Anti-PD l1 antibodies formulations

Abstract

Liquid pharmaceutical formulations comprising anti-PD-L1 antibodies for subcutaneous administration are provided.SOLUTION: Provided is a liquid pharmaceutical formulation comprising atezolizumab in a concentration of 100g / L to 150g / L, histidine-acetate in a concentration of 15mM to 25mM, sucrose in a concentration of 200mM to 280mM, polysorbate in a concentration of 0.04% (w / v) to 0.08% (w / v), methionine in a concentration of 5mM to 15mM, hyaluronidase in a concentration of 1000U / ml to / ml, and a pH of 5.6 to 6.0, wherein the liquid pharmaceutical formulation is for subcutaneous administration. 3000U.SELECTED DRAWING: Figure 9B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority benefit of U.S. Provisional Application No. 62 / 945,730, filed December 9, 2019, the entire contents of which are incorporated herein by reference.

[0002] Submitting a sequence listing as an ASCII text file The following submission in an ASCII text file is incorporated herein by reference in its entirety: Sequence Listing in computer readable form (CRF) (Filename: 146392049940SEQLIST.TXT, Recorded: November 22, 2020, Size: 9KB).

[0003] Technical Field The present invention provides liquid pharmaceutical formulations comprising anti-PD-L1 antibodies, such as liquid pharmaceutical formulations for subcutaneous administration. The present invention also provides methods for making such formulations and methods of using the same. [Background technology]

[0004] The pharmaceutical use of antibodies has increased over the past few years. In many cases, such antibodies are injected or infused via the intravenous (IV) route. Unfortunately, the amount of antibody that can be administered via the intravenous route is limited by the physicochemical properties of the antibody, particularly its solubility and stability in a suitable liquid formulation, and by the volume of the infusion. Alternative administration routes are subcutaneous or intramuscular injection. These injection routes require a high protein concentration in the final injected solution (Shire, SJ, Shahrokh, Z. et al., "Challenges in the development of high protein concentration formulations," J. Pharm. Sci. 2004;93(6):1390-1402; Roskos, LK, Davis CGet al., "The clinical pharmacology of therapeutic antibodies," Drug Development Research 2004;61(3):108-120). The use of one or more glycosaminoglycanase enzymes has been proposed to increase the interstitial space into which antibody formulations can be injected in order to increase the volume and thereby the therapeutic dose (WO 2006 / 091871).

[0005] It would be desirable to provide a highly concentrated and stable pharmaceutical formulation of a therapeutically active antibody for subcutaneous injection. The advantage of subcutaneous injection is that it can be administered to patients with short intervention times by a physician. Furthermore, patients can be trained to administer subcutaneous injections themselves. Typically, injections via the subcutaneous route are limited to approximately 2 ml. For patients requiring multiple doses, multiple unit doses of the formulation can be injected at multiple sites on the body surface. Currently, there are no highly concentrated and stable pharmaceutical anti-PD-L1 antibody formulations on the market that are suitable for subcutaneous administration. Therefore, it would be desirable to provide such a highly concentrated and stable pharmaceutical formulation of a therapeutically active antibody for subcutaneous injection. Injection of parenteral drugs into subcutaneous tissue is usually limited to volumes of less than 2 ml due to the viscoelastic resistance to hydraulic conductivity in this subcutaneous (SC) tissue, the backpressure generated during injection (Aukland K. and Reed R., "Interstitial-Lymphatic Mechanisms in the Control of Extracellular Fluid Volume," Physiology Reviews, 1993;73:1-78), and the perception of pain.

[0006] The preparation of highly concentrated protein formulations is extremely difficult, and each formulation must be adapted to the specific protein used, as each protein has a different aggregation behavior. Aggregates are suspected to cause immunogenicity of therapeutic proteins, at least in some cases. An immunogenic response to protein or antibody aggregates can result in neutralizing antibodies that neutralize the therapeutic protein or antibody. The immunogenicity of protein aggregates is believed to be the biggest problem associated with subcutaneous injections, whereby repeated administration increases the risk of an immune response.

[0007] PD-L1 is overexpressed in many cancers and is often associated with poor prognosis (Okazaki T et al., Intern. Iramim. 2007 19(7):813) (Thompson RH et al., Cancer Res 2006, 66(7):3381). Interestingly, the majority of tumor-infiltrating T lymphocytes, in contrast to T lymphocytes in normal tissues and peripheral blood, predominantly express PD-1, suggesting that upregulation of PD-1 on tumor-reactive T cells may contribute to impaired antitumor immune responses (Blood 2009 114(8):1537). This may be due to the exploitation of PD-L1 signaling mediated by PD-L1-expressing tumor cells interacting with PD-1-expressing T cells, resulting in attenuation of T cell activation and evasion of immune surveillance (Sharpe et al., Nat Rev 2002) (Keir ME et al., 2008 Annu. Rev. Immunol. 26:677). Thus, inhibition of PD-L1 / PD-1 interactions can enhance CD8+ T cell-mediated tumor killing.

[0008] The therapeutic targeting of PD-1, as well as other molecules that signal through interactions with PD-1, such as programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2), is an area of ​​considerable interest. Inhibition of PD-L1 signaling has been proposed as a means of enhancing T cell immunity for the treatment of cancer and infectious diseases, including acute and chronic (e.g., persistent) infections. Formulations of anti-PD-L1 antibodies that can be used for intravenous infusion have been disclosed (see U.S. Patent Application Publication No. 2016 / 0319022). However, optimal formulations of anti-PD-L1 antibodies suitable for subcutaneous injection have not yet been developed, leaving a significant unmet medical need.

[0009] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety, as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0010] In one aspect, provided herein is a liquid pharmaceutical formulation comprising a monoclonal anti-PD-L1 antibody at a concentration of about 100 g / L to about 150 g / L, histidine acetate at a concentration of about 15 mM to about 25 mM, sucrose at a concentration of about 200 mM to about 280 mM, polysorbate at a concentration of about 0.04% (w / v) to about 0.08% (w / v), methionine at a concentration of about 5 mM to about 15 mM, and a pH of about 5.6 to about 6.0, wherein the monoclonal antibody is (a) a light chain variable region comprising: (1) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 1); (2) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 2); (3) HVR-L3 containing the amino acid sequence QQYLYHPAT (SEQ ID NO: 3); and (b) a heavy chain variable region comprising: (1) HVR-H1 containing the amino acid sequence GFTFSDSWIH (SEQ ID NO: 4); (2) HVR-H2 containing the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 5); (3) HVR-H3 containing the amino acid sequence WPGGFDY (SEQ ID NO: 6) In some embodiments, the monoclonal antibody in the formulation is at a concentration of about 120 g / L to about 130 g / L. In some embodiments, the monoclonal antibody in the formulation is at a concentration of about 125 g / L. In some embodiments, the histidine acetate is at a concentration of about 17 mM to about 22 mM. In some embodiments, the histidine acetate is at a concentration of about 20 mM. In some embodiments, the sucrose is at a concentration of about 220 mM to about 260 mM. In some embodiments, the sucrose is at a concentration of about 240 mM. In some embodiments, the pH is about 5.8. In some embodiments, the polysorbate in the formulation is polysorbate 20. In some embodiments, the polysorbate is at a concentration of about 0.05% (w / v) to about 0.07% (w / v). In some embodiments, the polysorbate is at a concentration of about 0.06% (w / v). In some embodiments, the methionine is at a concentration of about 10 mM. In some embodiments, the formulation further comprises a hyaluronidase enzyme. In some embodiments, the hyaluronidase enzyme is recombinant human hyaluronidase (rHuPH20). In some embodiments, the hyaluronidase enzyme is at a concentration of about 1000 U / ml to about 3000 U / ml. In some embodiments, the hyaluronidase enzyme is at a concentration of about 2000 U / ml.

[0011] In one aspect, provided herein is a liquid pharmaceutical formulation comprising a monoclonal anti-PD-L1 antibody at a concentration of about 100 g / L to about 150 g / L, histidine acetate at a concentration of about 15 mM to about 25 mM, sucrose at a concentration of about 200 mM to about 280 mM, polysorbate at a concentration of about 0.01% (w / v) to about 0.03% (w / v), and a pH of about 5.3 to about 5.7, wherein the monoclonal antibody is (a) a light chain variable region comprising: (1) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 1); (2) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 2); (3) HVR-L3 containing the amino acid sequence QQYLYHPAT (SEQ ID NO: 3); and (b) a heavy chain variable region comprising: (1) HVR-H1 containing the amino acid sequence GFTFSDSWIH (SEQ ID NO: 4); (2) HVR-H2 containing the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 5); (3) HVR-H3 containing the amino acid sequence WPGGFDY (SEQ ID NO: 6) In some embodiments, the monoclonal antibody in the formulation is at a concentration of about 120 g / L to about 130 g / L. In some embodiments, the monoclonal antibody in the formulation is at a concentration of about 125 g / L. In some embodiments, the histidine acetate is at a concentration of about 17 mM to about 22 mM. In some embodiments, the histidine acetate is at a concentration of about 20 mM. In some embodiments, the sucrose is at a concentration of about 220 mM to about 260 mM. In some embodiments, the sucrose is at a concentration of about 240 mM. In some embodiments, the pH is about 5.5. In some embodiments, the polysorbate in the formulation is polysorbate 20. In some embodiments, the polysorbate is at a concentration of about 0.02% (w / v). In some embodiments, the formulation is mixed with a hyaluronidase enzyme prior to administration to a subject. In some embodiments, the hyaluronidase enzyme is recombinant human hyaluronidase (rHuPH20). In some embodiments, the concentration of the hyaluronidase enzyme in the mixture is about 1000 U / ml to about 3000 U / ml. In some embodiments, the concentration of the hyaluronidase enzyme in the mixture is about 2000 U / ml.

[0012] In some embodiments of the above-mentioned aspects or embodiments described herein, the monoclonal antibody is not subjected to prior lyophilization. In some embodiments of the above-mentioned aspects or embodiments described herein, the monoclonal antibody is a humanized antibody. In some embodiments of the above-mentioned aspects or embodiments described herein, the monoclonal antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8. In some embodiments of the above-mentioned aspects or embodiments described herein, the monoclonal antibody is a full-length antibody. In some embodiments of the above-mentioned aspects or embodiments described herein, the monoclonal antibody is an IgG1 antibody. In some embodiments of the above-mentioned aspects or embodiments described herein, the monoclonal antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:9 and a heavy chain comprising the amino acid sequence of SEQ ID NO:10. In some embodiments of the above-mentioned aspects or embodiments described herein, the monoclonal antibody is stored in a glass vial or a metal alloy container. In some embodiments of the above-mentioned aspects or embodiments described herein, the metal alloy is 316L stainless steel or Hastelloy. In some embodiments of the above aspects or embodiments described herein, the formulation is stable at 2-8°C for at least 6 months. In some embodiments of the above aspects or embodiments described herein, the formulation is stable at 2-8°C for at least 12 months. In some embodiments of the above aspects or embodiments described herein, the formulation is stable at 2-8°C for at least 24 months. In some embodiments of the above aspects or embodiments described herein, the antibody in the formulation retains at least about 80% of its biological activity after storage. In some embodiments of the above aspects or embodiments described herein, the biological activity is measured by antibody binding to PD-L1. In some embodiments of the above aspects or embodiments described herein, the formulation is sterile. In some embodiments of the above aspects or embodiments described herein, the formulation is suitable for administration to a subject.In some embodiments of the above aspects or embodiments described herein, the formulation is for subcutaneous administration.

[0013] Further provided herein is an article of manufacture comprising a container holding the liquid pharmaceutical formulation of any of the above aspects or embodiments. In some embodiments, the container is a glass vial or a metal alloy container. In some embodiments, the metal alloy is 316L stainless steel or Hastelloy.

[0014] Further provided herein is a kit comprising a container holding the liquid pharmaceutical formulation of any of the above aspects or embodiments.

[0015] Further provided herein is a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the liquid pharmaceutical formulation of any of the above aspects or embodiments, wherein the disease or disorder is selected from the group consisting of infectious diseases, cancer, and inflammatory diseases. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, and breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the subject is a human.

[0016] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows. [Brief explanation of the drawings]

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] [Figure 1A] 1 shows the levels of high molecular weight species (HMWS) of various drug substance (DS) formulations after multiple freeze-thaw cycles. [Figure 1B] Figure 1 shows the percentage of the main peak in ion exchange chromatography (IEC) of various drug substance (DS) formulations after multiple freeze-thaw cycles. [Figure 1C] Figure 1 shows the pre-peak sum of non-reducing capillary electrophoresis-SDS (NR CE-SDS) of various drug substance (DS) formulations after multiple freeze-thaw cycles. [Figure 2A] 1 shows the levels of acidic species for various DS formulations after up to one month at 25°C. [Figure 2B] The levels of basic species for various DS formulations are shown after up to one month at 25°C. [Figure 2C] The levels of HMWS for various DS formulations after up to one month at 25°C are shown. [Figure 3A] The HMWS levels of drug product (DP) formulations are shown after up to 3 months at 25°C. [Figure 3B] The percentage of the main peak in the SEC of the drug product (DP) formulation after up to 3 months at 25°C is shown. [Figure 4A] 1 shows the levels of acidic species in DP formulations after up to 3 months at 25°C. [Figure 4B] The levels of basic species in DP formulations are shown after up to 3 months at 25°C. [Figure 5A] Shown is the percentage of pre-peak in DP formulations after up to 3 months at 25°C. [Figure 5B] Shown is the percentage of the main peak of NR CE-SDS in the DP formulation after up to 3 months at 25°C. [Figure 6A] The levels of HMWS in the DP formulations are shown after up to one month at 40°C. [Figure 6B] The percentage of the main peak of SEC in the DP formulations after up to 1 month at 40° C. is shown. [Figure 6C]Figure 1 shows the NR CE-SDS pre-peak sum in DP formulations after up to 1 month at 40°C. [Figure 7A] 1 shows the levels of acidic species in DP formulations after up to 1 month at 40°C. [Figure 7B] 1 shows the levels of basic species in DP formulations after up to 1 month at 40°C. [Figure 7C] The percentage of the main peak of IEC in the DP formulations after up to 1 month at 40° C. is shown. [Figure 8A] 1 shows the stability of polysorbate 20 in various DP formulations at 40° C. [Figure 8B] Demonstrating the stability of polysorbate 20 in various DP formulations at 25°C for up to 3 months. [Figure 9A] rHuPH20 activity assay with various DP formulations at 25°C for up to 3 months. [Figure 9B] rHuPH20 activity assay with various DP formulations for up to 3 months at 25°C. [Figure 10A] 1 shows rHuPH20 activity in formulations containing different concentrations of polysorbate shaken for 24 hours. [Figure 10B] Figure 1 shows rHuPH20 activity in formulations containing different concentrations of polysorbate after 24 hours of shaking. Higher concentrations of polysorbate maintained higher levels of rHuPH20 activity under shaking at room temperature. [Figure 11] Figure 1 shows the viscosity of various DP formulations at temperatures between 5°C and 25°C. DETAILED DESCRIPTION OF THE INVENTION

[0019] I. Definition Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary widely. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "molecule" includes any combination of two or more such molecules, and so forth.

