 min, the flowrate was reduced to 0.5 mL min ^-1 . The elution was monitored at 280 nm for the detection of peaks. The assay variability was determined to be within 1%.

[0324] The chemical composition of each identified peak of the pembrolizumab drug substance reference sample (from fed-batch process) was determined by collecting samples of each peak, and performing peptide mapping and reverse phase liquid chromatography followed by mass-spectrometry according to methods identical to those in Example 3 with analysis performed by MS/MS. The main peak was determined to predominantly contain the antibody with the amino acid sequence set forth in SEQ ID NO:5 and SEQ ID NO: 11 for both light chains and heavy chains, respectively. Oxidation (for example, Methioninel05) and deamidation of asparagine residues (for example, N31, N52, N55, N59 or N61 in the heavy chain of SEQ ID NO: 11) of the foregoing antibody w as detected in the Acidic Variants peak. Deamidation of asparagine residues (for example, N384, N389 or N390 in the heavy chain of SEQ ID NO: 11) of the foregoing antibody was detected in the Acidicl peak. In the Basic 1 peak, antibodies with one heavy chain consisting of the amino acid sequence of SEQ ID NO: 11, one heavy chain consisting of the ammo acid sequence of SEQ ID NO: 12, and two light chains consisting of the amino acid sequence of SEQ ID NO: 5; or one heavy chain consisting of the amino acid sequence of SEQ ID NO: 11, one heavy chain consisting of the amino acid sequence of SEQ ID NO: 14, wherein the C-terminal leucine is alpha-ami dated, and two light chains consisting of the amino acid sequence of SEQ ID NO: 5 were detected.

[0325] The ion exchange chromatograms and % acidic species, main peak, and basic species from samples after Protein A chromatography (PAP) and Anion exchange chromatography (AEXP) according to procedures in Examples 1-2 are illustrated in Figures 6-7 and Table 4. The sum of the acidic species (acidic variants. Acidic 1 and Pre-Mam) for the PAP and AEXP samples are 7.86% and 8.76% respectively. The sum of the basic species (basic 1, basic variant A, basic2, basic variant B) for the PAP and AEXP samples are 16.95% , 18. 19% respectively. The ion exchange chromatograms and % acidic species, main peak, and basic species from pembrolizumab reference obtained by fed-batch method are illustrated in Figure 8 and Table 5. The sum of the acidic species (acidic variants, Acidic 1 and Pre-Main) for the reference sample is 16.56%. The sum of the basic species (basic 1, basic variant A, basic2, basic variant B) for the reference sample is 23.76%. The sum of the basic variants (basic variant A and basic variant B) for the reference sample is 6.23%. In summary', the pembrolizumab samples prepared by the continuous perfusion process of the invention had higher percentage of main species due to lower percentage of acidic species in the mixture. In other batches of pembrolizumab samples prepared by the continuous perfusion process substantially similar to Examples 1-2, the main peak is about 74-80%.

Table 4: Pembrolizumab obtained from the methods of the invention: acidic, main and basic species

Table 5: Pembrolizumab reference acidic, main and basic species

[0326] Figures 9-12 also provide a time course for the % total acidic species, % main species, % total basic species and % basic 1 species in cell-free permeate (PERM), after Protein A chromatography step (PAP), after anion exchange chromatography (AEXP) prepared according to procedures in Examples 1-2 in a time course by culture days.

Example 6: Hyaluronidase Activity Assay

[0327] Enzyme activity was determined by a turbidimetric assay on a Molecular Devices SpectraMax M5e microplate reader. Calibration curve standards and test samples were prepared via dilution with chilled Enzyme Diluent (20 mM sodium phosphate, 77 mM sodium chloride, 0.01 % bovine serum albumin (BSA) at pH 7.0 at 25°C) to the working concentrations outlined in Table 6.