[0020] The term "about" as used herein refers to a normal error range for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to a value or parameter with "about" includes (and describes) embodiments that are directed to that value or parameter itself.

[0021] Aspects and embodiments of the invention described herein are understood to include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0022] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient is in a form such that it is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is one that can be competently administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0023] A "sterile" formulation is sterile or free or essentially free of all viable microorganisms and their spores.

[0024] A "frozen" formulation is a formulation below 0°C. Generally, a frozen formulation is not freeze-dried, and is not lyophilized before or after. In some embodiments, a frozen formulation includes a frozen bulk drug for storage (in a stainless steel tank) or a frozen pharmaceutical product (in its final vial configuration).

[0025] A "stable" formulation is one in which the protein therein essentially retains its physical and / or chemical stability and / or biological activity upon storage. In some embodiments, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Deliver}' Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. Stability can be qualitatively and / or quantitatively assessed in a variety of ways, including assessing aggregate formation (e.g., using size exclusion chromatography, measuring turbidity, and / or visual inspection); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF), or capillary zone electrophoresis; amino- or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis comparing reduced and intact antibodies; peptide map (e.g., trypsin or LYS-C) analysis; assessing antibody biological activity or antigen-binding function, etc. Instability can include one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extensions, C-terminal processing, glycosylation changes, etc.

[0026] A protein "retains its physical stability" in a pharmaceutical formulation if it shows little or no signs of aggregation, precipitation, and / or denaturation upon visual inspection of color and / or clarity, or as measured by UV light scattering or size exclusion chromatography.

[0027] A protein "retains its chemical stability" in a pharmaceutical formulation if the chemical stability at a given time is such that the protein is considered to still retain its biological activity, as defined below. Chemical stability can be assessed by detecting and quantifying chemically modified forms of the protein. Chemical modifications may include size changes (e.g., clipping) and can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical modifications include charge changes (e.g., occurring as a result of deamidation), which can be assessed, for example, by ion exchange chromatography or icIEF.

[0028] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is at least about 60% (within the error of the assay) of the biological activity exhibited at the time the pharmaceutical formulation was prepared, as determined in an assay (e.g., an antigen binding assay). Other "biological activity" assays for antibodies are detailed herein below.

[0029] As used herein, the "biological activity" of a monoclonal antibody includes the ability of the antibody to bind to an antigen and produce a measurable biological response that can be measured in vitro or in vivo.

[0030] As used herein, a "deamidated" monoclonal antibody is one in which one or more asparagine residues thereof has been derivatized, eg, to aspartic acid or iso-aspartic acid.

[0031] As used herein, an "oxidized" monoclonal antibody is one in which one or more tryptophan residues and / or one or more methionines have been oxidized.

[0032] As used herein, a "glycosylated" monoclonal antibody is one in which one or more lysine residues thereof are glycosylated.

[0033] An antibody that is "prone to deamidation" is one that contains one or more residues that have been shown to be prone to deamidation.

[0034] An "oxidation-prone" antibody is one that contains one or more residues that are known to be prone to oxidation.

[0035] An "aggregation-prone" antibody is one that is found to aggregate with one or more other antibody molecules, particularly upon freezing and / or shaking. A "fragmentation-prone" antibody is one that is found to be cleaved into two or more fragments, for example at its hinge region.

[0036] By "reducing deamidation, oxidation, aggregation, or fragmentation" is intended to prevent or reduce the amount of deamidation, oxidation, aggregation, or fragmentation compared to a monoclonal antibody formulated in a different formulation.

[0037] The antibody to be formulated may be essentially pure and, desirably, essentially homogeneous (e.g., free from contaminating proteins, etc.). An "essentially pure" antibody refers to a composition comprising at least about 90% by weight, preferably at least about 95% by weight, of antibody based on the total weight of protein in the composition, and an "essentially homogeneous" antibody refers to a composition comprising at least about 99% by weight of antibody based on the total weight of protein in the composition.

[0038] "Isotonic" means that the formulation has essentially the same osmotic pressure as human blood. Isotonic formulations typically have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured, for example, using a vapor pressure osmometer or a freezing osmometer.

[0039] As used herein, "buffer" refers to a buffered solution that resists changes in pH due to the action of its acid-base conjugate components. In some embodiments, buffers of the present invention have a pH in the range of about 4.5 to about 7.0, preferably about 5.6 to about 7.0, e.g., 5.6 to 6.9, 5.7 to 6.8, 5.8 to 6.7, 5.9 to 6.6, 5.9 to 6.5, 6.0, 6.0 to 6.4, or 6.1 to 6.3. In one embodiment, the buffer has a pH of 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. For example, sodium phosphate is an example of a buffer that controls the pH in this range.

[0040] "Surfactant" as used herein refers to surface active agents, for example, nonionic surfactants.Examples of surfactants herein include polysorbates (for example, polysorbate 20 and polysorbate 80); poloxamers (for example, poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, Myristyl betaine or cetyl betaine; lauramidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearamidopropyl betaine (e.g., lauramidopropyl); myristamidopropyl dimethylamine, palmidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and MONAQUAT TM series (Mona Industries, Inc., Paterson, NJ); polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). In one embodiment, the surfactant herein is polysorbate 20.

[0041] In the pharmacological sense, in the context of the present invention, a "therapeutically effective amount" of an antibody refers to an amount effective to prevent or treat the disorder for which the antibody is effective in treating. A "disorder" is any condition that would benefit from treatment with the antibody. This includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0042] A "preservative" is, for example, a compound that can be optionally included in a formulation to substantially reduce bacterial activity, thus facilitating the production of a multi-purpose formulation. Examples of possible preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. In one embodiment, the preservative herein is benzyl alcohol.

[0043] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cells being treated during clinical pathology. Desirable effects of treatment include slowing the rate of disease progression, amelioration or alleviation of the condition, and remission, or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are alleviated or eliminated, including, but not limited to, reduced proliferation (or destruction) of cancerous cells, a decrease in symptoms caused by the disease, an improvement in the quality of life of those afflicted with the disease, a reduction in the dose of other drugs required to treat the disease, a delay in disease progression, and / or an increase in the individual's survival time.

[0044] As used herein, "delaying disease progression" means to postpone, prevent, slow, retard, stabilize, and / or prolong the onset of a disease (e.g., cancer). Such a delay can be of varying duration depending on the history of the disease and / or the individual being treated. As will be apparent to one skilled in the art, a sufficient or significant delay can effectively encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, can be delayed.

[0045] An "effective amount" is at least the minimum amount necessary to achieve measurable improvement or prevention of a particular disorder. The effective amount herein may vary depending on factors such as the patient's condition, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in an individual. An effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the treatment. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and complications and intermediate pathological phenotypes manifesting during disease development. For therapeutic use, beneficial or desired results include clinical results such as alleviation of one or more symptoms caused by the disease, improving the quality of life of a person suffering from the disease, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, e.g., via targeting, delaying disease progression, and / or prolonging survival. In the case of cancer or tumors, an effective amount of a drug may be effective in reducing the number of cancer cells; shrinking tumor size; inhibiting (i.e., slowing to some extent, and preferably stopping) the infiltration of cancer cells into peripheral organs; inhibiting (i.e., slowing to some extent, and preferably stopping) tumor metastasis, inhibiting to some extent tumor growth, and / or alleviating to some extent one or more symptoms associated with the disorder. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in the clinical field, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of the administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be or is achieved.

[0046] As used herein, "in conjunction with" refers to the administration of one treatment in addition to another therapeutic modality. Thus, "in conjunction with" refers to the administration of one therapeutic modality to an individual before, during, or after the administration of the other therapeutic modality.

[0047] A "disorder" is any condition that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0048] The terms "cell proliferative disorder" and "proliferative disorder" refer to diseases involving some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

[0049] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

[0050] The terms "cancer" and "cancerous" 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, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial cell squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung cancer, including adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer and gastrointestinal stromal cancer. cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic These include, but are not limited to, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (e.g., associated with brain tumors), Meigs' syndrome, brain cancer, and head and neck cancer, and associated metastases. In some embodiments, cancers suitable for treatment with the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid cancer, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma.In some embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer (including metastatic forms of these cancers).

[0051] Chemotherapeutic agents are chemicals useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (Cytoxan®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altrateamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins ( especially bullatacin and bullatacinone; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including synthetic analogs topotecan (Hycamtin®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopoletin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (its analogs dozelesin, carzelesin, and bizelesin synthetic analogs; podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethrin nitrogen mustards such as thiamin, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 11 and calicheamicin omega 11 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicins, including dynemicin A; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (Adriamycin®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), pegylated liposomal doxorubicin (including CAELYX® and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones and 5-fluorouracil (5-FU); combretastatins; folic acid analogs such as ribozymes, ... denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine;Diazicon; Elformitin; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidynin; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerin; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; 2-Ethylhydrazide; Procarbazine; PSK® Polysaccharide Complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ), albumin-engineered nanoparticle formulations of paclitaxel (ABRAXANE; TM) and docetaxel (TAXOTERE®, Rhome-Poulene Rorer, Antony, France); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum drugs such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas that prevent tubulin polymerization to form microtubules, such as vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylmethylnitine (DMFO); retinoids such as retinoic acid (VELBAN®). xarotene (TARGRETIN®); bisphosphonates, including clodronate (e.g., BONEFOS® or OSTAC®), etidronic acid (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R) (e.g., erlotinib (Tarceva®)). TM)); and VEGF-A, which reduces cell proliferation; vaccines, such as the THERATOPE® vaccine and gene therapy vaccines, such as the ALLOVECTIN® vaccine, the LEUVECTIN® vaccine, and the VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., Celecoxib, orafenib, ABT510; BCL-2 inhibitors, such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors; tyrosine kinase inhibitors; serine-threonine kinase inhibitors, such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors, such as lonafarnib (SCH6636, SARASAR®). TM and pharmaceutically acceptable salts, acids, or derivatives of the foregoing, as well as combinations of two or more of the foregoing, such as CHOP (an abbreviation for cyclophosphamide, doxorubicin, vincristine, and prednisolone combination therapy), and FOLFOX (oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN TM ), and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0052] Chemotherapeutic agents, as defined herein, include "anti-hormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block or inhibit the action of hormones that can promote cancer growth. They may themselves be hormones, including, but not limited to, antiestrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON.cndot. toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestany, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in the proliferation of adherent cells, such as PKC-alpha, Raf, and H-Ras; ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; vinorelbine and esperamicin (see U.S. Pat. No. 4,675,187), and pharmaceutically acceptable salts, acids, and derivatives of any of the above; and combinations of two or more of the above.

[0053] As used herein, a "growth inhibitory agent" refers to a compound or composition that inhibits cell growth either in vitro or in vivo. In one embodiment, the growth inhibitory agent is a growth inhibitory antibody that prevents or reduces proliferation of cells expressing the antigen to which the antibody binds. In another embodiment, the growth inhibitory agent can significantly reduce the proportion of cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a location other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. These agents that arrest G1 also cause S-phase arrest, such as DNA alkylating agents, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found, for example, on page 13, in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antitineoplastic drugs" by Murakami et al. (WB Saunders, Philadelphia, 1995). Taxanes (paclitaxel and docetaxel) are anticancer drugs, both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules derived from tubulin dimers, stabilize microtubules by preventing depolymerization, and inhibit mitosis in cells.

[0054] "Radiation therapy" refers to the use of directed gamma or beta radiation to induce sufficient damage to cells to limit their ability to function normally or to destroy them completely. It will be appreciated that there are many methods known in the art for determining dose and duration of treatment. A typical treatment is given as a single administration, with typical doses ranging from 10 to 200 units (Gray) per day.

[0055] A "subject" or "individual" for purposes of treatment refers to any animal classified as a mammal, including humans, and domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, etc. In some embodiments, the mammal is a human.

[0056] The term "antibody" as used herein is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

[0057] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with experimental, diagnostic, or therapeutic uses of the antibody, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95%, and in some embodiments greater than 99%, by weight of the antibody, as determined, for example, by the Lowry assay; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, by using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, for example, using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0058] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains a variable domain (V) at one end. H ) followed by multiple constant domains. Each light chain has a variable domain (V L ) at one end and a constant domain at the other end. The light chain constant domain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0059] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain is the C H 1. C H 2 and C H It contains three domains (collectively called CH) and a light chain CHL (or CL) domain.

[0060] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain is H The variable domain of the light chain is sometimes referred to as "V L These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0061] The term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light-chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration, connected by three HVRs that form loops that connect, and in some cases form part of, the beta-sheet structure. The HVRs within each chain are held together in close proximity by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participating in antibody-dependent cellular toxicity.

[0062] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0063] As used herein, the term "isotype" or "subclass" of IgG means any of the subclasses of immunoglobulins defined by the chemical and antigenic properties of their constant regions.

[0064] Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al. Cellular and Mol. Immunology, 4th ed. (WB Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or noncovalent association of the antibody with one or more other proteins or peptides.

[0065] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody in its substantially intact form, as described below, and not to antibody fragments. These terms specifically refer to antibodies comprising a heavy chain containing an Fc region.

[0066] A "naked antibody" for purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0067] An "antibody fragment" includes a portion of an intact antibody, preferably including its antigen-binding region. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0068] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, named for its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0069] An "Fv" is the minimum antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimeric" structure similar to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0070] Fab fragments contain heavy and light chain variable domains, and also contain the light chain constant domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which one or more cysteine ​​residues in the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0071] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a general review of scFvs, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0072] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are described in further detail, for example, in EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0073] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of distinct antibodies. In some embodiments, such monoclonal antibodies typically comprise an antibody comprising a polypeptide sequence that binds to a target, where the target-binding polypeptide sequence has been obtained by a process that includes selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It is understood that the selected target-binding sequence can be further modified, e.g., to improve affinity for the target, humanize the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and that antibodies comprising modified target-binding sequences are also monoclonal antibodies of the invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0074] 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 used in accordance with the present invention can be produced using, for example, hybridoma techniques (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA techniques (see U.S. Pat. No. 4,816,567), phage display techniques (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 222:581-597 (1992)), and the like. al.,J.Mol.Biol.338(2):299-310(2004);Lee et al.,J.Mol.Biol.340(5):1073-1093(2004);Fellouse,Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004);and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), and techniques for producing human or human-like antibodies in animals having some or all of the immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol.7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and These antibodies can be produced by a variety of techniques, including immunoprecipitation (see Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0075] As used herein, monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies, in which the antigen-binding region of the antibody is derived from an antibody generated, for example, by immunizing macaque monkeys with the antigen of interest.

[0076] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient or donor antibody. These modifications can be made to further refine antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being those of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0077] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques disclosed herein for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The methods described in Cole et al., Monoclonal antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouse, which have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled (e.g., XENOMOUSE TM (See U.S. Patent Nos. 6,075,181 and 6,150,584 for related technology. See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via human B cell hybridoma technology.

[0078] A "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than it has for a homolog of that antigen from a second mammalian species. Typically, a species-dependent antibody "specifically binds" to a human antigen (e.g., about 1 x 10 -7 M or less, approximately 1×10 -8 M or less, or about 1 x 10 -9The species-dependent antibody (having a binding affinity (Kd) value of M or less) has a binding affinity for a homolog of the antigen from a second non-human mammalian species that is at least about 50-fold weaker, or at least about 500-fold weaker, or at least about 1000-fold weaker than the binding affinity for the human antigen. The species-dependent antibody can be any of the various types of antibodies defined above, but may also be a humanized antibody or a human antibody.

[0079] As used herein, the terms "hypervariable region," "HVR," or "HV" refer to the region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Typically, antibodies contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). In natural antibodies, H3 and L3 exhibit the highest diversity among the six HVRs, and H3 in particular is thought to play a unique role in conferring superior specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0080] Several HVR delineations are used and encompassed herein. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are listed below.

[0081] TIFF2026009902000002.tif90170

[0082] HVRs may include the following "extended HVRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) of the VL, and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) of the VH. The variable domain residues are numbered according to Kabat et al. (see above) for each of these definitions.

[0083] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0084] The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and may contain inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues may be determined for a given antibody by aligning the antibody's sequence with the "standard" Kabat numbered sequence at the regions of homology.

[0085] The Kabat numbering system is generally used when referring to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in immunoglobulin heavy chain constant regions (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the numbering of residues in a human IgG1 EU antibody.

[0086] The term "linear antibody" refers to the antibodies described in Zapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0087] As used herein, the terms "specifically binds to" or "specific for" refer to a measurable and reproducible interaction, e.g., binding between a target and an antibody, that is determinative of the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for a longer duration than it binds to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In some embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In other embodiments, specific binding can include, but does not require, exclusive binding.

[0088] II. Antibody Formulation and Preparation In some embodiments, provided herein is a liquid pharmaceutical formulation comprising an anti-PD-L1 antibody described herein, e.g., a liquid pharmaceutical formulation for subcutaneous administration. In some embodiments, the formulation comprises an anti-PD-L1 antibody (e.g., a monoclonal antibody), sucrose, a buffer, and a surfactant, and the pH of the formulation is about 5.0 to about 6.5. In some embodiments, the formulation further comprises methionine. In some embodiments, the anti-PD-L1 antibody described herein in the formulation is at a concentration of about 100 g / L to about 150 g / L. In some embodiments, the buffer is histidine (e.g., histidine acetate). In some embodiments, the buffer in the formulation is at a concentration of about 15 mM to about 25 mM. In some embodiments, the sucrose in the formulation is about 200 mM to about 280 mM. In some embodiments, the surfactant in the formulation is polysorbate (e.g., polysorbate 20). In some embodiments, the polysorbate in the formulation is at a concentration of about 0.005% (w / v) to about 0.08% (w / v). In some embodiments, the formulation comprises methionine at a concentration of about 5 mM to about 15 mM. In some embodiments, the formulation has a pH of about 5.0 to about 6.3. In some embodiments, provided herein is a liquid pharmaceutical formulation comprising an anti-PD-L1 antibody described herein at a concentration of about 100 g / L to about 150 g / L, histidine acetate at a concentration of about 15 mM to about 25 mM, sucrose at a concentration of about 200 mM to about 280 mM, polysorbate at a concentration of about 0.04% (w / v) to about 0.08% (w / v), methionine at a concentration of about 5 mM to about 15 mM, and a pH of about 5.6 to about 6.0. In some embodiments, the formulation further comprises a hyaluronidase enzyme (e.g., recombinant human hyaluronidase (rHuPh20)). In some embodiments, the formulation comprises a hyaluronidase enzyme (e.g., rHuPh20) at a concentration of about 1000 U / ml to about 3000 U / ml. In some embodiments, the formulation is sterile. In some embodiments, the formulation is suitable for administration to a subject. In some embodiments, the formulation is for subcutaneous administration.

[0089] In some embodiments, provided herein is a liquid pharmaceutical formulation comprising an anti-PD-L1 antibody described herein at a concentration of about 100 g / L to about 150 g / L, histidine acetate at a concentration of about 15 mM to about 25 mM, sucrose at a concentration of about 200 mM to about 280 mM, polysorbate at a concentration of about 0.01% (w / v) to about 0.03% (w / v), and a pH of about 5.3 to about 5.7. In some embodiments, the formulation is sterile. In some embodiments, the formulation is suitable for administration to a subject. In some embodiments, the formulation is for subcutaneous administration.

[0090] In some embodiments, the antibody in the formulation is stable for at least about 6 months, at least about 12 months, at least about 18 months, at least 2 years, at least 3 years, or at least 4 years at −20° C. In some embodiments, the antibody in the formulation is stable for at least about 6 months, at least about 12 months, at least about 18 months, at least 2 years, or at least 3 years at 2-8° C. In some embodiments, after storage, the antibody retains at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of its biological activity (e.g., target binding or therapeutic efficacy) exhibited prior to storage, i.e., at the time the pharmaceutical formulation was prepared.

[0091] In some embodiments, the formulation is stable for at least about 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, or more days at about 40° C. In some embodiments, the formulation is stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, or more weeks at about 40° C. In some embodiments, the formulation is stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months at about 25° C. In some embodiments, the formulations are stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months at about 5° C. In some embodiments, the formulations are stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months at about −20° C. In some embodiments, the formulation is stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months at 5° C. or −20° C. Further, in some embodiments, the formulation is stable after freezing (e.g., to −20° C., −40° C., or −70° C.) and thawing the formulation, for example, 1, 2, 3, 4, or 5 cycles of freezing and thawing.

[0092] A. Anti-PD-L1 antibody In some embodiments, the antibody in the formulation is an anti-PD-L1 antibody. PD-L1 (programmed cell death 1 ligand 1), also known as PDL1, B7-H1, B7-4, CD274, and B7-H, is a transmembrane protein whose interaction with PD-1 inhibits T cell activation and cytokine production. In some embodiments, the anti-PD-L1 antibodies described herein bind to human PD-L1. Examples of anti-PDL1 antibodies that can be formulated using the formulations described herein are described in PCT Patent Application Publication No. WO 2010 / 077634, U.S. Pat. No. 8,217,149, and U.S. Pat. Application Publication No. 2016 / 0319022, which are incorporated herein by reference.

[0093] In some embodiments, the anti-PD-L1 antibody can inhibit the binding between PD-L1 and PD-1 and / or the binding between PD-L1 and B7-1. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments, the anti-PD-L1 antibody is a full-length antibody. In some embodiments, the anti-PD-L1 antibody is a humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody.

[0094] The anti-PD-L1 antibodies described in WO 2010 / 077634, U.S. Pat. No. 8,217,149, and U.S. Patent Application Publication No. 2016 / 0319022 may be formulated in the formulations described herein.

[0095] In some embodiments, the anti-PD-L1 antibodies in the formulations described herein are: (a) a light chain variable region comprising: (1) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 1); (2) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 2); (3) HVR-L3 containing the amino acid sequence QQYLYHPAT (SEQ ID NO: 3); and (b) a heavy chain variable region comprising: (1) HVR-H1 containing the amino acid sequence GFTFSDSWIH (SEQ ID NO: 4); (2) HVR-H2 containing the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 5); (3) HVR-H3 containing the amino acid sequence WPGGFDY (SEQ ID NO: 6) Includes:

[0096] In a further embodiment, the anti-PD-L1 antibody in the formulation described herein comprises a heavy chain and a light chain sequence, wherein: (a) the heavy chain variable region sequence has at least 85% sequence identity to the heavy chain variable region sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 8); or (b) the light chain variable region sequence has at least 85% sequence identity to the light chain variable region sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK (SEQ ID NO: 7).

[0097] In some embodiments, the monoclonal antibody in the formulation comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the monoclonal antibody in the formulation comprises a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a light chain variable region having the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8.

[0098] In a still further specific aspect, the antibody further comprises a human or mouse constant region. In a further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In a further particular aspect, the human constant region is IgG1. In a further aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In a further aspect, the mouse constant region is IgG2A. In a further particular aspect, the antibody has reduced or minimal effector function. In a further particular aspect, the minimal effector function is due to an "effector-less Fc mutation" or aglycosylation. In a further embodiment, the effector-less Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0099] In a further embodiment, the anti-PD-L1 antibody in the formulation described herein comprises a heavy chain and a light chain sequence, wherein: (a) The heavy chain sequence is: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP has at least 85% sequence identity to ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 10); or (b) The light chain sequence has at least 85% sequence identity to the light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9).

[0100] In some embodiments, provided is an isolated anti-PD-L1 antibody comprising heavy and light chain sequences, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, provided is an isolated anti-PD-L1 antibody comprising heavy and light chain sequences, wherein the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, provided is an isolated anti-PD-L1 antibody comprising heavy and light chain sequences, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:9, and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:10.

[0101] In some embodiments, provided is an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO:9, and the heavy chain comprises the amino acid sequence of SEQ ID NO:10.

[0102] In some embodiments, the anti-PD-L1 antibodies in the formulations described herein are: (a) a light chain variable region comprising: (1) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 1); (2) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 2); (3) HVR-L3 containing the amino acid sequence QQYLYHPAT (SEQ ID NO: 3); and (b) a heavy chain variable region comprising: (1) HVR-H1 containing the amino acid sequence GFTFSDSWIH (SEQ ID NO: 4); (2) HVR-H2 containing the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 5); (3) HVR-H3 containing the amino acid sequence WPGGFDY (SEQ ID NO: 6) In some embodiments, the anti-PD-L1 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the monoclonal antibody in the formulation comprises a light chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a light chain variable region having the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a heavy chain variable region having the amino acid sequence of SEQ ID NO:8.

[0103] In some embodiments, the anti-PD-L1 antibodies in the formulations described herein are: (a) a light chain variable region comprising: (1) HVR-L1 containing the amino acid sequence RASQDVSTAVA (SEQ ID NO: 1); (2) HVR-L2 containing the amino acid sequence SASFLYS (SEQ ID NO: 2); (3) HVR-L3 containing the amino acid sequence QQYLYHPAT (SEQ ID NO: 3); and (b) a heavy chain variable region comprising: (1) HVR-H1 containing the amino acid sequence GFTFSDSWIH (SEQ ID NO: 4); (2) HVR-H2 containing the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 5); (3) HVR-H3 containing the amino acid sequence WPGGFDY (SEQ ID NO: 6) In some embodiments, the anti-PD-L1 antibody comprises heavy and light chain sequences, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, provided is an isolated anti-PD-L1 antibody comprising heavy and light chain sequences, wherein the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, provided is an isolated anti-PD-L1 antibody comprising heavy and light chain sequences, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:9, and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:10.

[0104] In some embodiments, the isolated anti-PD-L1 antibody is an oxidized monoclonal antibody. In some embodiments, the oxidized monoclonal antibody in the formulation comprises a light chain comprising the amino acid sequence of SEQ ID NO:9 and a heavy chain comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the oxidized monoclonal antibody in the formulation comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:10, wherein one or more of W33, W50, or W101 are oxidized. In some embodiments, the oxidized monoclonal antibody in the formulation comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:10, wherein one or more of M253 and M429 are oxidized. In some embodiments, the oxidized monoclonal antibody retains at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of its biological activity (e.g., target binding or therapeutic efficacy) exhibited prior to storage, i.e., at the time the pharmaceutical formulation was prepared.

[0105] In some embodiments, the isolated anti-PD-L1 antibody is a glycosylated monoclonal antibody. In some embodiments, the glycosylated monoclonal antibody in the formulation comprises a light chain comprising the amino acid sequence of SEQ ID NO: 9 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the glycosylated monoclonal antibody in the formulation comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10, wherein one or more of the lysines are glycosylated. In some embodiments, the glycosylated monoclonal antibody in the formulation comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10, wherein K65 is glycosylated.