Table 6. Example Working Concentrations Used in Activity Assay

50 pL of diluted standard and samples were transferred to a clear-bottom 96-well plate (Plate 1) in triplicate. 50 pL of Enzyme Diluent solution was added in dedicated wells on the plate as a blank control. The plate was sealed and incubated at 37°C for 10 minutes. After incubation, 50 pL of 37°C Hyaluronic Acid Solution (0.06 % hyaluronic acid 300 mM phosphate at pH 5.35 at 37°C) was added to each well containing solution with a multichannel pipette. The plate was sealed then incubated at 37°C for exactly 45 minutes with shaking at 600 rpm. Prior to removing Plate 1 from incubation, a second plate (Plate 2) was prepared with 200 pL of Acidic Albumin Solution (24 mM sodium acetate, 79 mM acetic acid, 0.1 % BSA at pH 3.75 at 25°C) in each well creating the same well layout as Plate 1. After the exact 45-mmute incubation of Plate 1, 40 E of solution from each well was removed with a multi-channel pipette and added to the corresponding wells in Plate 2 (containing Acidic Albumin Solution). Plate 2 was incubated at 25°C for 20 minutes within the plate chamber of the microplate reader. The microplate reader was set to read absorbance at 600 nm after 5 seconds of shaking. After the 20-minute incubation, absorbance at 600 nm was read for each well.

Generating Calibration Curve

[0328] Triplicate absorbance values from calibration curve standards were averaged and subtracted by the average absorbance of the blank. The absolute value of the resulting corrected absorbance was plotted vs the calibration curve standard volumetric activity values (1 , 12, 10, 7.5, 5, and 2 Units/mL, for example). The plot was fit with a first order polynomial (Figure 14) and the resulting equation of the fit was used to determine the enzymatic activity of the test samples.

Calculation of enzymatic activity’ of test samples

[0329] Triplicate absorbance values were averaged and subtracted by the average absorbance of the blank. The absolute value of the resulting corrected absorbance was entered into the fit equation from the calibration curve to determine the calculated activity for the test samples. Any corrected absorbance value that fell outside the calibration curve was discarded. Values were corrected for dilution by multiplying the assay volumetric activity by the dilution factor used to prepare the solutions. For example, if the estimated enzymatic activity of the formulation or standard stock solution was 1500 Umts/mL and was diluted to 12, 10, and 7.5 Units/mL for analysis, the dilution factors would have been 125, 150, and 200 respectively. Final calculated enzyme activity values were averaged and reported in Units/mL.

Example 7 : Evaluation of the stability of Recombinant Human Hyaluronidase PH20 variant fragment 2 with Pembrolizumab CPP after light stress

[0330] Co-formulated samples containing Pembrolizumab manufactured by the continuous perfusion process (CPP) and PH20 variant fragment 2 in 7% w/v sucrose, 0.02% w/v PS-80, 10 mM methionine in 10 mM Histidine buffer at pH 5.5 were prepared at the concentrations shown in Table 7. Table 7. Co-formulations of Pembrolizumab CPP and PH20 variant fragment 2 used in light stress study

[0331] Samples were filled into 2R vials with a 1.6 mL fill volume. 1 vial of each sample was subjected to a cumulative light exposure of 240 Klux-hr CWL, equivalent to 0.20X ICH CWL, where IX ICH = 1200 klux-hr and 10 Watt*hr/m ² UV, equivalent to 0.05X ICH UV, where IX ICH = 200 Watt*hr/m ² , A second vial of each sample was covered with aluminum foil and stored at 2-8°C as a control. Enzyme activity test was performed to evaluate enzyme stability under light stress across the Pembrolizumab CPP concentration range listed in Table 7 (Figure 15). The data indicates at concentrations between 25 - 165 mg/mE Pembrolizumab CPP, there was an enhanced retention of PH20 variant fragment 2 enzymatic activity and stability following light stress in the presence of Pembrolizumab CPP compared to samples of PH20 variant fragment 2 alone. In addition, retention of PH20 variant fragment 2 activity upon light stress displays a dependence on Pembrolizumab CPP concentration. The data indicates that at concentrations equal or greater than 75 mg/mL Pembrolizumab CPP, the enhancement of PH20 Variant Fragment 2 enzyme activity was unexpectedly higher compared to lower concentrations of Pembrolizumab CPP.