[0106] In some embodiments, the isolated anti-PD-L1 antibody is aglycosylated.

[0107] In some embodiments, the anti-PD-L1 antibody is atezolizumab (TECENTRIQ®).

[0108] In any of the embodiments herein, the isolated anti-PDL1 antibody can bind to human PDL1, for example, human PD-L1 set forth in UniProtKB / Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof.

[0109] In still further embodiments, provided is an isolated nucleic acid encoding any of the antibodies described herein. In some embodiments, the nucleic acid further comprises a vector suitable for expressing the nucleic acid encoding any of the aforementioned anti-PD-L1 antibodies. In still further specific aspects, the vector is in a host cell suitable for expressing the nucleic acid. In even more specific aspects, the host cell is a eukaryotic or prokaryotic cell. In even more specific aspects, the eukaryotic cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

[0110] Antibodies or antigen-binding fragments thereof can be produced using methods known in the art, such as by methods comprising culturing a host cell containing nucleic acid encoding any of the aforementioned anti-PD-L1 antibodies or antigen-binding fragments in a form suitable for expression under conditions suitable for producing such antibody or fragment, and recovering the antibody or fragment.

[0111] B. Antibody Preparation In general, various methodologies for preparing antibodies for research, testing, and clinical use are well established in the art. The antibodies in the formulations are prepared using techniques available in the art for producing antibodies, exemplary methods of which are described in WO 2010 / 077634, U.S. Pat. No. 8,217,149, and U.S. Patent Application Publication No. 2016 / 0319022.

[0112] C. Biologically active antibodies Antibodies generated as described above can be subjected to one or more "biological activity" assays to select antibodies with properties beneficial from a therapeutic standpoint, or to select formulations and conditions that retain the biological activity of the antibody. The antibody can be tested for its ability to bind to the intended antigen. For example, in the case of an anti-PD-L1 antibody, the antigen-binding properties of the antibody can be assessed in an assay that detects its ability to bind to PD-L1. In some embodiments, the binding properties of the antibody can be determined, for example, by saturation binding, ELISA, and / or competitive assays (e.g., RIA). The antibody can also be subjected to other biological activity assays, for example, to evaluate its effectiveness as a therapeutic agent. Such assays are known in the art and depend on the antibody's target antigen and intended use. For example, the biological effect of antibody-mediated PD-L1 blockade can be assessed using CD8+ T cells, a lymphocytic choriomeningitis virus (LCMV) mouse model, and / or a syngeneic tumor model, such as those described in U.S. Pat. No. 8,217,149.

[0113] To screen for antibodies that bind to a specific epitope on an antigen of interest (e.g., antibodies that block the binding of an exemplary anti-PDL1 antibody to PD-L1), a conventional cross-blocking assay, such as that described in *Antibodies, A Laboratory Manual*, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping, such as that described in *Champe et al., J. Biol. Chem. 270:1388-1394 (1995), can be performed to determine whether an antibody binds to the epitope of interest.

[0114] D. Preparation of Formulations After preparing the antibody of interest (e.g., techniques for producing antibodies that can be formulated as disclosed herein are described in detail below and are known in the art), a pharmaceutical formulation containing it is prepared. In some embodiments, the formulated antibody has not been subjected to prior lyophilization, and the formulations of interest herein are aqueous formulations. In some embodiments, the formulations are for subcutaneous administration. In some embodiments, the antibody is a full-length antibody. In one embodiment, the antibody in the formulation is an antibody fragment such as F(ab')2, which may require addressing issues not encountered with full-length antibodies (e.g., clipping of the antibody to Fab). The therapeutically effective amount of antibody present in the formulation is determined, for example, by considering the desired dosage and one or more modes of administration. About 100 g / L to about 150 g / L, or about 110 g / L to about 140 g / L, or about 120 g / L to about 130 g / L are exemplary concentrations of the antibodies described herein in the formulation. In some embodiments, the antibody in the formulation is at a concentration of about 100 g / L to about 150 g / L. In some embodiments, the antibody in the formulation is at a concentration of about 110 g / L to about 140 g / L. In some embodiments, the antibody in the formulation is at a concentration of about 120 g / L to about 130 g / L. In some embodiments, the antibody in the formulation is at a concentration of about 100 g / L. In some embodiments, the antibody in the formulation is at a concentration of about 105 g / L. In some embodiments, the antibody in the formulation is at a concentration of about 110 g / L. In some embodiments, the antibody in the formulation is at a concentration of about 115 g / L. In some embodiments, the antibody in the formulation is at a concentration of about 120 g / L. In some embodiments, the antibody in the formulation is at a concentration of about 125 g / L. In some embodiments, the antibody in the formulation is at a concentration of about 130 g / L. In some embodiments, the antibody in the formulation is at a concentration of about 135 g / L. In some embodiments, the antibody in the formulation is at a concentration of about 140 g / L, hi some embodiments, the antibody in the formulation is at a concentration of about 145 g / L, hi some embodiments, the antibody in the formulation is at a concentration of about 150 g / L.Exemplary concentrations of the antibodies described herein in the formulation are 100 g / L to 150 g / L, or 110 g / L to 140 g / L, or 120 g / L to 130 g / L. In some embodiments, the antibody in the formulation is at a concentration of 100 g / L to 150 g / L. In some embodiments, the antibody in the formulation is at a concentration of 110 g / L to 140 g / L. In some embodiments, the antibody in the formulation is at a concentration of 120 g / L to 130 g / L. In some embodiments, the antibody in the formulation is at a concentration of 100 g / L. In some embodiments, the antibody in the formulation is at a concentration of 105 g / L. In some embodiments, the antibody in the formulation is at a concentration of 110 g / L. In some embodiments, the antibody in the formulation is at a concentration of 115 g / L. In some embodiments, the antibody in the formulation is at a concentration of 120 g / L. In some embodiments, the antibody in the formulation is at a concentration of 125 g / L. In some embodiments, the antibody in the formulation is at a concentration of 130 g / L. In some embodiments, the antibody in the formulation is at a concentration of 135 g / L. In some embodiments, the antibody in the formulation is at a concentration of 140 g / L. In some embodiments, the antibody in the formulation is at a concentration of 145 g / L. In some embodiments, the antibody in the formulation is at a concentration of 150 g / L.

[0115] Liquid pharmaceutical formulations are prepared comprising antibodies in a pH buffered solution. The buffers of the present invention have a pH ranging from about 5.0 to about 6.5. In some embodiments, the pH ranges from about 5.3 to about 6.0, from about 5.6 to about 6.0, from about 5.7 to about 5.9, from about 5.3 to about 5.7, from about 5.4 to about 5.6, from about 5.5 to about 5.8, from about 5.0 to about 6.0, from about 5.1 to about 5.8, from about 5.2 to about 5.8, from about 5.3 to about 5.8, or from about 5.4 to about 5.8. In some embodiments of the present invention, the formulation has a pH of 5.2 or about 5.2. In some embodiments of the present invention, the formulation has a pH of 5.3 or about 5.3. In some embodiments of the present invention, the formulation has a pH of 5.4 or about 5.4. In some embodiments of the invention, the formulation has a pH of 5.5 or about 5.5. In some embodiments of the invention, the formulation has a pH of 5.6 or about 5.6. In some embodiments of the invention, the formulation has a pH of 5.7 or about 5.7. In some embodiments of the invention, the formulation has a pH of 5.8 or about 5.8. In some embodiments of the invention, the formulation has a pH of 5.9 or about 5.9. In some embodiments of the invention, the formulation has a pH of 6.0 or about 6.0. Examples of buffers that control the pH within this range include histidine (e.g., L-histidine) or sodium acetate. In some embodiments, the buffer contains histidine acetate or sodium acetate at a concentration of about 15 mM to about 25 mM. In some embodiments, the buffer contains histidine acetate or sodium acetate at a concentration of about 15 mM to about 25 mM, about 16 mM to about 25 mM, about 17 mM to about 25 mM, about 18 mM to about 25 mM, about 19 mM to about 25 mM, about 20 mM to about 25 mM, about 21 mM to about 25 mM, about 22 mM to about 25 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM. In some embodiments, the buffer contains histidine acetate at a concentration of about 15 mM to about 25 mM.In some embodiments of the invention, the buffer contains histidine acetate or sodium acetate at a concentration of about 15 mM to about 25 mM, about 16 mM to about 25 mM, about 17 mM to about 25 mM, about 18 mM to about 25 mM, about 19 mM to about 25 mM, about 20 mM to about 25 mM, about 21 mM to about 25 mM, about 22 mM to about 25 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM. In some embodiments of the invention, the buffer contains histidine acetate at a concentration of about 20 mM. In one embodiment, the buffer is histidine acetate in an amount of about 20 mM at pH 5.0. In one embodiment, the buffer is histidine acetate at pH 5.1 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.2 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.3 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.4 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.5 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.6 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.7 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.8 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.9 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.0 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.1 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.2 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.3 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.4 in an amount of about 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.5 in an amount of about 20 mM.In one embodiment, the buffer is histidine acetate at pH 5.0 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.1 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.2 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.3 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.4 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.5 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.6 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.7 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.8 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 5.9 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.0 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.1 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.2 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.3 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.4 in an amount of 20 mM. In one embodiment, the buffer is histidine acetate at pH 6.5 in an amount of 20 mM.

[0116] The formulation further comprises sucrose in an amount of about 200 mM to about 280 mM. In some embodiments, the sucrose in the formulation is about 210 mM to about 280 mM, about 220 mM to about 280 mM, about 230 mM to about 280 mM, about 240 mM to about 280 mM, about 200 mM to about 270 mM, about 200 mM to about 260 mM, about 200 mM to about 240 mM, about 210 mM to about 270 mM, about 220 mM to about 260 mM, about 230 mM to about 250 mM, or about 235 mM to about 245 mM. In some embodiments, the sucrose in the formulation is about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 235 mM, about 240 mM, about 245 mM, about 250 mM, about 260 mM, about 270 mM, or about 280 mM. In some embodiments, the sucrose in the formulation is about 240 mM. The formulation further comprises sucrose in an amount from 200 mM to 280 mM. In some embodiments, the sucrose in the formulation is 210 mM to 280 mM, 220 mM to 280 mM, 230 mM to 280 mM, 240 mM to 280 mM, 200 mM to 270 mM, 200 mM to 260 mM, 200 mM to 240 mM, 210 mM to 270 mM, 220 mM to 260 mM, 230 mM to 250 mM, or 235 mM to 245 mM. In some embodiments, the sucrose in the formulation is 200 mM, 210 mM, 220 mM, 230 mM, about 235 mM, 240 mM, 245 mM, 250 mM, 260 mM, 270 mM, or 280 mM. In some embodiments, the sucrose in the formulation is 240 mM.

[0117] In some embodiments, a surfactant is added to the antibody formulation. Exemplary surfactants include non-ionic surfactants such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188, etc.). The amount of surfactant added is such that the surfactant reduces aggregation of the formulated antibody and / or minimizes particle formation in the formulation and / or reduces adsorption. For example, the surfactant may be present in the formulation in an amount of about 0.005% (w / v) to about 0.08% (w / v). In some embodiments, the surfactant (e.g., polysorbate 20) is present in an amount of about 0.005% to about 0.07%, about 0.005% to about 0.065%, about 0.005% to about 0.06%, about 0.01% to about 0.08%, about 0.015% to about 0.08%, about 0.02% to about 0.08%, about 0.01% to about 0.03%, about 0.01% to about 0.025%, about 0.01% to about 0.02%, about 0.015% to about 0.03%, about 0.02% to about In some embodiments, the surfactant (e.g., polysorbate 20) is about 0.02% (w / v). In some embodiments, the surfactant (e.g., polysorbate 20) is about 0.06% (w / v). In some embodiments, the surfactant (e.g., polysorbate 20) is 0.02% (w / v). In some embodiments, the surfactant (e.g., polysorbate 20) is 0.06% (w / v). In some embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.01% or about 0.01%. In some embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.015% or about 0.015%. In some embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.02% or about 0.02%.In some embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.025% or about 0.025%. In some embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.03% or about 0.03%. In some embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.05% or about 0.055%. In some embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.055% or about 0.055%. In some embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.06% or about 0.06%. In some embodiments, the surfactant (e.g., polysorbate 20) is present in the formulation in an amount of 0.065% or about 0.065%. In some embodiments, the surfactant (eg, polysorbate 20) is present in the formulation in an amount at or about 0.07%.

[0118] In some embodiments, methionine is added to the antibody formulation. In some embodiments, the methionine in the formulation is about 1 mM to about 20 mM, about 5 mM to about 15 mM, about 6 mM to about 14 mM, about 7 mM to about 13 mM, about 8 mM to about 12 mM, about 9 mM to about 11 mM, about 8 mM to about 13 mM, about 8 mM to about 11 mM, about 8 mM to about 10 mM, about 9 mM to about 13 mM, about 9 mM to about 12 mM, or about 9 mM to about 10 mM. In some embodiments, the methionine in the formulation is 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, or about 15 mM. In certain embodiments, the methionine in the formulation is about 10 mM. In some embodiments, the methionine in the formulation is 1 mM to 20 mM, 5 mM to 15 mM, 6 mM to 14 mM, 7 mM to 13 mM, 8 mM to 12 mM, 9 mM to 11 mM, 8 mM to 13 mM, 8 mM to 11 mM, 8 mM to 10 mM, 9 mM to 13 mM, 9 mM to 12 mM, or 9 mM to 10 mM. In some embodiments, the methionine in the formulation is 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM. In certain embodiments, the methionine in the formulation is 10 mM.

[0119] In some embodiments, a hyaluronan-degrading enzyme (i.e., hyaluronidase) or hyaluronan synthesis inhibitor is added to, mixed with, or co-administered with the antibody formulations described herein prior to administration. Hyaluronan (hyaluronic acid; HA) is a glycosaminoglycan found primarily in mammalian connective tissue, skin, cartilage, and synovial fluid. In connective tissue, the water of hydration associated with hyaluronan creates a hydrated matrix between tissues. HA is found in the extracellular matrix of many cells, particularly in soft connective tissue. Hyaluronidase is an enzyme that degrades hyaluronan.

[0120] Glycosaminoglycans (GAGs) are complex linear polysaccharides of the extracellular matrix (ECM). GAGs are characterized by repeating disaccharide units of N-substituted hexosamine and uronic acid (in the case of hyaluronan (HA), chondroitin sulfate (CS), chondroitin (C), dermatan sulfate (DS), heparan sulfate (HS), and heparin (H)), or galactose (in the case of keratan sulfate (KS)). With the exception of HA, all GAGs exist covalently bound to core proteins. Structurally, GAGs and their core proteins are referred to as proteoglycans (PGs).

[0121] HA is found in the extracellular matrix of many cells, particularly in soft connective tissues. Various physiological functions have been assigned to HA, for example, in water and plasma protein homeostasis (Laurent TC et al., FASEB J., 1992;6:2397-2404). HA production increases in proliferating cells and appears to be involved in mitosis. HA production also appears to be involved in locomotion and cell migration. HA is thought to play an important role in cell regulation, development, and differentiation (Laurent et al., supra). HA has been widely used in clinical medicine. Its tissue protective and rheological properties have proven useful in ophthalmic surgery (e.g., to protect the corneal endothelium during cataract surgery). Hyaluronan protein interactions also participate in the structure of the extracellular matrix or "stroma."

[0122] Hyaluronidases are a group of generally neutral or acid-active enzymes found throughout the animal kingdom. Hyaluronidases differ with respect to substrate specificity and mechanism of action (WO 2004 / 078140). There are three general classes of hyaluronidases: 1. Mammalian hyaluronidase (EC 3.2.1.35), an endo-beta-N-acetylhexosaminidase with tetra- and hexasaccharides as its main end products, has both hydrolytic and transglycosidase activity and can degrade hyaluronan and chondroitin sulfate (CS), typically C4-S and C6-S. 2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan to various degrees into CS and DS. They are endo-beta-N-acetylhexosaminidases that act primarily by beta-elimination to generate disaccharide end products. 3. Hyaluronidases (EC 3.2.1.36) from leeches, other parasites, and crustaceans are endo-beta-glucuronidases that generate tetra- and hexasaccharide end products through hydrolysis of beta 1-3 bonds.

[0123] Mammalian hyaluronidases can be further divided into two groups: neutral-active enzymes and acid-active enzymes. There are six hyaluronidase-like genes in the human genome: HYAL1, HYAL2, HYAL3, HYAL4, HYALP1, and PH20 / SPAM1. HYALP1 is a pseudogene, and HYAL3 has been shown to retain enzymatic activity toward some known substrates. HYAL4 is a chondroitinase and exhibits little activity toward hyaluronan. HYAL1 is the prototypical acid-active enzyme, and PH20 is the prototypical neutral-active enzyme. Acid-active hyaluronidases, such as HYAL1 and HYAL2, generally lack catalytic activity at neutral pH (i.e., pH 7). For example, HYAL1 has little catalytic activity in vitro above pH 4.5 (Frost, IG, and Stern, R., "A microtiter-based assay for hyaluronidase activity not requiring specialized reagents," Anal. Biochemistry, 1997;251:263-269). HYAL2 is an acid-active enzyme with extremely low specific activity in vitro.

[0124] Hyaluronidase-like enzymes are also characterized by those that are usually locked to the plasma membrane via a glycosylphosphatidylinositol anchor, such as human HYAL2 and human PH20 (Danilkovitch-Miagkova et al., Proc. Natl. Acad. Sci. USA, 2003;100(8):4580-4585; Phelps et al., Science 1988;240(4860):1780-1782), and those that are usually soluble, such as human HYAL1 (Frost, IGet al., "Purification, cloning, and expression of human plasma hyaluronidase," Biochem. Biophys. Res. Commun. 1997;236(1):10-15). However, there is variability between species: for example, bovine PH20 is very loosely bound to the plasma membrane and is not fixed via a phospholipase-sensitive anchor (Lalancette et al, Biol Reprod., 2001;65(2):628-36). These unique properties of bovine hyaluronidase have led to the development of a soluble bovine testicular hyaluronidase enzyme extract for clinical use (Wydase TM , Hyalase TM ) enabled the synthesis of PH20. Other PH20 species are lipid-anchored enzymes that are typically insoluble without the use of detergents or lipases. For example, human PH20 is anchored to the plasma membrane via a GPI anchor. Attempts to create human PH20 DNA constructs that would not incorporate a lipid anchor into the polypeptide have resulted in catalytically inactive or insoluble enzymes (Arming et al., Eur. J. Biochem., 1997;1;247(3):810-4). Naturally occurring macaque sperm hyaluronidase is found in both soluble and membrane-bound forms. The 64 kDa membrane-bound form retains enzymatic activity at pH 7.0, whereas the 54 kDa form is active only at pH 4.0 (Cherr et al., Dev. Biol., 1996;10;175(1):142-53). Thus, soluble forms of PH20 often lack enzymatic activity under neutral conditions.

[0125] In accordance with the teachings of WO 2006 / 091871 and U.S. Patent No. 7,767,429, small amounts of soluble hyaluronidase glycoprotein (sHASEGP) can be incorporated into formulations to facilitate the administration of therapeutic agents to subcutaneous tissue. By rapidly depolymerizing HA in the extracellular space, sHASEGP reduces interstitial viscosity, thereby increasing hydraulic conductivity and allowing larger volumes to be safely and comfortably administered into tissues. The increased hydraulic conductivity induced by sHASEGP through reduced interstitial viscosity allows for greater dispersion and increased systemic bioavailability of SC-administered therapeutic agents.

[0126] When injected into the subcutaneous tissue, hyaluronan depolymerization by sHASEGP is localized to the injection site in the SC tissue. Experimental evidence indicates that sHASEGP is locally inactivated in the interstitial space with a half-life of 13 to 20 minutes in mice, and no systemic absorption into the blood is detected after a single intravenous administration to CD-I mice. Within the vascular compartment, sHASEGP demonstrates half-lives of 2.3 and 5 minutes in mice and cynomolgus monkeys, respectively, at doses up to 0.5 mg / kg. The rapid clearance of sHASEGP, combined with the continuous synthesis of hyaluronidase substrates within the SC tissue, results in a transient, locally active penetration enhancement with respect to other co-injected molecules, an effect that is fully reversible within 24 to 48 hours after administration (Bywaters GL, et al., "Reconstitution of the dermal barrier to dye spread after hyaluronidase injection," Br. Med. J., 1951;2(4741):1178-1183).

[0127] In addition to its effect on local dispersion, sHASEGP also acts as an absorption enhancer. Macromolecules larger than 16 kilodaltons (kDa) are largely excluded from absorption through capillaries via diffusion, with most absorption occurring through the draining lymph nodes. Thus, subcutaneously administered macromolecules, such as therapeutic antibodies (molecular weight approximately 150 kDa), must traverse the interstitial matrix before reaching the draining lymphatics for subsequent absorption into the vascular compartment. By increasing local dispersion, sHASEGP increases the rate of absorption (Ka) of many macromolecules. This results in a significantly higher peak blood concentration (C) compared to SC administration in the absence of sHASEGP. max ) and in some cases increased bioavailability (Bookbinder LH, et al., “A recombinant human enzyme for enhanced interstitial transport of therapeutics”, J. Control. Release 2006;114:230-241).

[0128] Hyaluronidase products of animal origin have been used clinically for over 60 years, primarily to enhance the dispersion and absorption of other co-administered drugs and for subcutaneous infusion (SC injection / infusion of large volumes of fluids) (Frost GI, "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery, 2007;4:427-440). The mechanism of action of hyaluronidase is described in detail in the following publications: Duran-Reynolds F., "A spreading factor in certain snake venoms and its relationship to their mode of action," CR Soc Biol Paris, 1938;69-81; Chain E., "A mucolytic enzyme in testes extracts," Nature 1939;977-978; Weissmann B., "The transglycosylative action of testicular hyaluronidase," J. Biol. Chem., 1955;216:783-94; Tammi, R., Saamanen, AM, Maibach, HI, Tammi M., "Degradation of newly synthesized high molecular mass hyaluronan in the epidermal and dermal compartments of human skin in organs." culture”,J.Invest.Dermatol.1991;97:126-130;Laurent,UBG,Dahl,LB,Reed,RK,”Catabolism of hyaluronan in rabbit skin takes place locally,in lymph nodes and liver”,Exp.Physiol.1991;76:695-703;Laurent,TCand Fraser,JRE"Degradation of Bioactive Substances: Physiology and Pathophysiology," Henriksen, JH (Ed) CRC Press, Boca Raton, FL; 1991. pp. 249-265; Harris, EN, et al., "Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE)," J. Biol. Chem. 2004; 279:36201-36209; Frost, GI, "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration," Expert Opinion on Drug Delivery, 2007; 4:427-440. Hyaluronidase products approved in EU member states include Hylase® "Dessau" and Hyalase®. Hyaluronidase products of animal origin approved in the United States include Vitrase. TM , Hydase TM , and Amphadase TM Includes:

[0129] The safety and efficacy of hyaluronidase products have been widely established. The most significant safety risk identified is hypersensitivity and / or allergenicity, which is thought to be related to the lack of purity of animal-derived preparations (Frost, GI, "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration," Expert Opinion on Drug Delivery, 2007;4:427-440). It should be noted that there are differences in the approved dosages of animal-derived hyaluronidase between the UK, Germany, and the US. In the UK, the usual dose as an adjuvant for subcutaneous or intramuscular injection is 1500 units, added directly to the injection. In the US, the usual dose used for this purpose is 150 units. For subcutaneous injection, hyaluronidase is used to subcutaneously administer relatively large volumes of fluid. In the UK, 1500 units of hyaluronidase are usually given with each 500 to 1000 ml of fluid for subcutaneous use. In the US, 150 units per liter of subcutaneous infusion solution is considered appropriate. In Germany, 150 to 300 units are considered appropriate for this purpose. In the UK, the diffusion of local anesthetic is accelerated by the addition of 1500 units. In Germany and the US, 150 units are considered appropriate for this purpose. Despite differences in dosage (the UK dosage is 10 times greater than the US dosage), no significant differences have been reported in the safety profiles of animal-derived hyaluronidase products marketed in the US and the UK, respectively. On December 2, 2005, Halozyme Therapeutics Inc. introduced recombinant human hyaluronidase, rHuPH20 (HYLENEX TM ) received FDA approval. The FDA approved HYLENEX at a dose of 150 units for SC administration in the following conditions: TM Approved: -As an adjuvant to increase the absorption and distribution of other injected drugs -Subcutaneous injection -As an adjunct to SC urography to improve reabsorption of radiopaque agents.

[0130] As part of that regulatory information, it was established that rHuPH20 retains the same properties of enhancing the dispersion and absorption of other injected drugs as previously approved animal-derived hyaluronidase preparations, but with an improved safety profile. Notably, the use of recombinant human hyaluronidase (rHuPH20) minimizes the risk of contamination with animal pathogens and the potential risk of transmissible spongiform encephalopathies compared to animal-derived hyaluronidase.

[0131] Soluble hyaluronidase glycoprotein (sHASEGP), methods for its preparation, and its use in pharmaceutical compositions are described in WO 2004 / 078140. Detailed experimental work outlined below has shown that the claimed formulation surprisingly has favorable storage stability and meets all requirements necessary for approval by health authorities.

[0132] The hyaluronidase enzyme in the formulations of the present invention is believed to enhance the delivery of anti-PD-L1 antibodies into the systemic circulation, for example, by increasing the absorption of the active agent (its action as a penetration enhancer). The hyaluronidase enzyme is also believed to increase the delivery of therapeutic anti-PD-L1 antibodies into the systemic circulation via the subcutaneous route of administration by reversible hydrolysis of hyaluronan, an extracellular component of SC interstitial tissue. Hydrolysis of hyaluronan in the subcutaneous tissue temporarily opens channels within the interstitial space of the SC tissue, thereby improving the delivery of therapeutic anti-PD-L1 antibodies into the systemic circulation. In addition, administration has been shown to reduce pain and volume-induced SC tissue swelling in humans.

[0133] When administered locally, hyaluronidase has its entire effect locally. In other words, hyaluronidase is inactivated and metabolized locally within minutes, and has not been observed to have systemic or long-term effects. The rapid inactivation of hyaluronidase within minutes upon entry into the bloodstream virtually precludes the possibility of conducting comparable biodistribution studies between different hyaluronidase products. This characteristic also minimizes any potential for overall safety concerns, as hyaluronidase products cannot act at distant sites. A unifying feature of all hyaluronidase enzymes provided herein is their ability to depolymerize hyaluronan, regardless of differences in chemical structure, species source, tissue source, or batches of pharmaceutical products sourced from the same species and tissue. They are unusual in the fact that their activity (except for potency) is the same despite their different structures. The hyaluronidase enzymes of the formulations of the present invention are characterized by their lack of adverse effects on the molecular integrity of the anti-PD-L1 antibodies in the stable pharmaceutical formulations described herein. Furthermore, hyaluronidase enzymes only regulate the delivery of anti-PD-L1 antibodies into the systemic circulation and do not possess properties that provide or contribute to the therapeutic effects of systemically absorbed anti-PD-L1 antibodies. Hyaluronidase enzymes are not systemically bioavailable and do not adversely affect the molecular integrity of anti-PD-L1 antibodies under the recommended storage conditions of stable pharmaceutical formulations according to the invention. Therefore, hyaluronidase enzymes are considered additives in anti-PD-L1 antibody formulations according to the invention. Because they do not exert a therapeutic effect, hyaluronidase enzymes represent a component of the pharmaceutical formulation separate from the therapeutically active anti-PD-L1 antibody. Several suitable hyaluronidase enzymes according to the invention are known in the art. In some embodiments, the enzyme is a human hyaluronidase enzyme, such as the enzyme known as rHuPH20. rHuPH20 is a member of a family of neutral and acid-active β-1,4 glycosyl hydrolases that depolymerize hyaluronan by hydrolysis of the β-1,4 bond between the Ci position of N-acetylglucosamine and the C4 position of glucuronic acid.Hyaluronan is a polysaccharide found in connective tissues, such as subcutaneous interstitial tissue, and in the intracellular matrix of some specialized tissues, such as the umbilical cord and vitreous humor. Hyaluronan hydrolysis temporarily reduces the viscosity of interstitial tissue, promoting the dispersion of injected fluids or localized filtrates or exudates, thereby facilitating their absorption. The effect of hyaluronidase is local and reversible with complete reconstitution of tissue hyaluronan occurring within 24 to 48 hours (Frost, GI, "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration," Expert Opinion on Drug Delivery, 2007;4:427-440). Increased connective tissue permeability through hyaluronan hydrolysis correlates with the effectiveness of hyaluronidase in enhancing the dispersion and absorption of co-administered molecules.