[0332] Surprisingly, increased retention of enzymatic activity of PH20 Variant Fragment 2 in the presence of Pembrolizumab CPP is not observed when the enzyme is co-formulated with Pembrolizumab reference from fed-batch process. Figure 16 shows calculated activity data for PH20 Variant Fragment 2 co-formulated with 165 mg/mL Pembrolizumab CPP or 165 mg/mL Pembrolizumab reference as well as the activity of PH20 Variant Fragment 2 alone after light stress. Compared to control samples, Pembrolizumab CPP co-formulations retain enzymatic activity while both Pembrohzumab reference co-formulation and enzyme-only samples indicate an approximate 33% loss in activity after light stress.

[0333] % Metl05 Oxidation (pre-peak 1+2+3) and total %acidic species (acidic 1+acidic variants +pre-main) of Pembrohzumab reference and Pembrohzumab CPP after light stress are compared to samples without stress, respectively (Figure 17). Pembrohzumab reference shows higher base levels of Metl05 oxidation and acidic species compared to Pembrohzumab CPP.

Metl05 oxidation and acidic species increase to a greater degree for Pembrohzumab reference samples after light stress compared to Pembrolizumab CPP.

[0334] For determination of % Metl05 oxidation, high performance hydrophobic interaction chromatography (HP-HIC) substantially identical to Example 3 was used to separate and quantitate oxidized products from the non-oxidized molecule. For the % acidic species determination, the IEX method in Example 5 was used.

Example 8: Evaluation of the stability of Recombinant Human Hyaluronidase PH20 variant fragment 2 with Pembrohzumab CPP after stainless steel exposure

[0335] Test formulations of PH20 variant fragment 2 co-formulated with pembrohzumab CPP or Pembrohzumab reference were prepared in polyethylene terephthalate copolyester glycol modified (PETG) bottles (125 mL) with the compositions outlined in Table 8. Each formulation was exposed to SS solid cylinders at room temperature for 24 - 72 hours. All formulations were filtered using 0.22 pm PES filter. Samples were staged on stability (protected from tight) for 3 months at 25°C to assess the PH20 variant fragment 2 activity.

Table 8. Co-formulations of Pembrohzumab CPP and Pembrohzumab reference and PH20 variant fragment 2 exposed to stainless steel [0336] Interestingly, enzyme activity of PH20 Variant Fragment 2 co-formulated with Pembrolizumab CPP was retained equivalently to co-formulations with Pembrolizumab reference after 3 month/25°C storage and was additionally comparable to activity levels of the initial samples (Figure 18). After storage at 3 months/25°C, enzyme-only sample that were exposed to stainless steel show decreased activity relative to the initial timepoint. The enzyme activity in co-formulated samples with Pembrolizumab CPP was not impacted through the temperatures and timepoints studied, indicating enhanced enzyme stability and activity in the presence of Pembrolizumab CPP.

[0337] The chemical stability properties of Pembrolizumab CPP were also monitored, specifically on charge variants and oxidation under various stresses. (Figures 19 and 20) Pembrolizumab CPP %Metl05 oxidation and %acidic species increases after light stress and after SS exposure at 3 months 25°C incubation, regardless in the presence and absence of PH20 variant fragment 2.

[0338] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g. Genbank sequences or GenelD entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference' each and every individual publication, database entry (e.g. Genbank sequences or GenelD entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. To the extent that the references provide a definition for a claimed term that conflicts with the definitions provided in the instant specification, the definitions provided in the instant specification shall be used to interpret the claimed invention.