[0134] The human genome contains multiple hyaluronidase genes. Only the PH20 gene product retains effective hyaluronidase activity and acts as a spreading agent under physiological extracellular conditions; acid-active hyaluronidases lack this property. rHuPH20 is the first and only recombinant human hyaluronidase enzyme currently available for therapeutic use. The human genome contains multiple hyaluronidase genes; only the PH20 gene product retains effective hyaluronidase activity and acts as a spreading agent under physiological extracellular conditions. The naturally occurring human PH20 protein has a lipid anchor attached to amino acids at the carboxy terminus that anchor it to the plasma membrane. The rHuPH20 enzyme developed by Halozyme is a truncated deletion strain lacking such amino acids at the carboxy terminus responsible for lipid binding. It produces a soluble, neutral pH-active enzyme similar to the protein found in bovine testis preparations. The rHuPH20 protein is synthesized with a 35 amino acid signal peptide that is removed from the N-terminus during secretion. The mature rHuPH20 protein contains an authentic N-terminal amino acid sequence orthologous to that found in several bovine hyaluronidase preparations.

[0135] PH20 hyaluronidase, including animal-derived PH20 and recombinant human rHuPH20, depolymerizes hyaluronan by hydrolysis of the β-1,4 bond between the C1 position of N-acetylglucosamine and the C4 position of glucuronic acid. Tetrasaccharides are the smallest digestion product (Weissmann, B., "The transglycosylative action of testicular hyaluronidase," J. Biol. Chem., 1955;216:783-94). This N-acetylglucosamine / glucuronic acid structure is not found in the N-linked glycans of recombinant biological products, and therefore rHuPH20 will not affect the glycosylation of antibodies formulated with it. The rHuPH20 enzyme itself possesses six N-linked glycans per molecule with a core structure similar to that found in monoclonal antibodies. As expected, these N-linked structures do not change over time, confirming the absence of enzymatic activity of rHuPH20 on these N-linked glycan structures. The short half-life of rHuPH20 and the constant synthesis of hyaluronan ensure that the enzyme's action on tissues is brief and localized.

[0136] The hyaluronidase enzyme additive in the subcutaneous formulation of the present invention can be prepared using recombinant DNA technology. This ensures that the same protein (identical amino acid sequence) is always obtained, avoiding allergic reactions caused by contaminating proteins that co-purify during extraction from tissue. In some embodiments, the hyaluronidase enzyme used in the formulation of the present invention is a human enzyme, such as rHuPH20. rHuPH20 (HYLENEX TM The amino acid sequence of ) is well known and available under CAS Registry Number 757971-58-7. The approximate molecular weight is 61 kDa (see also U.S. Patent No. 7,767,429).

[0137] Multiple structural and functional comparisons have been performed between natural-source mammalian hyaluronidases and PH-20 cDNA clones from humans and other mammals. The PH-20 gene is the gene used for the recombinant product rHuPH20; however, the recombinant drug product is a 447-amino acid truncated version of the complete protein encoded by the PH-20 gene. Structural similarity in amino acid sequence rarely exceeds 60% in any comparison. Functional comparisons demonstrate that the activity of rHuPH20 is highly similar to that of previously approved hyaluronidase products. This information is consistent with clinical findings over the past 50 years, and the clinical safety and efficacy of hyaluronidase units are comparable, regardless of the source of the hyaluronidase. The use of rHuPH20 in anti-PD-L1 antibody SC formulations according to the present invention may allow for the administration of higher drug doses and potentially enhance the absorption of subcutaneously administered anti-PD-L1 antibodies, such as atezolizumab, into the systemic circulation.

[0138] The use of small amounts of soluble hyaluronidase glycoproteins (sHASEGPs) has been shown to facilitate subcutaneous injection of therapeutic proteins and antibodies; see WO 2006 / 091871. The addition of such soluble hyaluronidase glycoproteins (as combined formulations or by coadministration) has been shown to facilitate the administration of therapeutic agents to the subcutaneous tissue. By rapidly depolymerizing hyaluronan HA in the extracellular space, sHASEGPs reduce interstitial viscosity, thereby increasing hydraulic conductivity and allowing larger volumes to be safely and comfortably administered into the subcutaneous tissue. The increased hydraulic conductivity induced by sHASEGPs through reduced interstitial viscosity allows for greater dispersion and increased systemic bioavailability of SC-administered therapeutic agents.

[0139] In some embodiments, the formulations described herein comprise an effective amount of at least one hyaluronidase enzyme (e.g., rHuPH20), for example, in an amount of about 1000 U / ml to about 5000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 1000 U / ml to about 4000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 1000 U / ml to about 3000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 1000 U / ml to about 2000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 2000 U / ml to about 4000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 2000 U / ml to about 3000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 1500 U / ml to about 3000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 1500 U / ml to about 2500 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 1500 U / ml to about 2000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 2000 U / ml to about 2500 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 1750 U / ml to about 2250 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 1900 U / ml to about 2100 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 1950 U / ml to about 2050 U / ml.In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of about 2000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 1000 U / ml to 4000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 1000 U / ml to 3000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 1000 U / ml to 2000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 2000 U / ml to 4000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 2000 U / ml to 3000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 1500 U / ml to 3000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 1500 U / ml to 2500 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 1500 U / ml to 2000 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 2000 U / ml to 2500 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 1750 U / ml to 2250 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 1900 U / ml to 2100 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 1950 U / ml to about 2050 U / ml. In some embodiments, the hyaluronidase enzyme (e.g., rHuPH20) is present in the formulation at a concentration of 2000 U / ml.

[0140] Liquid pharmaceutical formulations of the invention containing hyaluronidase enzyme are particularly suitable for subcutaneous injection. Those skilled in the art will appreciate that such formulations containing an anti-PD-L1 antibody and hyaluronidase enzyme may be provided for administration in the form of a single combined formulation, or alternatively, in the form of two separate formulations that can be mixed immediately prior to subcutaneous injection. Alternatively, the anti-PD-L1 antibody and hyaluronidase enzyme can be administered as separate injections at different sites in the body, for example, sites directly adjacent to each other. The therapeutic agents present in formulations according to the invention can also be injected as a continuous infusion, for example, by injecting the hyaluronidase enzyme first, followed by the anti-PD-L1 antibody formulation. The injections can also be performed in the reverse order, i.e., by injecting the anti-PD-L1 antibody formulation first, followed by the hyaluronidase enzyme. When the anti-PD-L1 antibody and hyaluronidase enzyme are administered as separate injections, one or both of the proteins should be provided with a buffer, one or more stabilizers, and a non-ionic surfactant at the concentrations specified in the dependent claims, except for the hyaluronidase enzyme, which may be provided in, for example, an L-histidine / HCl buffer at a pH of about 6.5, 100-150 mM NaCl, and 0.01-0.1% (w / v) polysorbate 20 or polysorbate 80. In one embodiment, the anti-PD-L1 antibody is provided with a buffer, one or more stabilizers, and a non-ionic surfactant at the concentrations specified herein.

[0141] As noted above, hyaluronidase enzyme may be considered an additional additive in the anti-PD-L1 antibody formulation. The hyaluronidase enzyme may be added to the anti-PD-L1 antibody formulation during manufacture, or may be added immediately prior to injection. Alternatively, the hyaluronidase enzyme may be provided as a separate injection. When provided as a separate injection, the hyaluronidase enzyme may be provided in a separate vial, either in lyophilized form that must be reconstituted with an appropriate diluent prior to subcutaneous injection, or in a liquid formulation provided by the manufacturer. The anti-PD-L1 antibody formulation and hyaluronidase enzyme may be procured as separate entities, or may be provided as a kit containing both injection components and appropriate instructions for their subcutaneous administration. Appropriate instructions for reconstitution and / or administration of one or both of the formulations may also be provided.

[0142] Accordingly, the present invention also provides a highly concentrated and stable pharmaceutical formulation of a pharmaceutically active anti-PD-L1 antibody, or a pharmaceutical composition comprising an admixture of such an antibody and a suitable amount of at least one hyaluronidase enzyme, in the form of a kit containing both injection components and suitable instructions for their subcutaneous administration.

[0143] A further aspect of the invention relates to an injection device comprising a liquid pharmaceutical formulation according to the invention. Such formulations may consist of a mixture of a pharmaceutically active anti-PD-L1 antibody or such antibody molecule as outlined herein with suitable excipients, and may additionally include a hyaluronidase enzyme, either as a combined formulation or as a separate formulation to be co-administered.

[0144] In some embodiments, provided herein is a liquid pharmaceutical formulation comprising a monoclonal anti-PD-L1 antibody described herein at a concentration of about 100 g / L to about 150 g / L, histidine acetate at a concentration of about 15 mM to about 25 mM, sucrose at a concentration of about 200 mM to about 280 mM, polysorbate at a concentration of about 0.04% (w / v) to about 0.08% (w / v), methionine at a concentration of about 5 mM to about 15 mM, and hyaluronidase enzyme at a concentration of about 1000 U / ml to about 3000 U / ml, and having a pH of about 5.6 to about 6.0. In some embodiments, the formulation is sterile. In some embodiments, the formulation is suitable for administration to a subject. In some embodiments, the formulation is for subcutaneous administration.

[0145] In some embodiments, provided herein is a liquid pharmaceutical formulation comprising a monoclonal anti-PD-L1 antibody described herein at a concentration of about 125 g / L, histidine acetate at a concentration of about 20 mM, sucrose at a concentration of about 240 mM, polysorbate 20 at a concentration of about 0.06% (w / v), methionine at a concentration of about 10 mM, rHuPH20 at a concentration of about 2000, and a pH of about 5.8. In some embodiments, the formulation is sterile. In some embodiments, the formulation is suitable for administration to a subject. In some embodiments, the formulation is for subcutaneous administration.

[0146] In some embodiments, provided herein is a liquid pharmaceutical formulation comprising a monoclonal anti-PD-L1 antibody described herein at a concentration of about 100 g / L to about 150 g / L, histidine acetate at a concentration of about 15 mM to about 25 mM, sucrose at a concentration of about 200 mM to about 280 mM, polysorbate at a concentration of about 0.01% (w / v) to about 0.03% (w / v), and a pH of about 5.3 to about 5.7. In some embodiments, the formulation is mixed with a hyaluronidase enzyme prior to administration to a subject. In some embodiments, the concentration of the hyaluronidase enzyme in the mixture is about 1000 U / ml to about 3000 U / ml. In some embodiments, the formulation is sterile. In some embodiments, the formulation is suitable for administration to a subject. In some embodiments, the formulation is for subcutaneous administration.

[0147] In some embodiments, provided herein is a liquid pharmaceutical formulation comprising a monoclonal anti-PD-L1 antibody described herein at a concentration of about 125 g / L, histidine acetate at a concentration of about 20 mM, sucrose at a concentration of about 240 mM, polysorbate 20 at a concentration of about 0.02% (w / v), and a pH of about 5.5. In some embodiments, the formulation is mixed with rHuPH20 prior to administration to a subject. In some embodiments, the concentration of rHuPH20 in the mixture is about 2000 U / L. In some embodiments, the formulation is sterile. In some embodiments, the formulation is suitable for administration to a subject. In some embodiments, the formulation is for subcutaneous administration.

[0148] In one embodiment, the formulation contains the agents defined above (e.g., antibody, buffer, sucrose, and / or surfactant) and is essentially free of one or more preservatives, such as benzyl alcohol, phenol, m-cresol, chlorobutanol, and benzethonium Cl. In another embodiment, a preservative may be included in the formulation, particularly when the formulation is a multi-dose formulation. The concentration of the preservative may range from about 0.1% to about 2%, for example, from about 0.5% to about 1%. One or more other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), may be included in the formulation, provided that they do not adversely affect the desired properties of the formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include: additional buffering agents; cosolvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters, and / or salt-forming counterions. Exemplary pharmaceutically acceptable carriers herein further include intercalating drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International). Some exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0149] The formulations herein may also contain two or more proteins as necessary for the particular indication being treated, e.g., those with complementary activities that do not adversely affect the other protein. For example, an antibody, if it is anti-PD-L1, may be combined with another agent (e.g., a chemotherapeutic agent and an anti-neoplastic agent).

[0150] In some embodiments, the physical stability, chemical stability, or biological activity of the antibody in the formulation is assessed or measured. Any method known in the art, including those described in the Examples herein, may be used to assess the stability and biological activity of the antibody in the formulation. For example, the stability of the antibody in the formulation can be measured by, but is not limited to, size exclusion chromatography (SEC or SE-HPLC), imaging capillary isoelectric focusing (ICIEF), peptide mapping, small-volume light obscuration (HIAC) assay, and capillary electrophoresis (CE) techniques, such as CE-sodium dodecyl sulfate (CE-SDS) and CE-glycan analysis. In some embodiments, the antibody in the formulation is stable at -20°C for at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 14 months, at least about 16 months, at least about 18 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, at least about 24 months, at least about 3 years, or at least about 4 years. In some embodiments, the antibody in the formulation is stable at 2°C to 8°C (e.g., 5°C) for at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 14 months, at least about 16 months, at least about 18 months, at least about 20 months, at least about 21 months, at least about 22 months, at least about 23 months, or at least about 24 months. In some embodiments, the stability of the antibody (i.e., antibody monomer) in the formulation after storage is measured by size exclusion chromatography. In some embodiments, the stability of the antibody (i.e., antibody monomer) in the formulation after storage is measured by imaging capillary isoelectric focusing. In some embodiments, the percent of antibody monomer in the formulation compared to total protein (e.g., including antibody and aggregates) is greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% after storage at -20°C for at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months.In some embodiments, the percent of antibody monomer in the formulation (e.g., compared to antibody and aggregates) is greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% after storage at 2°C to 8°C (e.g., 5°C) for at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months. In some embodiments, the percent of antibody monomer in the formulation (e.g., compared to antibody and aggregates) is greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% after shaking at room temperature (e.g., about 15°C to 25°C) for at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, or at least about 24 hours. In some embodiments, the percent of total aggregates (e.g., high molecular weight species and low molecular weight species) in the formulation is less than any of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% after storage at -20°C for at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months. In some embodiments, the percent of total aggregates (e.g., high molecular weight species and low molecular weight species) in the formulation is less than any of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% after storage at 2°C to 8°C (e.g., 5°C) for at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months.In some embodiments, the percent of total aggregates (e.g., high molecular weight species and low molecular weight species) in the formulation is less than any of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% after shaking at room temperature (e.g., about 15° C. to 25° C.) for at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, or at least about 24 hours. In any of the embodiments herein, the stable formulations can be stored in glass vials, metal alloy containers, or intravenous (IV) bags. In some embodiments, the metal alloy is 316L stainless steel or Hastelloy.

[0151] Formulations to be used for in vivo administration should be sterile, which is readily accomplished by filtration through sterile filtration membranes, either before or after preparation of the formulation.

[0152] III. Methods of Treatment and Administration of Antibody Formulations The formulation is administered to a mammal, e.g., a human, in need of treatment with the antibody according to known methods, such as intravenous administration (as a bolus or by continuous infusion over a period of time), intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, buccal, topical, or by inhalation routes. In one embodiment, the formulation is administered to the mammal by intravenous administration. For such purposes, the formulation can be injected, for example, using a syringe or via an intravenous line. In one embodiment, the formulation is administered to the mammal by subcutaneous administration.

[0153] The appropriate dosage of antibody (a "therapeutically effective amount") will depend, for example, on the condition being treated, the severity and course of the condition, whether the antibody is being administered prophylactically or therapeutically, previous therapy, the patient's medical history and response to the antibody, the type of antibody used, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments, and may be administered to the patient at any time from the time of diagnosis. The antibody may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question.

[0154] As a general proposition, a therapeutically effective amount of an antibody administered to a human, whether administered as a single dose or multiple doses, will be in the range of about 0.01 to about 50 mg / kg / kg of patient body weight. In some embodiments, the antibody is used at a daily dose of, for example, about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg. In some embodiments, the antibody is administered at 15 mg / kg. However, other dosing regimens may be useful. In one embodiment, an anti-PD-L1 antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg on day 1 of a 21-day cycle. The dose may be administered in a single dose or multiple doses (e.g., two or three doses), such as by infusion. The dose of an antibody administered in combination therapy may be reduced compared to monotherapy. The progress of this therapy is easily monitored by conventional techniques.

[0155] Formulations containing the anti-PD-L1 antibodies described herein can be used in a variety of in vitro and in vivo diagnostic and therapeutic applications. For example, the antibody-containing formulations can be administered to a subject or individual to treat a disease or disorder (e.g., a disease or disorder mediated by the interaction of PD-1 and PD-L1).

[0156] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is locally advanced or metastatic. In some embodiments, the cancer is selected from the group consisting of solid tumors, hematological cancers, bladder cancer, brain cancer, breast cancer, colon cancer, colorectal cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, renal cancer, salivary gland cancer, stomach cancer, thymic reticular carcinoma, thyroid cancer, and squamous cell carcinoma of the head and neck. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is small cell lung cancer. In some embodiments, the cancer is urothelial carcinoma. In some embodiments, the cancer is breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer. In some embodiments, the subject or individual being treated has PD-L1 positive cancer cells (eg, as detected by IHC).

[0157] In some embodiments, the disease or disorder is an infectious disease. In some embodiments, the infectious disease is a persistent infection. In some embodiments, the infectious disease is a viral infection, a bacterial infection, a fungal infection, a helminth infection, or a protozoan infection. In some embodiments, the viral infection is selected from the group consisting of cytomegalovirus, Epstein-Barr virus, hepatitis B, hepatitis C virus, herpes virus, measles virus, influenza, human immunodeficiency virus, human T-lymphotropic virus, lymphocytic choriomeningitis virus, respiratory syncytial virus, and / or rhinovirus. In some embodiments, the bacterial infection is selected from the group consisting of Helicobacter spp., Mycobacterium spp., Porphyromonas spp., Chlamydia spp., Salmonella spp., Listeria spp., Streptococcus spp., Haemophilus spp., Neisseria spp., Klebsiella spp., Borrelia spp., Bacteroides spp., and Treponema spp. In some embodiments, the protozoal infection is selected from the group consisting of Leishmania spp., Plasmodium falciparum, Schistosoma spp., Toxoplasma spp., Trypanosoma spp., and Cestoda spp. In some embodiments, the fungal infection is selected from the group consisting of blastomycosis, coccidioidomycosis, histoplasmosis, candidiasis, cryptococcosis, aspergillosis, mucormycosis, and pneumocystis.

[0158] In some embodiments, the disease or disorder is an inflammatory disease, hi some embodiments, the inflammatory disease is selected from the group consisting of acute disseminated encephalomyelitis, Addison's disease, Alzheimer's disease, ankylosing spondylitis, antiphospholipid syndrome, atherosclerosis, autoimmune hemolytic anemia, autoimmune hepatitis, arthritis, Behcet's disease, Berger's disease, bullous pemphigoid, celiac disease, Chagas' disease, cholangitis, Crohn's disease, dermatomyositis, type 1 diabetes, glomerulonephritis, Goodpasture's syndrome, graft-versus-host disease, Graves' disease, and Gila syndrome. The disease is selected from the group consisting of: urticaria, urticaria, hyper-IgE syndrome, idiopathic thrombocytopenic purpura, lupus erythematosus, lupus nephritis, multiple sclerosis, myasthenia gravis, organ transplant rejection, Parkinson's disease, pemphigus, pernicious anemia, polymyositis, primary biliary cirrhosis, psoriasis, Raynaud's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, temporal arteritis, thyroiditis, ulcerative colitis, uveitis, vasculitis, and Wegener's granulomatosis.

[0159] In some embodiments, the antibody-containing formulations may be administered to a subject or individual in conjunction with another therapeutic agent to treat a disease or disorder. For example, to treat cancer, the anti-PD-L1 antibody formulations described herein may be administered in conjunction with another anti-cancer treatment (e.g., chemotherapy or a different antibody therapy).

[0160] IV. Manufactured Articles or Kits In another embodiment of the present invention, an article of manufacture or kit is provided that includes a container holding a liquid pharmaceutical formulation of the present invention, and optionally provides instructions for its use. Suitable containers include, for example, bottles, vials, bags, and syringes. The container can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloy (e.g., stainless steel or Hastelloy). An exemplary container is a 300 cc metal alloy container (e.g., for storage at -20°C). Another exemplary container can be a 10-50 cc glass vial (e.g., for storage at 2-8°C). For example, the container can be a 10 cc, 15 cc, 20 cc, or 50 cc glass vial. The container holds the formulation, and a label on or associated with the container can display instructions for use. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further comprises one or more additional pharmaceutical agents (e.g., chemotherapeutic agents and anti-neoplastic agents). Suitable containers for the one or more pharmaceutical agents include, for example, bottles, vials, bags, and syringes.

[0161] The specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0162] The present invention will be more fully understood by reference to the following examples. However, the examples should not be construed as limiting the scope of the present invention. It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art in light thereof, which are to be included within the spirit and scope of this application and the scope of the claims. [Example]

[0163] Example 1: Stability of Drug Substance (DS) Formulation For DS studies, the formulations were filled into stainless steel mini-cans and placed under appropriate storage conditions to evaluate frozen storage and promote stability of atezolizumab. To formulate the DS for study, the atezolizumab ultrafiltration diafiltration pool was buffer exchanged into an appropriate buffer system (e.g., histidine acetate, histidine hydrochloride, histidine acetate with arginine), and then polysorbate 20 and methionine were added to the ultrafiltration diafiltration material to formulate the DS.

[0164] DS stability after multiple freeze-thaw cycles Figures 1A-1C show the levels of high molecular weight species (HMWS) (Figure 1A), the percentage of the main peak in ion exchange chromatography (IEC) (Figure 1B), and the pre-peak sum in non-reducing capillary electrophoresis-SDS (NR CE-SDS) (Figure 1C) for various DS formulations after multiple freeze-thaw cycles. All formulations contained 150 mg / ml or 125 mg / ml atezolizumab (indicated as 150 mg or 125 mg in the figure), 20 mM histidine acetate (HA) or histidine hydrochloride (HCl), 10 mM methionine, and 0.06% (w / v) polysorbate 20. Unless otherwise noted, all formulations were at pH 5.5. Formulations containing low sucrose concentrations (e.g., 100 mM) were insufficient to maintain stability through multiple freeze-thaw cycles at high protein concentrations. As a result of these experiments, a sucrose concentration of 240 mM was selected for formulations composed of histidine acetate or histidine hydrochloride to support five freeze / thaw (F / T) cycles.

[0165] DS stability at 25℃ Figures 2A-2C show the levels of acidic species (Figure 2A), basic species (Figure 2B), and HMWS (Figure 2C) for various DS formulations after 1 month at 25°C. All formulations contained 125 mg / ml atezolizumab (denoted as 125 mg in the figure), 20 mM histidine acetate (HA) or histidine hydrochloride (HCl), 10 mM methionine, and 0.06% (w / v) polysorbate 20. IEC showed a lower percentage of acidic and a higher percentage of basic in the pH 5.5 formulations.

[0166] Example 2: Stability of Drug Product (DP) Formulations For DP stability studies, the formulations were filled into glass vials and placed under appropriate storage conditions to evaluate the stability of atezolizumab in different buffer systems and additives. To formulate, the atezolizumab ultrafiltration diafiltration pool was buffer exchanged into an appropriate buffer system (e.g., histidine acetate, histidine hydrochloride, histidine acetate with arginine), and then polysorbate 20, methionine, and recombinant human hyaluronidase were added to the ultrafiltration diafiltration material to formulate the DP.

[0167] DP stability at 25°C Figures 3A-3B show the HMWS levels (Figure 3A) and the percentage of the main SEC peak (Figure 3B) for DP formulations after 3 months at 25°C. All formulations contained 125 mg / ml atezolizumab (shown as 125 mg in the figure), 20 mM histidine acetate or histidine hydrochloride, 240 mM sucrose, 10 mM methionine, 0.06% polysorbate 20, and 2000 U / ml recombinant human hyaluronidase (rHuPH20). The formulation containing histidine acetate at pH 5.8 had a higher percentage of the main SEC peak compared to the other two formulations (Figure 3B). The formulation containing histidine hydrochloride at pH 5.5 had a higher percentage of HMWS compared to the other two formulations (Figure 3A). Overall, the histidine acetate formulations were slightly slower to degrade by SEC when compared to the histidine hydrochloride formulations.

[0168] Figures 4A-4B show the levels of acidic species (Figure 4A) and basic species (Figure 4B) in DP formulations after 3 months at 25°C. All formulations contained 125 mg / ml atezolizumab (denoted as 125 mg in the figure), 20 mM histidine acetate or histidine hydrochloride, 240 mM sucrose, 10 mM methionine, 0.06% polysorbate 20, and 2000 U / ml recombinant human hyaluronidase (rHuPH20). Overall, the main peak degradation rates of IECs were similar among all three formulations. However, the formation of acidic and basic species differed among the three formulations. The formulation containing histidine acetate at pH 5.8 had a lower percentage of basic species compared to the other two formulations (Figure 4B). Formulations containing histidine acetate had a higher percentage of acidic species compared to formulations containing histidine hydrochloride (Figure 4A).

[0169] Figures 5A-5B show the percentage of the pre-peak (Figure 5A) and the main peak of NR CE-SDS (Figure 5B) in the DP formulations after up to 3 months at 25° C. All formulations contained 125 mg / ml atezolizumab (shown as 125 mg in the figure), 20 mM histidine acetate or histidine hydrochloride, 240 mM sucrose, 10 mM methionine, 0.06% polysorbate 20, and 2000 U / ml recombinant human hyaluronidase (rHuPH20).

[0170] DP stability at 40℃ Figures 6A-6C show the levels of HMWS (Figure 6A), the percentage of the main peak in SEC (Figure 6B), and the total NR-CE-SDS pre-peak (Figure 6C) in DP formulations after 1 month at 40°C. All formulations contained 150 mg / ml or 125 mg / ml atezolizumab (indicated as 150 mg or 125 mg in the figure), 200-240 mM sucrose, 10 mM methionine, 0.06% polysorbate 20, and 2000 U / ml recombinant human hyaluronidase (rHuPH20). The formulation containing 125 mg / ml atezolizumab and histidine acetate had lower HMWS (Figure 6A), a higher percentage of the main peak (Figure 6B), and a lower total NR-CE-SDS pre-peak (Figure 6C) than the other formulations. With increasing protein concentration, HMWS also increased. From the viewpoint of pH, higher pH (e.g., 5.8) reduced HMWS formation and fragmentation. The addition of arginine also contributed to the increase in HMWS. Although arginine could increase solubility, it could not maintain the physical stability of atezolizumab (e.g., increased HMWS).

[0171] Figures 7A-7C show the levels of acidic species (Figure 7A), basic species (Figure 7B), and the percentage of the main peak of IEC (Figure 7C) in DP formulations after 1 month at 40°C. All formulations contained 150 mg / ml or 125 mg / ml atezolizumab (denoted as 150 mg or 125 mg in the figure), 200-240 mM sucrose, 10 mM methionine, 0.06% polysorbate 20, and 2000 U / ml recombinant human hyaluronidase (rHuPH20). Formulations containing histidine acetate buffer had higher levels of acidic species compared to formulations containing histidine hydrochloride buffer or histidine acetate + arginine buffer (Figure 7A). The formulation containing 125 mg / ml atezolizumab and histidine acetate at pH 5.8 had lower levels of basic species compared to the other formulations (Figure 7B).

[0172] Based on the product stability results presented, a formulation containing 125 mg / ml atezolizumab and histidine acetate at pH 5.8 was selected for formulation of atezolizumab.

[0173] Example 3: Stability of Polysorbate 20 Figures 8A-8B show the stability of polysorbate 20 in various DP formulations for up to 3 months at 40°C (Figure 8A) and 25°C (Figure 8B). All formulations contained 125 mg / ml atezolizumab (shown as 125 mg in the figure), 20 mM histidine acetate or histidine hydrochloride, 240 mM sucrose, 10 mM methionine, 0.06% polysorbate 20, and 2000 U / ml recombinant human hyaluronidase (rHuPH20). Polysorbate 20 showed less degradation in the pH 5.8 formulation with histidine acetate compared to the other two formulations at both 40°C and 25°C after 3 months.

[0174] Data from the 25° C. experiment described above was used to calculate the theoretical amount of polysorbate 20 loss after 6 months at 25° C. As shown in the table below, polysorbate 20 is expected to show less degradation after 6 months in the pH 5.8 formulation with histidine acetate compared to the other two formulations. TIFF2026009902000003.tif41170

[0175] In addition to atezolizumab product stability, the pH 5.8 formulation composed of histidine acetate most effectively maintained polysorbate 20 stability.

[0176] Example 4: Activity of rHuPH20 Figures 9A-9B show rHuPH20 activity assays using various DP formulations at 25°C for up to 3 months. All formulations contained 150 mg / ml or 125 mg / ml atezolizumab (denoted as 150 mg or 125 mg in the figures), 20 mM histidine acetate or histidine hydrochloride, 240 mM sucrose, 10 mM methionine, 0.06% polysorbate 20, and 2000 U / ml recombinant human hyaluronidase (rHuPH20). Formulations containing histidine acetate at pH 5.8 maintained rHuPH20 activity at higher levels than formulations containing histidine acetate at pH 5.5. Increasing the pH also increased the stability of rHuPH20 at 25°C. The histidine hydrochloride formulation provided better stability of rHuPH20 compared to the other formulations at accelerated conditions (Figure 9B), but histidine hydrochloride was not suitable for atezolizumab. A slight decrease in rHuPH20 activity was observed for the histidine acetate formulation at accelerated conditions, but this was not observed upon storage at 5°C. Consequently, a pH 5.8 formulation composed of histidine acetate was selected for atezolizumab.

[0177] Figures 10A-10B show rHuPH20 activity in formulations containing different concentrations of polysorbate after 24 hours of shaking. Higher concentrations of polysorbate maintained higher levels of rHuPH20 activity under shaking at room temperature. A minimum of 0.03% (w / v) polysorbate 20 is required to prevent loss of rHuPH20 due to shaking. Considering polysorbate 20 release criteria and possible polysorbate degradation during shelf life, a polysorbate 20 level of 0.06% (w / v) was selected for the formulation.

[0178] Example 5: Viscosity of Pharmaceutical (DP) Formulations Figure 11 shows the viscosity of various DP formulations at temperatures between 5°C and 25°C. All formulations contained 127-128 mg / ml atezolizumab, 20 mM histidine acetate or histidine hydrochloride, 240 mM sucrose, 10 mM methionine, 0.06% polysorbate 20, and 2000 U / ml recombinant human hyaluronidase (rHuPH20). The formulation containing histidine hydrochloride had the highest viscosity at all temperatures evaluated.

[0179] Based on the screening of formulations described in these Examples, a DP formulation containing 125 mg / ml atezolizumab, 20 mM histidine acetate, 240 mM sucrose, 10 mM methionine, 0.06% polysorbate 20, and 2000 U / ml recombinant human hyaluronidase (rHuPH20) at pH 5.8 was selected for the atezolizumab formulation for subcutaneous administration.

Claims

1. 1. A liquid pharmaceutical formulation comprising a monoclonal anti-PD-L1 antibody at a concentration of about 100 g / L to about 150 g / L, histidine acetate at a concentration of about 15 mM to about 25 mM, sucrose at a concentration of about 200 mM to about 280 mM, polysorbate at a concentration of about 0.04% (w / v) to about 0.08% (w / v), methionine at a concentration of about 5 mM to about 15 mM, and a pH of about 5.6 to about 6.0, wherein the monoclonal antibody is (a) a light chain variable region comprising: (1) HVR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO: 1); (2) HVR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO: 2); (3) HVR-L3 comprising the amino acid sequence QQYLYHPAT (SEQ ID NO: 3); and (b) a heavy chain variable region comprising: (1) HVR-H1 comprising the amino acid sequence GFTFSDSWIH (SEQ ID NO: 4); (2) HVR-H2 comprising the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 5); (3) HVR-H3 containing the amino acid sequence WPGGFDY (SEQ ID NO: 6) 10. A liquid pharmaceutical formulation comprising:

2. 10. The liquid pharmaceutical formulation of claim 1, wherein the monoclonal antibody in the formulation is at a concentration of about 120 g / L to about 130 g / L.

3. 10. The liquid pharmaceutical formulation of claim 1, wherein the monoclonal antibody in the formulation is at a concentration of about 125 g / L.

4. 4. The liquid pharmaceutical formulation of claim 1, wherein the histidine acetate is at a concentration of about 17 mM to about 22 mM.

5. 4. The liquid pharmaceutical formulation of claim 1, wherein the histidine acetate is at a concentration of about 20 mM.

6. 6. The liquid pharmaceutical formulation of claim 1, wherein the sucrose is at a concentration of about 220 mM to about 260 mM.

7. 6. The liquid pharmaceutical formulation of claim 1, wherein the sucrose is at a concentration of about 240 mM.

8. 8. The liquid pharmaceutical formulation of claim 1, wherein the pH is about 5.

8.

9. 9. A liquid pharmaceutical formulation according to any one of claims 1 to 8, wherein the polysorbate in the formulation is polysorbate 20.

10. 10. The liquid pharmaceutical formulation of claim 1, wherein the polysorbate is at a concentration of about 0.05% (w / v) to about 0.07% (w / v).

11. 10. The liquid pharmaceutical formulation of claim 1, wherein the polysorbate is at a concentration of about 0.06% (w / v).

12. 12. The liquid pharmaceutical formulation of any one of claims 1 to 11, wherein methionine is at a concentration of about 10 mM.

13. 13. The liquid pharmaceutical formulation of any one of claims 1 to 12, further comprising a hyaluronidase enzyme.

14. 14. The liquid pharmaceutical formulation of claim 13, wherein the hyaluronidase enzyme is recombinant human hyaluronidase (rHuPH20).

15. 15. The liquid pharmaceutical formulation of claim 13 or 14, wherein the hyaluronidase enzyme is at a concentration of about 1000 U / ml to about 3000 U / ml.

16. 15. The liquid pharmaceutical formulation of claim 13 or 14, wherein the hyaluronidase enzyme is at a concentration of about 2000 U / ml.

17. 1. A liquid pharmaceutical formulation comprising a monoclonal anti-PD-L1 antibody at a concentration of about 100 g / L to about 150 g / L, histidine acetate at a concentration of about 15 mM to about 25 mM, sucrose at a concentration of about 200 mM to about 280 mM, polysorbate at a concentration of about 0.01% (w / v) to about 0.03% (w / v), and a pH of about 5.3 to about 5.7, wherein the monoclonal antibody is (a) a light chain variable region comprising: (1) HVR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO: 1); (2) HVR-L2 comprising the amino acid sequence SASFLYS (SEQ ID NO: 2); (3) HVR-L3 comprising the amino acid sequence QQYLYHPAT (SEQ ID NO: 3); and (b) a heavy chain variable region comprising: (1) HVR-H1 comprising the amino acid sequence GFTFSDSWIH (SEQ ID NO: 4); (2) HVR-H2 comprising the amino acid sequence AWISPYGGSTYYADSVKG (SEQ ID NO: 5); (3) HVR-H3 containing the amino acid sequence WPGGFDY (SEQ ID NO: 6) 10. A liquid pharmaceutical formulation comprising:

18. 18. The liquid pharmaceutical formulation of claim 17, wherein the monoclonal antibody in the formulation is at a concentration of about 120 g / L to about 130 g / L.

19. 18. The liquid pharmaceutical formulation of claim 17, wherein the monoclonal antibody in the formulation is at a concentration of about 125 g / L.

20. 20. The liquid pharmaceutical formulation of any one of claims 17 to 19, wherein the histidine acetate is at a concentration of about 17 mM to about 22 mM.

21. 20. The liquid pharmaceutical formulation of any one of claims 17 to 19, wherein the histidine acetate is at a concentration of about 20 mM.

22. 22. The liquid pharmaceutical formulation of any one of claims 17 to 21, wherein the sucrose is at a concentration of about 220 mM to about 260 mM.

23. 22. The liquid pharmaceutical formulation of any one of claims 17 to 21, wherein the sucrose is at a concentration of about 240 mM.

24. 24. The liquid pharmaceutical formulation of any one of claims 17 to 23, having a pH of about 5.

5.

25. 25. A liquid pharmaceutical formulation according to any one of claims 17 to 24, wherein the polysorbate in the formulation is polysorbate 20.

26. 26. The liquid pharmaceutical formulation of any one of claims 17 to 25, wherein the polysorbate is at a concentration of about 0.02% (w / v).

27. 27. The liquid pharmaceutical formulation of any one of claims 17 to 26, which is mixed with a hyaluronidase enzyme prior to administration to a subject.

28. 28. The liquid pharmaceutical formulation of claim 27, wherein the hyaluronidase enzyme is recombinant human hyaluronidase (rHuPH20).

29. 29. The liquid pharmaceutical formulation of claim 27 or 28, wherein the concentration of the hyaluronidase enzyme in the mixture is from about 1000 U / ml to about 3000 U / ml.

30. 29. The liquid pharmaceutical formulation of claim 27 or 28, wherein the concentration of the hyaluronidase enzyme in the mixture is about 2000 U / ml.

31. 31. The liquid pharmaceutical formulation of any one of claims 1 to 30, wherein the monoclonal antibody has not been subjected to prior lyophilization.

32. 32. The liquid pharmaceutical formulation of any one of claims 1 to 31, wherein the monoclonal antibody is a humanized antibody.

33. 33. The liquid pharmaceutical formulation of any one of claims 1 to 32, wherein the monoclonal antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

8.

34. 34. The liquid pharmaceutical formulation of any one of claims 1 to 33, wherein the monoclonal antibody is a full-length antibody.

35. 35. The liquid pharmaceutical formulation of claim 34, wherein the monoclonal antibody is an IgG1 antibody.

36. 36. The liquid pharmaceutical formulation of any one of claims 1 to 35, wherein the monoclonal antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 9 and a heavy chain comprising the amino acid sequence of SEQ ID NO:

10.

37. 37. The liquid pharmaceutical formulation of any one of claims 1 to 36, wherein the monoclonal antibody is stored in a glass vial or a metal alloy container.

38. 38. The liquid pharmaceutical formulation of claim 37, wherein the metal alloy is 316L stainless steel or Hastelloy.

39. 39. The liquid pharmaceutical formulation of any one of claims 1 to 38, which is stable at 2-8°C for at least 6 months.

40. 39. The liquid pharmaceutical formulation of any one of claims 1 to 38, which is stable at 2-8°C for at least 12 months.

41. 39. The liquid pharmaceutical formulation of any one of claims 1 to 38, which is stable at 2-8°C for at least 24 months.

42. 42. The liquid pharmaceutical formulation of any one of claims 39 to 41, wherein the antibody in the formulation retains at least about 80% of its biological activity after storage.

43. 43. The liquid pharmaceutical formulation of claim 42, wherein the biological activity is measured by antibody binding to PD-L1.

44. 44. The liquid pharmaceutical formulation of any one of claims 1 to 43, which is sterile.

45. 45. A liquid pharmaceutical formulation according to any one of claims 1 to 44, suitable for administration to a subject.

46. 46. ​​The liquid pharmaceutical formulation of any one of claims 1 to 45, which is for subcutaneous administration.

47. 47. An article of manufacture comprising a container holding the liquid pharmaceutical formulation of any one of claims 1 to 46.

48. 48. The article of manufacture of claim 47, wherein the container is a glass vial or a metal alloy container.

49. 49. The article of manufacture of claim 48, wherein the metal alloy is 316L stainless steel or Hastelloy.

50. 47. A kit comprising a container holding the liquid pharmaceutical formulation of any one of claims 1 to 46.

51. 50. A method of treating a disease or disorder in a subject comprising administering to the subject an effective amount of the liquid pharmaceutical formulation of any one of claims 1 to 46, wherein the disease or disorder is selected from the group consisting of infectious diseases, cancer, and inflammatory diseases.

52. 52. The method of claim 51, wherein the disease or disorder is cancer.

53. 53. The method of claim 52, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, urothelial carcinoma, and breast cancer.

54. 54. The method of claim 53, wherein the breast cancer is triple-negative breast cancer.

55. 55. The method of any one of claims 51 to 54, wherein the subject is a human.

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